Image-based differential multiplexing for multiplexed detection of DNA mutations

Abstract

Provided herein are methods and kits for detecting the presence of DNA mutations in the KRAS, BRAF, CTNNB1, and APC genes. The methods and kits employ microcarriers, each of which has a probe specific for a DNA mutation in the KRAS, BRAF, CTNNB1, or APC gene and an identifier unique to the probe sequence. After isolating and amplifying DNA from a sample, hybridization of the amplified DNA with a probe specific for a DNA mutation coupled to the microcarrier indicates the presence of a DNA mutation in the sample. Because each microcarrier can be identified by detecting the identifier, a multiplex screening assay for multiple mutations in each of the KRAS, BRAF, CTNNB1, and APC genes is provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 432,534, filed December 9, 2016, and U.S. Application Serial No. 15 / 836,809, filed December 8, 2017, each of which is incorporated herein by reference in its entirety.

[0003] Submit sequence listing as ASCII text file

[0004] The contents of the following submitted ASCII text file are incorporated herein by reference in their entirety: Computer Readable Form (CRF) of a Sequence Listing (File Name: 695502001241SEQLIST.txt, Record Date: December 11, 2017, Size: 121 KB). Technical Field

[0005] The present invention provides a method for multiplex detection of DNA mutations in samples using microcarriers, and a kit related thereto. The microcarriers are encoded with an identifier and include probes for detecting target DNA mutations. Background Art

[0006] Early detection is a crucial factor in reducing the number of deaths attributable to cancer, as the growth and metastasis of more advanced tumors are associated with increased mortality. Most cases of colorectal cancer are sporadic rather than hereditary, but specific mutations in several genes and even types are common in these sporadic cases. For example, the KRAS gene is believed to be mutated in 30-50% of colorectal cancers (Mundade, R. et al. (2014) Oncoscience 1:400-6). Although existing technologies such as colonoscopy and sigmoidoscopy are effective in detecting many types of early colorectal cancer, they suffer from low patient acceptance due to their invasive nature. Alternatives such as stool DNA testing have been tested (Carethers, JM (2014) Clin. Gastroenterol. Hepatol. 12:377-81), but comprehensive tests that simultaneously examine multiple genes with a high level of accuracy are needed rather than single gene tests.

[0007] Immunology and molecular diagnostic assays play a key role in research and clinical settings. It is often necessary to assay a small set of multiple targets to obtain meaningful or bird's-eye-view results to facilitate research or clinical decision-making. This is especially true in the era of genomics and proteomics, where it is believed that abundant genetic markers and / or biomarkers influence or predict specific disease states. In theory, the determination of multiple targets can be achieved by testing each target in parallel or sequentially in different reaction vessels (i.e., multiple singleplexing). However, assays using singleplexing strategies are not only generally cumbersome, but also generally require large sample volumes, especially when the number of targets to be analyzed is large.

[0008] Multiplex determination measures multiple analytes (two or more) simultaneously in a single determination. Multiplex determination is commonly used in high-throughput screening settings, in which many samples can be analyzed at one time. The ability to simultaneously determine multiple analytes and to determine many samples in parallel is a hallmark of multiplex detection, and is also the reason why such determinations have become powerful tools in the field ranging from drug discovery to functional genomics to clinical diagnostics. In contrast to the monoplex method, by merging all targets in the same reaction vessel, since each sample only processes one reaction vessel, the determination is far less troublesome and much easier to perform. Therefore, the volume of the required test sample can be significantly reduced, which is especially important when samples (e.g., tumor tissue, cerebrospinal fluid, or bone marrow) are difficult to remove in large quantities and / or are invasive. Equally important is the ability to reduce reagent costs and greatly improve determination throughput.

[0009] Many mensuration of complex macromolecular samples are made up of two steps.In the first step, the reagent that can specifically capture target macromolecule is attached on the solid surface.These immobilized molecules can be used for capturing target macromolecule from complex sample by various methods such as hybridization (for example, in the mensuration based on DNA, RNA).In the second step, the complex of detection molecule and capture molecule is hatched together with target, thus emission signal, such as fluorescence or other electromagnetic signal.Then the amount of target by the intensity quantification of those signals.

[0010] Can carry out multiple determination by utilizing multiple capture agents, every kind of capture agent has specificity to different target macromolecules.In the array multiple determination based on chip, every kind of capture agent (for example, single-stranded oligonucleotide probe) is attached to the predetermined position on chip.The amount of the multiple targets in composite sample is measured by measuring the signal of detection molecule at each position of capture agent corresponding to a type.In suspension array multiple determination, microparticle or microcarrier are suspended in the determination solution.These microparticles or microcarrier contain recognition element, and it can be embedded, printed, or otherwise produced by one or more elements of microparticle / microcarrier.Every kind of capture agent is fixed on the particle with identical ID, and the signal emission from the detection molecule on the particle surface with specific ID reflects the amount of corresponding target.

[0011] A kind of application that multiple determination is particularly suitable for is the detection of DNA mutation.Especially, detection of mutations related to cancer can contribute to the early diagnosis of cancer and the detection of tumors in a small and / or not easily found position by conventional diagnostic tools (such as colonoscopy).See, for example, U.S. Patent No. 7,833,757.However, existing diagnostic technology is usually expensive or time-consuming.The method of using continuous, independent determination method to detect multiple gene mutations is time-consuming, and lacks consistency if different determination types are used (see Schneider, M. etc. (2011) Cancers 3:91-105).Multiple determination techniques such as analog coding microcarriers are applied to this problem and can provide a cheaper, faster determination with more accurate results, while enabling multiple screening of many mutations known to be related to tumorigenesis in a single determination.

[0012] Therefore, there is a need to apply a robust and sensitive multiplex assay system to the problem of screening for DNA mutations. This provides a mechanism for multiplexing many DNA mutations in a single assay.

[0013] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. Summary of the Invention

[0014] In order to meet this need, provided herein is a method and a test kit for using a unique identifier-encoded microcarrier, wherein the microcarrier includes a probe for detecting a DNA mutation (e.g., a mutation associated with colorectal cancer). These microcarriers can be used for multiple determinations, wherein each of the microcarriers includes a probe for detecting a specific DNA mutation (e.g., a mutation in KRAS, BRAF, CTNNB1 or APC genes) and an identifier for associating the microcarrier with its associated probe. Methods and test kits disclosed herein can be used for, for example, monitoring colorectal cancer, monitoring the response to the treatment of colorectal cancer, and / or early screening / detection of colorectal cancer.

[0015] Thus, in one aspect, provided herein is a method for detecting the presence of DNA mutations in the KRAS, BRAF, CTNNB1, and APC genes, the method comprising: (a) isolating DNA from a sample; (b) amplifying the isolated DNA by polymerase chain reaction (PCR) using primer pairs specific for the loci of one or more DNA mutations in each of the KRAS, BRAF, CTNNB1, and APC genes; (c) hybridizing the amplified DNA with at least four probes, the at least four probes comprising one or more probes specific for DNA mutations in each of the KRAS, BRAF, CTNNB1, and APC genes, wherein each of the at least four probes is coupled to a microcarrier, and wherein each of the microcarriers comprises an identifier corresponding to the probe coupled thereto; (d) detecting the presence or absence of hybridization of the amplified DNA to the at least four probes, wherein hybridization between the amplified DNA and one of the probes indicates the presence of a DNA mutation corresponding to the probe; (e) detecting an identifier of the microcarrier; and (f) correlating the detected identifier of the microcarrier with the detected presence or absence of hybridization of the amplified DNA to the corresponding probe of the microcarrier. Also provided herein are methods for detecting the presence of DNA mutations in the KRAS, BRAF, CTNNB1, and APC genes, the methods comprising: (a) amplifying isolated DNA by polymerase chain reaction (PCR) using primer pairs specific for the loci of one or more DNA mutations in each of the KRAS, BRAF, CTNNB1, and APC genes; (b) hybridizing the amplified DNA with at least four probes, the at least four probes comprising one or more probes specific for DNA mutations in each of the KRAS, BRAF, CTNNB1, and APC genes, wherein each of the at least four probes is coupled to a microcarrier, and wherein each of the microcarriers comprises an identifier corresponding to the probe coupled thereto; (c) detecting the presence or absence of hybridization of the amplified DNA to the at least four probes, wherein hybridization between the amplified DNA and one of the probes indicates the presence of a DNA mutation corresponding to the probe; (d) detecting an identifier for the microcarrier; and (e) correlating the detected identifier for the microcarrier with the detected presence or absence of hybridization of the amplified DNA to the corresponding probe of the microcarrier. In some embodiments, the KRAS, BRAF, CTNNB1, and APC genes are human genes. In some embodiments, step (b) comprises amplifying the isolated DNA by PCR in the presence of at least four blocking nucleic acids, wherein each of the at least four blocking nucleic acids hybridizes to a wild-type DNA locus corresponding to one of the DNA mutations in the KRAS, BRAF, CTNNB1, or APC gene and prevents amplification of the wild-type DNA locus.In some embodiments, each of the at least four blocking nucleic acids comprises: a single-stranded oligonucleotide that hybridizes to the corresponding wild-type DNA locus; and a 3' terminal portion that blocks the extension of the single-stranded oligonucleotide. In some embodiments, the 3' terminal portion comprises one or more reverse deoxythymidines. In some embodiments, each of the at least four blocking nucleic acids comprises one or more modified nucleotides selected from the group consisting of: locked nucleic acid (LNA), peptide nucleic acid (PNA), hexose nucleic acid (HNA), threose nucleic acid (TNA), glycol nucleic acid (GNA) and cyclohexenyl nucleic acid (CeNA). In some embodiments, the one or more DNA mutations in the KRAS gene include one or more DNA mutations of a KRAS protein encoding a G12D, G12V, G12S or G13D mutation. In some embodiments, at least one of the at least four blocking nucleic acids comprises the sequence TACGCCACCAGCT (invdT). n , wherein n is 1, 2 or 3 (SEQ ID NO: 3); TTGGAGCTGGTGGCGTA (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 142); GCTGGTGGCGTAGGCA (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 143); GCTGGTGGCGTAGGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 144) or TTGGAGCTGGTGGCGT (invdT) n , wherein n is 1, 2, or 3 (SEQ ID NO: 145), wherein the italicized nucleic acids represent locked nucleic acids. In some embodiments, the one or more DNA mutations in the BRAF gene include one or more DNA mutations encoding a BRAF protein with a V600E mutation. In some embodiments, at least one of the at least four blocking nucleic acids comprises the sequence GAGATTTCACTGTAGC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 10); GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 146); GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 147); GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 148) or GAGATTTCACTGTAGC (invdT) n, wherein n is 1, 2, or 3 (SEQ ID NO: 149), wherein the italicized nucleic acids represent locked nucleic acids. In some embodiments, the one or more DNA mutations in the CTNNB1 gene include at least a first CTNNB1 mutation encoding a CTNNB1 protein with a T41A or T41I mutation. In some embodiments, at least one of the at least four blocking nucleic acids comprises the sequence GCCACTACCACAGCT(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 15); TGCCACTACCACAG (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 150); CACTACCACAGCTCC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 151); GCCACTACCACAGCT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 152) or GCCACTACCACAGCT (invdT) n , wherein n is 1, 2, or 3 (SEQ ID NO: 153), wherein the italicized nucleic acids represent locked nucleic acids. In some embodiments, the one or more DNA mutations in the CTNNB1 gene include at least a first CTNNB1 mutation encoding a CTNNB1 protein with an S45F or S45P mutation. In some embodiments, at least one of the at least four blocking nucleic acids comprises the sequence GCTCCTTCTCTGAGT(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 20); TCCTTCTCTGAGTGG (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 174); GCTCCTTCTCTGAGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 175); TCCTTCTCTGAGTGG(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 176) or GCTCCTTCTCTGAGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 177), wherein italicized nucleic acids represent locked nucleic acids. In some embodiments, the one or more DNA mutations in the APC gene include at least a first APC mutation encoding an APC protein with a Q1367* mutation. In some embodiments, at least one of the at least four blocking nucleic acids comprises GTGCTCAGACACC(invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 33); GTGCTCAGACACC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 158); AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 159); AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 160) or AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 161), wherein italicized nucleic acids represent locked nucleic acids. In some embodiments, the one or more DNA mutations in the APC gene include at least a first APC mutation encoding an APC protein with an R1450* mutation. In some embodiments, at least one of the at least four blocking nucleic acids comprises the sequence CTTCTCGCTTGGTT(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 37); GTACTTCTCGCTTGGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 162); CTTCTCGCTTGGTT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 163); GTACTTCTCGCTTGGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 164) or GTACTTCTCGCTTGGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 165), wherein italic nucleic acids represent locked nucleic acids. In some embodiments, the one or more DNA mutations in the APC gene include at least a first APC mutation encoding an APC protein with an E1309 frameshift mutation. In some embodiments, at least one of the at least four blocking nucleic acids comprises the sequence CTTTTCTTTTATTTCTGC(invdT) n, wherein n is 1, 2, or 3 (SEQ ID NO: 29); CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 154); CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 155); CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 156) or CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 157), wherein italicized nucleic acids represent locked nucleic acids. In some embodiments, the one or more DNA mutations in the APC gene include at least a first APC mutation encoding an APC protein with an S1465 frameshift mutation. In some embodiments, at least one of the at least four blocking nucleic acids comprises the sequence CCACTCTCTCTCTTTTCAGC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 25); TAGGTCCACTCTCTCTCTTTTCAGCA (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 166); TAGGTCCACTCTCTCTCTTTTCAGCA (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 167); CCACTCTCTCTCTTTTCAGC (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 168) or TAGGTCCACTCTCTCTCTTTTCAGCA (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 169), wherein italic nucleic acids represent locked nucleic acids. In some embodiments, the one or more DNA mutations in the APC gene include at least a first APC mutation encoding an APC protein with a T1556 frameshift mutation. In some embodiments, at least one of the at least four blocking nucleic acids comprises the sequence CAATAGTTTTTTCTGCC (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 41); GAATCAATAGTTTTTTCTGCCTC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 170); TCAGAATCAATAGTTTTTTCTG(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 171); GAATCAATAGATTTTACTGCCTC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 172) or AATCAATAGTTTTTCTGCCTC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 173), wherein italic nucleic acids represent locked nucleic acids. In some embodiments, each primer pair comprises a primer coupled to a detection reagent. In some embodiments, the detection reagent comprises a fluorescent detection reagent, and wherein in step (d), detecting the presence or absence of hybridization of the amplified DNA to the at least four probes comprises fluorescent imaging of the fluorescent detection reagent. In some embodiments, the detection reagent comprises biotin, and wherein detecting the presence or absence of hybridization of the amplified DNA to the at least four probes in step (d) comprises: (1) after hybridization in step (c), contacting the microcarrier with streptavidin conjugated to a signal-emitting entity; and (2) detecting a signal from the signal-emitting entity associated with the microcarrier. In some embodiments, the signal-emitting entity comprises phycoerythrin (PE). In some embodiments, detecting the identifier of the microcarrier in step (e) comprises bright field imaging of the identifier.

[0016] In some embodiments, the one or more DNA mutations in the KRAS gene include KRAS mutations encoding KRAS proteins that are G12D, G12V, G12S, and G13D mutants. In some embodiments, probes specific for one or more DNA mutations in the KRAS gene include four probes comprising the sequences GGAGCTGATGG (SEQ ID NO: 4), GGAGCTGTTGG (SEQ ID NO: 5), TGGAGCTAGTGG (SEQ ID NO: 6), and TGGAGCTGGTGACGT (SEQ ID NO: 7), and wherein each of the four probes is coupled to a microcarrier having a different identifier. In some embodiments, each of the four probes also comprises 8 nucleotides at the 5' end, wherein the 8 nucleotides at the 5' end are adenine or thymine nucleotides, and wherein each of the four probes comprises at least 24 total nucleotides. In some embodiments, the four probes include: (1) a first probe comprising a sequence selected from the group consisting of GGAGCTGATGG (SEQ ID NO:4), AGCTGATGGCGTA (SEQ ID NO:178), TGGAGCTGATGGCG (SEQ ID NO:179), TGGAGCTGATGG (SEQ ID NO:180), and GCTGATGGCGTA (SEQ ID NO:181); (2) a second probe comprising a sequence selected from the group consisting of GGAGCTGTTGG (SEQ ID NO:5), TGGAGCTGTTGGTGGC (SEQ ID NO:182), GGAGCTGTTGGTG (SEQ ID NO:183), TGGAGCTGTTGGT (SEQ ID NO:184), and TGGAGCTGTaGGTGG (SEQ ID NO:185); (3) a third probe comprising a sequence selected from the group consisting of TTGGAGCTAGTGGCGTA (SEQ ID NO:186), GCTAGTGGCGTAGGC (SEQ ID NO:187). NO:187), AGCTAGTGGCGT (SEQ ID NO:188), GTTGGAGCTAGTGG (SEQ ID NO:189) and GGAGCTAGTGG (SEQ ID NO:190);and (4) a fourth probe comprising a sequence selected from the group consisting of GGTGACGTAGGCAA (SEQ ID NO: 191), TGACGTAGGCAAGAG (SEQ ID NO: 192), GCTGGTGACGTAGG (SEQ ID NO: 193), AGCTGGTGACGTAG (SEQ ID NO: 194), and GGAGCTGGTGACGT (SEQ ID NO: 195); and wherein each of the four probes is coupled to a microcarrier having a different identifier. In some embodiments, the four probes include: (1) a first probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTAAGGAGCTGATGG (SEQ ID NO:47), TTTTTTTTTTTTAGCTGATGGCGTA (SEQ ID NO:74), TTTTTTTTTTTATGGAGCTGATGGCG (SEQ ID NO:75), TTTTTTTTTTTTATGGAGCTGATGG (SEQ ID NO:76), and TTTTTTTTTTTTTGCTGATGGCGTA (SEQ ID NO:77); (2) a second probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTTAAGGAGCTGTTGG (SEQ ID NO:48), TTTTTTTTTATGGAGCTGTTGGTGGC (SEQ ID NO:78), TTTTTTTTTTTAAGGAGCTGTTGGTG (SEQ ID NO:79), TTTTTTTTTTTATGGAGCTGTTGGT (SEQ ID NO:80). NO:80) and TTTTTTTTTATGGAGCTGTAGGTGG (SEQ ID NO:81); (3) a third probe comprising a sequence selected from the group consisting of TTTTTTTTTTTATGGAGCTAGTGG (SEQ ID NO:49), TTTTTTTTTTGGAGCTAGTGGCGTA (SEQ ID NO:82), TTTTTAATTTGCTAGTGGCGTAGGC (SEQ ID NO:83), TTTTTTTTTATTTAGCTAGTGGCGT (SEQ ID NO:84), TTTTTTTTTTTGTTGGAGCTAGTGG (SEQ ID NO:85), and TTTTTTTTTTTTTAAGGAGCTAGTGG (SEQ ID NO:86);and (4) a fourth probe comprising a sequence selected from the group consisting of TTTTTTTTTATGGAGCTGGTGACGT (SEQ ID NO: 50), TTTTTTTTAAAGGTGACGTAGGCAA (SEQ ID NO: 87), TTTTTTTTTATGACGTAGGCAAGAG (SEQ ID NO: 88), TTTTTTTTTTTGCTGGTGACGTAGG (SEQ ID NO: 89), TTTTTTTTTTAAGCTGGTGACGTAG (SEQ ID NO: 90), and TTTTTTTTTAAGGAGCTGGTGACGT (SEQ ID NO: 91); and wherein each of the four probes is coupled to a microcarrier having a different identifier. In some embodiments, step (b) comprises amplifying the isolated DNA by PCR using a primer pair comprising the sequences GTACTGGTGGAGTATTTGATAGTG (SEQ ID NO: 1) and ATCGTCAAGGCACTCTTGCCTAC (SEQ ID NO: 2). In some embodiments, step (b) comprises amplifying the isolated DNA by PCR in the presence of a blocking nucleic acid comprising the sequence: TACGCCACCAGCT(invdT); n , wherein n is 1, 2 or 3 (SEQ ID NO: 3); TTGGAGCTGGTGGCGTA (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 142); GCTGGTGGCGTAGGCA (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 143); GCTGGTGGCGTAGGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 144) or TTGGAGCTGGTGGCGT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 145), wherein the italicized nucleic acids represent locked nucleic acids.

[0017] In some embodiments, the one or more DNA mutations in the BRAF gene include two or more BRAF mutations encoding V600E mutated BRAF proteins. In some embodiments, the probes specific for one or more DNA mutations in the BRAF gene include two probes comprising the following sequences: TCTAGCTACAGAGAAAT (SEQ ID NO: 11) and GTCTAGCTACAGAAAAAT (SEQ ID NO: 12), and wherein each of the two probes is coupled to a microcarrier having a different tag. In some embodiments, each of the two probes also comprises 8 nucleotides at the 5' end, wherein the 8 nucleotides at the 5' end are adenine or thymine nucleotides, and wherein each of the two probes comprises at least 24 total nucleotides. In some embodiments, the probes include: (1) a first probe comprising a sequence selected from the group consisting of: TACAGAGAAATCTCGAT (SEQ ID NO: 196), TACAGAGAAATCTC (SEQ ID NO: 197), CTAGCTACAGAGAAAT (SEQ ID NO: 198), CTAGCTACAGAGAAA (SEQ ID NO: 199), and TCTAGCTACAGAG (SEQ ID NO: 200); and (2) a second probe comprising a sequence selected from the group consisting of: GTCTAGCTACAGAAAAATC (SEQ ID NO: 201), GTCTAGCTACAGAAAAAT (SEQ ID NO: 12), TAGCTACAGAAAAA (SEQ ID NO: 202), TCTAGCTACAGAAAAAT (SEQ ID NO: 203), and TCTAGCTACAGAAAAATC (SEQ ID NO: 204); and wherein each of the two probes is coupled to a microcarrier having a different identifier.In some embodiments, the probes include: (1) a first probe comprising a sequence selected from the group consisting of TTTTTTAATTTCTAGCTACAGAGAAAT (SEQ ID NO:51), TTTTTTTTTATACAGAGAAATCTCGAT (SEQ ID NO:92), TTTTTTTTTAATTTACAGAGAAATCTC (SEQ ID NO:93), TTTTTTAATTACTAGCTACAGAGAAAT (SEQ ID NO:94), TTTTTTTAATTACTAGCTACAGAGAAA (SEQ ID NO:95), and TTTTTTTTTTAATTTCTAGCTACAGAG (SEQ ID NO:96); and (2) a second probe comprising a sequence selected from the group consisting of TTTTTTTATGTCTAGCTACAGAAAAAT (SEQ ID NO:52), TTTTATGTCTAGCTACAGAAAAATC (SEQ ID NO:97), TTTTTTTTATTTTTAGCTACAGAAAAA (SEQ ID NO:98), TTTTTTTATTTCTAGCTACAGAAAAAT (SEQ ID NO:99). NO: 99) and TTTTTTTTATTCTAGCTACAGAAAAATC (SEQ ID NO: 100); and wherein each of the two probes is coupled to a microcarrier having a different identifier. In some embodiments, step (b) comprises amplifying the isolated DNA by PCR using a primer pair comprising the sequence GGACCCACTCCATCGAGATTT (SEQ ID NO: 8) and CAGATATATTTCTTCATGAAGACCTCACAGTAA (SEQ ID NO: 9). In some embodiments, step (b) comprises amplifying the isolated DNA by PCR in the presence of a blocking nucleic acid comprising the following sequence: GAGATTTCACTGTAGC (invdT). n , wherein n is 1, 2 or 3 (SEQ ID NO: 10); GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 146); GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 147); GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 148) or GAGATTTCACTGTAGC (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 149), wherein the italicized nucleic acids represent locked nucleic acids.

[0018] In some embodiments, the one or more DNA mutations in the CTNNB1 gene include CTNNB1 mutations encoding CTNNB1 proteins that are T41A, T41I, S45F, and S45P mutants. In some embodiments, probes specific for one or more DNA mutations in the CTNNB1 gene include four probes comprising the following sequences: AGGAGCTGTGGCAG (SEQ ID NO: 16), GGAGCTGTGATA (SEQ ID NO: 17), TTTACCACTCAGAAAAG (SEQ ID NO: 21), and TACCACTCAGAGGAG (SEQ ID NO: 22), and wherein each of the four probes is coupled to a microcarrier having a different identifier. In some embodiments, each of the four probes also comprises 8 nucleotides at the 5' end, wherein the 8 nucleotides at the 5' end are adenine or thymine nucleotides, and wherein each of the four probes comprises at least 24 total nucleotides. In some embodiments, the four probes include: (1) a first probe comprising a sequence selected from the group consisting of AGGAGCTGTGGCAGT (SEQ ID NO: 205), AGGAGCTGTGGCAGTG (SEQ ID NO: 206), GCTGTGGCAGTGGC (SEQ ID NO: 207), GCTGTGGCAGTGGCA (SEQ ID NO: 208), and AAGGAGCTGTGGCAG (SEQ ID NO: 209); (2) a second probe comprising a sequence selected from the group consisting of GGAGCTGTGATAGTGG (SEQ ID NO: 210), GAGCTGTGATAGTGGC (SEQ ID NO: 211), AGCTGTGATAGTGGCA (SEQ ID NO: 212), AGAAGGAGCTGTGATA (SEQ ID NO: 213), and GGAGCTGTGAT (SEQ ID NO: 214); and (3) a third probe comprising a sequence selected from the group consisting of ACTCAGAAAAGGAGCT (SEQ ID NO: 215). NO:215), TACCACTCAGAAAGGA (SEQ ID NO:216), TTTACCACTCAGAAAGGAG (SEQ ID NO:217), TTACCACTCAGAAAG (SEQ ID NO:218) and CAGAAAAGGAGCTGTG (SEQ ID NO:219);and (4) a fourth probe comprising a sequence selected from the group consisting of: ACTCAGAGGAGGAGC (SEQ ID NO:220), TTACCACTCAGAGGA (SEQ ID NO:221), TTACCACTCAGAGGAGG (SEQ ID NO:222), TTAACACTCAGAGGAG (SEQ ID NO:223), and TTACCAATCAGAGGAGG (SEQ ID NO:224); and wherein each of the four probes is coupled to a microcarrier having a different identifier. In some embodiments, the probes include: (1) a fourth probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTTAGGAGCTGTGGCAG (SEQ ID NO: 53), TTTTTTTTTTTAGGAGCTGTGGCAGTG (SEQ ID NO: 101), TTTTTTTTTTTAGCTGTGGCAGTGGC (SEQ ID NO: 102), TTTTTTTTTTTGCTGTGGCAGTGGCA (SEQ ID NO: 103), and TTTTTTTTTTTAAGGAGCTGTGGCAG (SEQ ID NO: 104); (2) a second probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTTGGAGCTGTGATA (SEQ ID NO: 54), TTTTTTTTTGGAGCTGTGATAGTGG (SEQ ID NO: 105), TTTTTTTTTGAGCTGTGATAGTGGC (SEQ ID NO: 106), TTTTTTTTTTTAGCTGTGATAGTGGCA (SEQ ID NO: 107). NO: 107), TTTTTTTTTAGAAGGAGCTGTGATA (SEQ ID NO: 108), and TTTTTTTTTTTTTTGGAGCTGTGAT (SEQ ID NO: 109); (3) a third probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTACCACTCAGAAAAG (SEQ ID NO: 55), TTTAATTTTACTCAGAAAAGGAGCT (SEQ ID NO: 110), TTTTTTTAATACCACTCAGAAAAGGA (SEQ ID NO: 111), TTTTTTTTACCACTCAGAAAAGGAG (SEQ ID NO: 112), TTTTTTTTATTACCACTCAGAAAAG (SEQ ID NO: 113), and TTTTTTTTTCAGAAAAGGAGCTGTG (SEQ ID NO: 114);and (4) a fourth probe comprising a sequence selected from the group consisting of TTTTTTTTTAATACCACTCAGAGGAG (SEQ ID NO:56), TTTTTTTTTAAAACTCAGAGGAGGAGC (SEQ ID NO:115), TTTTTTTTTTTATTACCACTCAGAGGA (SEQ ID NO:116), TTTTTTTTTATTACCACTCAGAGGAGG (SEQ ID NO:117), TTTTTTTTTTATTAACACTCAGAGGAG (SEQ ID NO:118), and TTTTTTTTTATTACCAATCAGAGGAGG (SEQ ID NO:119), and wherein each of the four probes is coupled to a microcarrier with a different identifier. In some embodiments, step (b) comprises amplifying the isolated DNA by PCR using a first primer pair comprising the sequence GGAATCCATTCTGGTGCCACT (SEQ ID NO: 13) and AGAAAATCCCTGTTCCCACTCATA (SEQ ID NO: 14) and a second primer pair comprising the sequence GGTGCCACTACCACAGCTCCT (SEQ ID NO: 18) and TCTCAAAACTGCATTCTGACTTTCA (SEQ ID NO: 19). In some embodiments, step (b) comprises amplifying the isolated DNA by PCR in the presence of the following blocking nucleic acids: a first blocking nucleic acid comprising the sequence: GCCACTACCACAGCT (invdT); n , wherein n is 1, 2 or 3 (SEQ ID NO: 15); TGCCACTACCACAG (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 150); CACTACCACAGCTCC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 151); GCCACTACCACAGCT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 152) or GCCACTACCACAGCT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 153); and a second blocking nucleic acid comprising the sequence: GCTCCTTCTCTGAGT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 20); TCCTTCTCTGAGTGG (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 174); GCTCCTTCTCTGAGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 175); TCCTTCTCTGAGTGG(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 176) or GCTCCTTCTCTGAGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 177), wherein italic nucleic acids represent locked nucleic acids.

[0019] In some embodiments, the one or more DNA mutations in the APC gene include APC mutations encoding APC proteins with Q1367*, R1450*, E1309 frameshift, S1465 frameshift, and T1556 frameshift mutations. In some embodiments, probes specific for one or more DNA mutations in the APC gene include five probes comprising the following sequences: ACTGCTGAAAAGAGAGAGT (SEQ ID NO: 26), GAAATAAAAGATTGG (SEQ ID NO: 30), TTTTGGGTGTCTAAG (SEQ ID NO: 34), CAAACCAAGTGAGAA (SEQ ID NO: 38), and AGAGGCAGAAAAAAACT (SEQ ID NO: 42), and wherein each of the five probes is coupled to a microcarrier having a different identifier. In some embodiments, each of the five probes further comprises 8 nucleotides at the 5' end, wherein the 8 nucleotides at the 5' end are adenine or thymine nucleotides, and wherein each of the five probes comprises at least 24 total nucleotides. In some embodiments, the probes include: (1) a first probe comprising a sequence selected from the group consisting of AAATAGCAGAAATAAAAG (SEQ ID NO: 225), GAAATAAAAGATTGGAA (SEQ ID NO: 226), AGAAATAAAAGATTG (SEQ ID NO: 227), GAAATAAATGAATGG (SEQ ID NO: 228), and CAGAAATAAAAGATT (SEQ ID NO: 229); (2) a second probe comprising a sequence selected from the group consisting of TTTGGGTGTCTAAG (SEQ ID NO: 230), GGGTGTCTAAGCACCACT (SEQ ID NO: 231), CTAAGCACCACTTTT (SEQ ID NO: 232), TTTTGGGTGTCTAA (SEQ ID NO: 233), and GGTGTCTAAGCACCA (SEQ ID NO: 234); (3) a third probe comprising a sequence selected from the group consisting of AAGTGAGAAGTACCTAA (SEQ ID NO: 235). NO:235), CAAACCAAGTGAGAA (SEQ ID NO:38), TCAAACCAAGTGAG (SEQ ID NO:236), ACCAAGTGAGAAGTA (SEQ ID NO:237) and AGCTCAAACCAAGTGAG (SEQ ID NO:238);(4) a fourth probe comprising a sequence selected from the group consisting of GCACCTACTGCTGAA (SEQ ID NO:239), ACCTACTGCTGAAAAG (SEQ ID NO:240), TGCTGAAAAGAGAGAGT (SEQ ID NO:241), ACTGCTGAAAAGAGAGAGT (SEQ ID NO:26), and CCTACTGCTGAAAAGAGA (SEQ ID NO:242); and (5) a fifth probe comprising a sequence selected from the group consisting of GCAGAAAAAAACTATTG (SEQ ID NO:243), AGAGGCAGAAAAAAACT (SEQ ID NO:42), CAGAAAAAAACTATTGATT (SEQ ID NO:244), AGAAAGAGGCAGAAAAAAACT (SEQ ID NO:245), and GAGGCAGAAAAAAACTA (SEQ ID NO:246); and wherein each of the five probes is coupled to a microcarrier having a different identifier. In some embodiments, the probes include: (1) a first probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTTGAAATAAAAGATTGG (SEQ ID NO:58), TTTTTTTTTTAAATAGCAGAAATAAAAG (SEQ ID NO:120), TTTTTTTTTTTGAAATAAAAGATTGGAA (SEQ ID NO:121), TTTTTTTTTTTTTTTGAAATAAAAGATTG (SEQ ID NO:122), TTTTTTTTTTTTTGAAATAAATGAATGG (SEQ ID NO:123), and TTTTTTTTTTTTTCAGAAATAAAAGATT (SEQ ID NO:124); (2) a second probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTTGGGTGTCTAAG (SEQ ID NO:59), TTTTTTTTTTTATTTGGGTGTCTAAG (SEQ ID NO:125), TTTTTTGGGTGTCTAAGCACCACT (SEQ ID NO:126). NO:126), TTTTTTTTTCTAAGCACCACTTTT (SEQ ID NO:127), TTTTTTTTTTTTTTGGGTGTCTAA (SEQ ID NO:128) and TTTTTTTTTGGTGTCTAAGCACCA (SEQ ID NO:129);(3) a third probe comprising a sequence selected from the group consisting of TTTTTTTTTTACAAACCAAGTGAGAA (SEQ ID NO:60), TTTTTTTTAAGTGAGAAGTACCTAA (SEQ ID NO: 130), TTTTTTTTTTTTCAAACCAAGTGAG (SEQ ID NO: 131), TTTTTTTTTTACCAAGTGAGAAGTA (SEQ ID NO: 132), and TTTTTTTTAGCTCAAACCAAGTGAG (SEQ ID NO: 133); (4) a fourth probe comprising a sequence selected from the group consisting of TTTTTTTTACTGCTGAAAAGAGAGAGT (SEQ ID NO: 57), TTTTTTTTTGCACCTACTGCTGAA (SEQ ID NO: 134), TTTTTTTTTTCCTACTGCTGAAAAG (SEQ ID NO: 135), TTTTTTTTTGCTGAAAAGAGAGAGT (SEQ ID NO: 136), and TTTTTTTTTCCTACTGCTGAAAAGAGA (SEQ ID NO: 137). NO: 137); and (5) a fifth probe comprising a sequence selected from the group consisting of TTTTTTTTTTAGAGGCAGAAAAAAACT (SEQ ID NO: 61), TTTTTTTTTTGCAGAAAAAAACTATTG (SEQ ID NO: 138), TTTTTTTTTTTCAGAAAAAAACTATTGATT (SEQ ID NO: 139), TTTTTTTTAGAAAGAGGCAGAAAAAAACT (SEQ ID NO: 140), and TTTTTTTTTTTGAGGCAGAAAAAAACTA (SEQ ID NO: 141); and wherein each of the five probes is coupled to a microcarrier having a different identifier. In some embodiments, step (b) comprises amplifying the isolated DNA by PCR using the following primer pairs: a first primer pair comprising the sequence TAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24); a second primer pair comprising the sequence TAGGATGTAATCAGACGACACAGGA (SEQ ID NO: 27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO: 28);a third primer pair comprising the sequence TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO: 31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO: 32); a fourth primer pair comprising the sequence GCAGAAGTAAAACACCTCCACCA (SEQ ID NO: 35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO: 36), wherein italicized nucleic acids represent locked nucleic acids; and a fifth primer pair comprising the sequence CAGGAAAATGACAATGGGAATG (SEQ ID NO: 39) and ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO: 40). In some embodiments, step (b) comprises amplifying the isolated DNA by PCR in the presence of the following blocking nucleic acids: a first blocking nucleic acid comprising the sequence: CCACTCTCTCTCTTTTCAGC (invdT); n , wherein n is 1, 2 or 3 (SEQ ID NO: 25); TAGGTCCACTCTCTCTCTTTTCAGCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 166); TAGGTCCACTCTCTCTCTTTTCAGCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 167); CCACTCTCTCTCTTTTCAGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 168) or TAGGTCCACTCTCTCTCTTTTCAGCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 169); a second blocking nucleic acid comprising the sequence: CTTTTCTTTTATTTCTGC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO:29); CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:154); CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:155); CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:156) or CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:157); a third blocking nucleic acid comprising the sequence: GTGCTCAGACACC(invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO:33); GTGCTCAGACACC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:158); AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:159); AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:160) or AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:161); a fourth blocking nucleic acid comprising the sequence: CTTCTCGCTTGGTT(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 37); GTACTTCTCGCTTGGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 162); CTTCTCGCTTGGTT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 163); GTACTTCTCGCTTGGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 164) or GTACTTCTCGCTTGGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 165); a fifth blocking nucleic acid comprising the sequence: CAATAGTTTTTTCTGCC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO:41); GAATCAATAGTTTTTTCTGCCTC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:170); TCAGAATCAATAGTTTTTTCTG(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:171); GAATCAATAGATTTTACTGCCTC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:172) or AATCAATAGTTTTTCTGCCTC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:173), wherein italic nucleic acids represent locked nucleic acids.

[0020] In some embodiments of any of the foregoing embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the method further comprises: amplifying the positive control DNA sequence using a primer pair specific for the positive control DNA sequence; contacting the amplified positive control gene sequence with a probe specific for the positive control gene sequence, wherein the probe specific for the positive control gene sequence is coupled to a microcarrier having an identifier corresponding to the positive control; detecting the presence or absence of hybridization between the amplified positive control DNA sequence and the probe specific for the positive control gene sequence; and detecting the identifier corresponding to the positive control. In some embodiments, the positive control DNA sequence comprises the sequence of a human leukocyte antigen gene. In some embodiments, the primer pair specific for the positive control DNA sequence comprises the sequence TGAGTGTTACTTCTTCCCACACTC (SEQ ID NO: 43) and ATTGCTTTTGCGCAATCCCT (SEQ ID NO: 44). In some embodiments, the probe specific for the positive control gene sequence comprises the sequence TTTTTTTTTTTTGGAGACGGTCTG (SEQ ID NO: 45). In some embodiments, the primer pair specific for the positive control DNA sequence comprises the sequence AATCCCATCACCATCTTCCA (SEQ ID NO: 71) and TGGACTCCACGACGTACTCA (SEQ ID NO: 72). In some embodiments, the probe specific for the positive control gene sequence comprises the sequence CTGTCTTCCACTCACTCC (SEQ ID NO: 73).

[0021] In some embodiments, the method further comprises: detecting the absence of hybridization of the amplified DNA to a microcarrier having an identifier corresponding to a negative control, wherein the microcarrier having an identifier corresponding to the negative control comprises a probe that does not hybridize to the amplified DNA; and detecting the identifier corresponding to the negative control. In some embodiments, the microcarrier having an identifier corresponding to the negative control comprises a probe comprising the sequence AATATAATATATATATA (SEQ ID NO: 46). In some embodiments, the identifier of the microcarrier comprises a digital barcode. In some embodiments, each of the microcarriers comprises: (i) a first photopolymer layer; (ii) a second photopolymer layer; and (iii) an intermediate layer between the first layer and the second layer, the intermediate layer having a coded pattern representing an identifier defined thereon, wherein the intermediate layer is partially substantially light-transmissive and partially substantially light-impermeable, representing a code corresponding to the microcarrier, wherein the outermost surface of the microcarrier comprises a photoresist photopolymer, and the photoresist photopolymer is functionalized with a probe specific for a DNA mutation, and wherein the microcarrier has approximately the same density as water. In some embodiments, the identifier of the microcarrier comprises an analog code. In some embodiments, each of the microcarriers comprises: (i) a substantially transparent polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other; (ii) a substantially opaque polymer layer, wherein the substantially opaque polymer layer is affixed to the first surface of the substantially transparent polymer layer and surrounds a central portion of the substantially transparent polymer layer, and wherein the substantially opaque polymer layer comprises a two-dimensional shape representing an analog code identifier, wherein the analog code represents the identifier; and (iii) a probe specific for a DNA mutation, wherein the probe is coupled to at least one of the first surface and the second surface of the substantially transparent polymer layer in at least the central portion of the substantially transparent polymer layer. In some embodiments, each of the microcarriers further comprises: (iv) a second substantially transparent polymer layer aligned with the first substantially transparent polymer layer, the second substantially transparent polymer layer having a central portion aligned with the central portion of the first substantially transparent polymer layer, wherein the second substantially transparent polymer layer is affixed to the second surface of the first substantially transparent polymer layer and does not extend beyond the two-dimensional shape of the first substantially transparent polymer layer; and (v) a substantially opaque magnetic layer surrounding the central portion of the first substantially transparent polymer layer between the substantially opaque polymer layer and the central portion of the substantially transparent polymer layer, wherein the substantially opaque magnetic layer is affixed between the first substantially transparent polymer layer and the second substantially transparent polymer layer. In some embodiments, each of the microcarriers further comprises an orientation indicator for orienting the analog code of the substantially opaque polymer layer.In some embodiments, the two-dimensional shape of the substantially opaque polymer layer comprises a gear shape comprising a plurality of gear teeth, and wherein the simulation code is represented by one or more aspects selected from the group consisting of: the height of one or more gear teeth in the plurality of gear teeth, the width of one or more gear teeth in the plurality of gear teeth, the number of gear teeth in the plurality of gear teeth, and the arrangement of one or more gear teeth within the plurality of gear teeth. In some embodiments, each of the microcarriers further comprises: (vi) one or more pillars protruding from a first surface of the first substantially transparent polymer layer, wherein the one or more pillars are not within a central portion of the first substantially transparent polymer layer; and / or (vii) one or more pillars protruding from a second surface of the first substantially transparent polymer layer or a surface of a second substantially transparent polymer layer that is not fixed to the first substantially transparent polymer layer, wherein the one or more pillars are not within a central portion of the first substantially transparent polymer layer or the second substantially transparent polymer layer. In some embodiments, the substantially transparent polymer of the first substantially transparent polymer layer or the second substantially transparent polymer layer comprises an epoxy polymer. In some embodiments, the epoxy polymer is SU-8. In some embodiments, the sample is a stool sample. In some embodiments, multiple stool samples are obtained from a patient. In some embodiments, multiple stool samples are obtained from a single stool specimen. In some embodiments, the methods of the present disclosure comprise detecting the presence or absence of hybridization of amplified DNA to a total of about 1 to about 1000 wells of a microcarrier / assay plate. In some embodiments, the methods of the present disclosure are used to detect colon cancer, rectal cancer, colorectal cancer, colon adenoma, rectal adenoma, or colorectal adenoma in, for example, a patient from whom a sample was collected.

[0022] Therefore, on the other hand, provided herein is a kit comprising at least four microcarriers, wherein each of the at least four microcarriers comprises: (i) a probe coupled to a microcarrier, wherein the probe is specific for a DNA mutation in a KRAS, BRAF, CTNNB1, or APC gene; and (ii) an identifier corresponding to the probe coupled thereto; wherein the kit comprises at least one microcarrier comprising a probe specific for a DNA mutation in a KRAS gene, at least one microcarrier comprising a probe specific for a DNA mutation in a BRAF gene, at least one microcarrier comprising a probe specific for a DNA mutation in a CTNNB1 gene, and at least one microcarrier comprising a probe specific for a DNA mutation in a APC gene. In some embodiments, the KRAS, BRAF, CTNNB1, and APC genes are human genes. In some embodiments, the kit further comprises: at least four blocking nucleic acids, wherein each of the at least four blocking nucleic acids hybridizes to a wild-type DNA locus corresponding to one of the DNA mutations in the KRAS, BRAF, CTNNB1, and APC genes. Each of the at least four blocking nucleic acids comprises: a single-stranded oligonucleotide that hybridizes to the corresponding wild-type DNA locus; and a 3' terminal portion that blocks the extension of the single-stranded oligonucleotide. In some embodiments, the 3' terminal portion comprises one or more inverted deoxythymidines. In some embodiments, each of the at least four blocking nucleic acids comprises one or more modified nucleotides selected from the group consisting of: locked nucleic acid (LNA), peptide nucleic acid (PNA), hexose nucleic acid (HNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), and cyclohexenyl nucleic acid (CeNA). In some embodiments, the DNA mutation in the KRAS gene includes one or more DNA mutations encoding KRAS proteins that are G12D, G12V, G12S, or G13D mutants. In some embodiments, at least one of the at least four blocking nucleic acids comprises the sequence TACGCCACCAGCT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 3); TTGGAGCTGGTGGCGTA (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 142); GCTGGTGGCGTAGGCA (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 143); GCTGGTGGCGTAGGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 144) or TTGGAGCTGGTGGCGT (invdT) n, wherein n is 1, 2, or 3 (SEQ ID NO: 145), wherein the italicized nucleic acids represent locked nucleic acids. In some embodiments, the DNA mutations in the BRAF gene include one or more DNA mutations encoding a BRAF protein with a V600E mutation. In some embodiments, at least one of the at least four blocking nucleic acids comprises the sequence GAGATTTCACTGTAGC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 10); GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 146); GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 147); GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 148) or GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2, or 3 (SEQ ID NO: 149), wherein italicized nucleic acids represent locked nucleic acids. In some embodiments, the DNA mutations in the CTNNB1 gene include at least a first CTNNB1 mutation encoding a CTNNB1 protein with a T41A or T41I mutation. In some embodiments, at least one of the at least four blocking nucleic acids comprises the sequence GCCACTACCACAGCT(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 15); TGCCACTACCACAG (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 150); CACTACCACAGCTCC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 151); GCCACTACCACAGCT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 152) or GCCACTACCACAGCT (invdT) n , wherein n is 1, 2, or 3 (SEQ ID NO: 153), wherein the italicized nucleic acids represent locked nucleic acids. In some embodiments, the DNA mutations in the CTNNB1 gene include at least a first CTNNB1 mutation encoding a CTNNB1 protein that is an S45F or S45P mutation. In some embodiments, at least one of the at least four blocking nucleic acids comprises the sequence GCTCCTTCTCTGAGT(invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 20); TCCTTCTCTGAGTGG (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 174); GCTCCTTCTCTGAGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 175); TCCTTCTCTGAGTGG(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 176) or GCTCCTTCTCTGAGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 177), wherein italicized nucleic acids represent locked nucleic acids. In some embodiments, the DNA mutation in the APC gene includes at least a first APC mutation encoding an APC protein with a Q1367* mutation. In some embodiments, at least one of the at least four blocking nucleic acids comprises the sequence GTGCTCAGACACC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 33); GTGCTCAGACACC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 158); AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 159); AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 160) or AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 161), wherein italic nucleic acids represent locked nucleic acids. In some embodiments, the DNA mutation in the APC gene includes at least a first APC mutation encoding an APC protein with an R1450* mutation. In some embodiments, at least one of the at least four blocking nucleic acids comprises the sequence CTTCTCGCTTGGTT(invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 37); GTACTTCTCGCTTGGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 162); CTTCTCGCTTGGTT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 163); GTACTTCTCGCTTGGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 164) or GTACTTCTCGCTTGGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 165), wherein italic nucleic acids represent locked nucleic acids. In some embodiments, the DNA mutation in the APC gene includes at least a first APC mutation encoding an APC protein with an E1309 frameshift mutation. In some embodiments, at least one of the at least four blocking nucleic acids comprises the sequence CTTTTCTTTTATTTCTGC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 29); CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 154); CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 155); CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 156) or CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 157), wherein italic nucleic acids represent locked nucleic acids. In some embodiments, the DNA mutation in the APC gene includes at least a first APC mutation encoding an APC protein with an S1465 frameshift mutation. In some embodiments, at least one of the at least four blocking nucleic acids comprises the sequence CCACTCTCTCTCTTTTCAGC(invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 25); TAGGTCCACTCTCTCTCTTTTCAGCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 166); TAGGTCCACTCTCTCTCTTTTCAGCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 167); CCACTCTCTCTCTTTTCAGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 168) or TAGGTCCACTCTCTCTCTTTTCAGCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 169), wherein italic nucleic acids represent locked nucleic acids. In some embodiments, the DNA mutation in the APC gene includes at least a first APC mutation encoding an APC protein with a T1556 frameshift mutation. In some embodiments, at least one of the at least four blocking nucleic acids comprises the sequence CAATAGTTTTTTCTGCC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 41); GAATCAATAGTTTTTTCTGCCTC (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 170); TCAGAATCAATAGTTTTTTCTG (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 171); GAATCAATAGATTTTACTGCCTC (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 172) or AATCAATAGTTTTTCTGCCTC (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 173), wherein italic nucleic acids represent locked nucleic acids. In some embodiments, the kit further comprises at least four primer pairs, wherein the kit comprises primer pairs specific for one or more DNA mutation loci in each of the KRAS, BRAF, CTNNB1 and APC genes. In some embodiments, each of the at least four primer pairs comprises a primer coupled to a detection reagent. In some embodiments, the detection reagent comprises a fluorescent detection reagent. In some embodiments, the detection reagent comprises biotin, and wherein the kit further comprises streptavidin conjugated to a signal emitting entity. In some embodiments, the signal emitting entity comprises phycoerythrin (PE).

[0023] In some embodiments, the DNA mutations in the KRAS gene include KRAS mutations encoding KRAS proteins that are G12D, G12V, G12S, and G13D mutants. In some embodiments, the kit comprises four probes comprising the sequences GGAGCTGATGG (SEQ ID NO: 4), GGAGCTGTTGG (SEQ ID NO: 5), TGGAGCTAGTGG (SEQ ID NO: 6), and TGGAGCTGGTGACGT (SEQ ID NO: 7), and wherein each of the four probes is coupled to a microcarrier having a different identifier. In some embodiments, the kit comprises: (1) a first probe comprising a sequence selected from the group consisting of GGAGCTGATGG (SEQ ID NO:4), AGCTGATGGCGTA (SEQ ID NO:178), TGGAGCTGATGGCG (SEQ ID NO:179), TGGAGCTGATGG (SEQ ID NO:180), and GCTGATGGCGTA (SEQ ID NO:181); (2) a second probe comprising a sequence selected from the group consisting of GGAGCTGTTGG (SEQ ID NO:5), TGGAGCTGTTGGTGGC (SEQ ID NO:182), GGAGCTGTTGGTG (SEQ ID NO:183), TGGAGCTGTTGGT (SEQ ID NO:184), and TGGAGCTGTaGGTGG (SEQ ID NO:185); (3) a third probe comprising a sequence selected from the group consisting of TTGGAGCTAGTGGCGTA (SEQ ID NO:186), GCTAGTGGCGTAGGC (SEQ ID NO:187), NO:187), AGCTAGTGGCGT (SEQ ID NO:188), GTTGGAGCTAGTGG (SEQ ID NO:189) and GGAGCTAGTGG (SEQ ID NO:190);and (4) a fourth probe comprising a sequence selected from the group consisting of GGTGACGTAGGCAA (SEQ ID NO: 191), TGACGTAGGCAAGAG (SEQ ID NO: 192), GCTGGTGACGTAGG (SEQ ID NO: 193), AGCTGGTGACGTAG (SEQ ID NO: 194), and GGAGCTGGTGACGT (SEQ ID NO: 195), and wherein each of the four probes is coupled to a microcarrier having a different identifier. In some embodiments, each of the four probes further comprises 8 nucleotides at the 5' end, wherein the 8 nucleotides at the 5' end are adenine or thymine nucleotides, and wherein each of the four probes comprises at least 24 total nucleotides. In some embodiments, the kit comprises: (1) a first probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTTTAAGGAGCTGATGG (SEQ ID NO:47), TTTTTTTTTTTTAGCTGATGGCGTA (SEQ ID NO:74), TTTTTTTTTTTATGGAGCTGATGGCG (SEQ ID NO:75), TTTTTTTTTTTTATGGAGCTGATGG (SEQ ID NO:76), and TTTTTTTTTTTTTGCTGATGGCGTA (SEQ ID NO:77); (2) a second probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTTAAGGAGCTGTTGG (SEQ ID NO:48), TTTTTTTTTATGGAGCTGTTGGTGGC (SEQ ID NO:78), TTTTTTTTTTTAAGGAGCTGTTGGTG (SEQ ID NO:79), TTTTTTTTTTTATGGAGCTGTTGGT (SEQ ID NO:80). NO:80) and TTTTTTTTTATGGAGCTGTAGGTGG (SEQ ID NO:81);(3) a third probe comprising a sequence selected from the group consisting of TTTTTTTTTTTATGGAGCTAGTGG (SEQ ID NO:49), TTTTTTTTTTGGAGCTAGTGGCGTA (SEQ ID NO:82), TTTTTAATTTGCTAGTGGCGTAGGC (SEQ ID NO:83), TTTTTTTTTATTTAGCTAGTGGCGT (SEQ ID NO:84), TTTTTTTTTTTGTTGGAGCTAGTGG (SEQ ID NO:85), and TTTTTTTTTTTTTAAGGAGCTAGTGG (SEQ ID NO:86); and (4) a fourth probe comprising a sequence selected from the group consisting of TTTTTTTTTTTATGGAGCTGGTGACGT (SEQ ID NO:50), TTTTTTTTAAAGGTGACGTAGGCAA (SEQ ID NO:87), TTTTTTTTTATGACGTAGGCAAGAG (SEQ ID NO:88), TTTTTTTTTTTGCTGGTGACGTAGG (SEQ ID NO:89). NO: 89), TTTTTTTTTTAAGCTGGTGACGTAG (SEQ ID NO: 90), and TTTTTTTTTAAGGAGCTGGTGACGT (SEQ ID NO: 91), and wherein each of the four probes is coupled to a microcarrier with a different identifier. In some embodiments, the kit further comprises a primer pair comprising the sequence GTACTGGTGGAGTATTTGATAGTG (SEQ ID NO: 1) and ATCGTCAAGGCACTCTTGCCTAC (SEQ ID NO: 2). In some embodiments, the kit further comprises a blocking nucleic acid comprising the following sequence: TACGCCACCAGCT (invdT); n , wherein n is 1, 2 or 3 (SEQ ID NO: 3); TTGGAGCTGGTGGCGTA (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 142); GCTGGTGGCGTAGGCA (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 143); GCTGGTGGCGTAGGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 144) or TTGGAGCTGGTGGCGT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 145), wherein the italicized nucleic acids represent locked nucleic acids.

[0024] In some embodiments, the DNA mutation in the BRAF gene includes two or more BRAF mutations encoding a V600E mutated BRAF protein. In some embodiments, the kit comprises two probes comprising the following sequences: TCTAGCTACAGAGAAAT (SEQ ID NO: 11) and GTCTAGCTACAGAAAAAT (SEQ ID NO: 12), and wherein each of the two probes is coupled to a microcarrier having a different identifier. In some embodiments, the kit comprises: (1) a first probe comprising a sequence selected from the group consisting of: TACAGAGAAATCTCGAT (SEQ ID NO: 196), TACAGAGAAATCTC (SEQ ID NO: 197), CTAGCTACAGAGAAAT (SEQ ID NO: 198), CTAGCTACAGAGAAA (SEQ ID NO: 199), and TCTAGCTACAGAG (SEQ ID NO: 200); and (2) a second probe comprising a sequence selected from the group consisting of: GTCTAGCTACAGAAAAATC (SEQ ID NO: 201), GTCTAGCTACAGAAAAAT (SEQ ID NO: 12), TAGCTACAGAAAAA (SEQ ID NO: 202), TCTAGCTACAGAAAAAT (SEQ ID NO: 203), and TCTAGCTACAGAAAAATC (SEQ ID NO: 204); and wherein each of the four probes is coupled to a microcarrier having a different identifier. In some embodiments, each of the two probes further comprises 8 nucleotides at the 5' end, wherein the 8 nucleotides at the 5' end are adenine or thymine nucleotides, and wherein each of the two probes comprises at least 24 total nucleotides.In some embodiments, the kit comprises: (1) a first probe comprising a sequence selected from the group consisting of TTTTTTAATTTCTAGCTACAGAGAAAT (SEQ ID NO:51), TTTTTTTTTATACAGAGAAATCTCGAT (SEQ ID NO:92), TTTTTTTTTAATTTACAGAGAAATCTC (SEQ ID NO:93), TTTTTTAATTACTAGCTACAGAGAAAT (SEQ ID NO:94), TTTTTTTAATTACTAGCTACAGAGAAA (SEQ ID NO:95), and TTTTTTTTTTAATTTCTAGCTACAGAG (SEQ ID NO:96); and (2) a second probe comprising a sequence selected from the group consisting of TTTTTTTATGTCTAGCTACAGAAAAAT (SEQ ID NO:52), TTTTATGTCTAGCTACAGAAAAATC (SEQ ID NO:97), TTTTTTTTATTTTTAGCTACAGAAAAA (SEQ ID NO:98), TTTTTTTATTTCTAGCTACAGAAAAAT (SEQ ID NO:99). NO: 99) and TTTTTTTTATTCTAGCTACAGAAAAATC (SEQ ID NO: 100); and wherein each of the two probes is coupled to a microcarrier having a different identifier. In some embodiments, the kit further comprises a primer pair comprising the sequence GGACCCACTCCATCGAGATTT (SEQ ID NO: 8) and CAGATATATTTCTTCATGAAGACCTCACAGTAA (SEQ ID NO: 9). In some embodiments, the kit further comprises a blocking nucleic acid comprising the following sequence: GAGATTTCACTGTAGC (invdT). n , wherein n is 1, 2 or 3 (SEQ ID NO: 10); GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 146); GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 147); GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 148) or GAGATTTCACTGTAGC (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 149), wherein the italicized nucleic acids represent locked nucleic acids.

[0025] In some embodiments, the DNA mutations in the CTNNB1 gene include CTNNB1 mutations that encode CTNNB1 proteins that are T41A, T41I, S45F, and S45P mutants. In some embodiments, the kit comprises four probes comprising the following sequences: AGGAGCTGTGGCAG (SEQ ID NO: 16), GGAGCTGTGATA (SEQ ID NO: 17), TTTACCACTCAGAAAAG (SEQID NO: 21), and TACCACTCAGAGGAG (SEQ ID NO: 22), and wherein each of the four probes is coupled to a microcarrier having a different identifier. In some embodiments, each of the four probes also comprises 8 nucleotides at the 5' end, wherein the 8 nucleotides at the 5' end are adenine or thymine nucleotides, and wherein each of the four probes comprises at least 24 total nucleotides. In some embodiments, the kit comprises: (1) a first probe comprising a sequence selected from the group consisting of AGGAGCTGTGGCAGT (SEQ ID NO: 205), AGGAGCTGTGGCAGTG (SEQ ID NO: 206), GCTGTGGCAGTGGC (SEQ ID NO: 207), GCTGTGGCAGTGGCA (SEQ ID NO: 208), and AAGGAGCTGTGGCAG (SEQ ID NO: 209); (2) a second probe comprising a sequence selected from the group consisting of GGAGCTGTGATAGTGG (SEQ ID NO: 210), GAGCTGTGATAGTGGC (SEQ ID NO: 211), AGCTGTGATAGTGGCA (SEQ ID NO: 212), AGAAGGAGCTGTGATA (SEQ ID NO: 213), and GGAGCTGTGAT (SEQ ID NO: 214); (3) a third probe comprising a sequence selected from the group consisting of ACTCAGAAAAGGAGCT (SEQ ID NO: 215). NO:215), TACCACTCAGAAAGGA (SEQ ID NO:216), TTTACCACTCAGAAAAGGAG (SEQ ID NO:217), TTACCACTCAGAAAG (SEQ ID NO:218) and CAGAAAAGGAGCTGTG (SEQ ID NO:219);and (4) a fourth probe comprising a sequence selected from the group consisting of: ACTCAGAGGAGGAGC (SEQ ID NO: 220), TTACCACTCAGAGGA (SEQ ID NO: 221), TTACCACTCAGAGGAGG (SEQ ID NO: 222), TTAACACTCAGAGGAG (SEQ ID NO: 223), and TTACCAATCAGAGGAGG (SEQ ID NO: 224); and wherein each of the four probes is coupled to a microcarrier having a different identifier. In some embodiments, each of the four probes further comprises 8 nucleotides at the 5' end, wherein the 8 nucleotides at the 5' end are adenine or thymine nucleotides, and wherein each of the four probes comprises at least 24 total nucleotides. In some embodiments, the kit comprises: (1) a first probe comprising a sequence selected from the group consisting of: TTTTTTTTTTTTTAGGAGCTGTGGCAG (SEQ ID NO: 53), TTTTTTTTTTTAGGAGCTGTGGCAGTG (SEQ ID NO: 101), TTTTTTTTTTTAGCTGTGGCAGTGGC (SEQ ID NO: 102), TTTTTTTTTTTGCTGTGGCAGTGGCA (SEQ ID NO: 103), and TTTTTTTTTTTAAGGAGCTGTGGCAG (SEQ ID NO: 104); (2) a second probe comprising a sequence selected from the group consisting of: TTTTTTTTTTTTTGGAGCTGTGATA (SEQ ID NO: 54), TTTTTTTTTGGAGCTGTGATAGTGG (SEQ ID NO: 105), TTTTTTTTTGAGCTGTGATAGTGGC (SEQ ID NO: 106), TTTTTTTTTTTAGCTGTGATAGTGGCA (SEQ ID NO: 107). NO:107), TTTTTTTTTGAAGGAGCTGTGATA (SEQ ID NO:108) and TTTTTTTTTTTTTTGGAGCTGTGAT (SEQ ID NO:109);(3) a third probe comprising a sequence selected from the group consisting of TTTTTTTTTTTACCACTCAGAAAAG (SEQ ID NO:55), TTTAATTTTACTCAGAAAAGGAGCT (SEQ ID NO:110), TTTTTTAATACCACTCAGAAAAGGA (SEQ ID NO:111), TTTTTTTTACCACTCAGAAAAGGAG (SEQ ID NO:112), TTTTTTTTATTACCACTCAGAAAAGGAG (SEQ ID NO:113), and TTTTTTTTTCAGAAAAGGAGCTGTG (SEQ ID NO:114); and (4) a fourth probe comprising a sequence selected from the group consisting of TTTTTTTTTTTAATACCACTCAGAGGAG (SEQ ID NO:56), TTTTTTTTTAAAACTCAGAGGAGGAGC (SEQ ID NO:115), TTTTTTTTTTTATTACCACTCAGAGGA (SEQ ID NO:116), TTTTTTTTTATTACCACTCAGAGGAG (SEQ ID NO:117). NO: 117), TTTTTTTTTTATTAACACTCAGAGGAG (SEQ ID NO: 118) and TTTTTTTTTATTACCAATCAGAGGAGG (SEQ ID NO: 119), and wherein each of the four probes is coupled to a microcarrier with a different identifier. In some embodiments, the kit further comprises a first primer pair comprising the sequence GGAATCCATTCTGGTGCCACT (SEQ ID NO: 13) and AGAAAATCCCTGTTCCCACTCATA (SEQ ID NO: 14), and a second primer pair comprising the sequence GGTGCCACTACCACAGCTCCT (SEQ ID NO: 18) and TCTCAAAACTGCATTCTGACTTTCA (SEQ ID NO: 19). In some embodiments, the kit further comprises the following blocking nucleic acids: a first blocking nucleic acid comprising the following sequence: GCCACTACCACAGCT (invdT); n , wherein n is 1, 2 or 3 (SEQ ID NO: 15); TGCCACTACCACAG (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 150); CACTACCACAGCTCC(invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 151); GCCACTACCACAGCT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 152) or GCCACTACCACAGCT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 153); a second blocking nucleic acid comprising the following sequence: GCTCCTTCTCTGAGT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 20); TCCTTCTCTGAGTGG (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 174); GCTCCTTCTCTGAGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 175); TCCTTCTCTGAGTGG(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 176) or GCTCCTTCTCTGAGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 177), wherein italic nucleic acids represent locked nucleic acids.

[0026] In some embodiments, the DNA mutations in the APC gene include APC mutations encoding APC proteins with Q1367*, R1450*, E1309 frameshift, S1465 frameshift, and T1556 frameshift mutations. In some embodiments, the kit comprises five probes comprising the sequences ACTGCTGAAAAGAGAGAGT (SEQ ID NO: 26), GAAATAAAAGATTGG (SEQ ID NO: 30), TTTTGGGTGTCTAAG (SEQ ID NO: 34), CAAACCAAGTGAGAA (SEQ ID NO: 38), and AGAGGCAGAAAAAAACT (SEQ ID NO: 42), and wherein each of the five probes is coupled to a microcarrier having a different identifier. In some embodiments, the kit comprises: (1) a first probe comprising a sequence selected from the group consisting of: AAATAGCAGAAATAAAAG (SEQ ID NO: 225), GAAATAAAAGATTGGAA (SEQ ID NO: 226), AGAAATAAAAGATTG (SEQ ID NO: 227), GAAATAAATGAATGG (SEQ ID NO: 228), and CAGAAATAAAAGATT (SEQ ID NO: 229); (2) a second probe comprising a sequence selected from the group consisting of: TTTGGGTGTCTAAG (SEQ ID NO: 230), GGGTGTCTAAGCACCACT (SEQ ID NO: 231), CTAAGCACCACTTTT (SEQ ID NO: 232), TTTTGGGTGTCTAA (SEQ ID NO: 233), and GGTGTCTAAGCACCA (SEQ ID NO: 234); (3) a third probe comprising a sequence selected from the group consisting of: AAGTGAGAAGTACCTAA (SEQ ID NO: 235). NO:235), CAAACCAAGTGAGAA (SEQ ID NO:38), TCAAACCAAGTGAG (SEQ ID NO:236), ACCAAGTGAGAAGTA (SEQ ID NO:237) and AGCTCAAACCAAGTGAG (SEQ ID NO:238);(4) a fourth probe comprising a sequence selected from the group consisting of GCACCTACTGCTGAA (SEQ ID NO:239), ACCTACTGCTGAAAAG (SEQ ID NO:240), TGCTGAAAAGAGAGAGT (SEQ ID NO:241), ACTGCTGAAAAGAGAGAGT (SEQ ID NO:26), and CCTACTGCTGAAAAGAGA (SEQ ID NO:242); and (5) a fifth probe comprising a sequence selected from the group consisting of GCAGAAAAAAACTATTG (SEQ ID NO:243), AGAGGCAGAAAAAAACT (SEQ ID NO:42), CAGAAAAAAACTATTGATT (SEQ ID NO:244), AGAAAGAGGCAGAAAAAAACT (SEQ ID NO:245), and GAGGCAGAAAAAAACTA (SEQ ID NO:246); and wherein each of the five probes is coupled to a microcarrier having a different identifier. In some embodiments, each of the five probes further comprises 8 nucleotides at the 5' end, wherein the 8 nucleotides at the 5' end are adenine or thymine nucleotides, and wherein each of the five probes comprises at least 24 total nucleotides. In some embodiments, the kit comprises: (1) a first probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTTGAAATAAAAGATTGG (SEQ ID NO:58), TTTTTTTTTTAAATAGCAGAAATAAAAG (SEQ ID NO:120), TTTTTTTTTTTGAAATAAAAGATTGGAA (SEQ ID NO:121), TTTTTTTTTTTTTTTGAAATAAAAGATTG (SEQ ID NO:122), TTTTTTTTTTTTTGAAATAAATGAATGG (SEQ ID NO:123), and TTTTTTTTTTTTTCAGAAATAAAAGATT (SEQ ID NO:124);(2) a second probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTTGGGTGTCTAAG (SEQ ID NO: 59), TTTTTTTTTATTTGGGTGTCTAAG (SEQ ID NO: 125), TTTTTTGGGTGTCTAAGCACCACT (SEQ ID NO: 126), TTTTTTTTTCTAAGCACCACTTTT (SEQ ID NO: 127), TTTTTTTTTTTTTTGGGTGTCTAA (SEQ ID NO: 128), and TTTTTTTTTGGTGTCTAAGCACCA (SEQ ID NO: 129); (3) a third probe comprising a sequence selected from the group consisting of TTTTTTTTTTTACAAACCAAGTGAGAA (SEQ ID NO: 60), TTTTTTTTAAGTGAGAAGTACCTAA (SEQ ID NO: 130), TTTTTTTTTTTTCAAACCAAGTGAG (SEQ ID NO: 131), TTTTTTTTTTACCAAGTGAGAAGTA (SEQ ID NO: 132). NO:132) and TTTTTTTTAGCTCAAACCAAGTGAG (SEQ ID NO:133); (4) a fourth probe comprising a sequence selected from the group consisting of TTTTTTTTACTGCTGAAAAGAGAGAGT (SEQ ID NO:57), TTTTTTTTTTGCACCTACTGCTGAA (SEQ ID NO:134), TTTTTTTTTACCTACTGCTGAAAAG (SEQ ID NO:135), TTTTTTTTTGCTGAAAAGAGAGAGT (SEQ ID NO:136), and TTTTTTTTTCCTACTGCTGAAAAGAGA (SEQ ID NO:137); and (5) a fifth probe comprising a sequence selected from the group consisting of TTTTTTTTTTAGAGGCAGAAAAAAACT (SEQ ID NO:61), TTTTTTTTTTGCAGAAAAAAACTATTG (SEQ ID NO:138), TTTTTTTTTTTCAGAAAAAAACTATTGATT (SEQ ID NO:139), TTTTTTTTTAGAAAGAGGCAGAAAAAAACT (SEQ ID NO:140). ID NO: 140) and TTTTTTTTTTTGAGGCAGAAAAAAACTA (SEQ ID NO: 141);And each of the five probes is coupled to a microcarrier with a different identifier. In some embodiments, the kit further comprises the following primer pairs: a first primer pair comprising the sequence TAAAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24); a second primer pair comprising the sequence TAGGATGTAATCAGACGACACAGGA (SEQ ID NO: 27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO: 28); a third primer pair comprising the sequence TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO: 31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO: 32); a fourth primer pair comprising the sequence GCAGAAGTAAAACACCTCCACCA (SEQ ID NO: 35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO: 36), wherein the italicized nucleic acids represent locked nucleic acids; and a fourth primer pair comprising the sequence CAGGAAAATGACAATGGGAATG (SEQ ID NO: 37). ID NO: 39) and ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO: 40). In some embodiments, the kit further comprises the following blocking nucleic acids: a first blocking nucleic acid comprising the following sequence: CCACTCTCTCTCTTTTCAGC (invdT); n , wherein n is 1, 2 or 3 (SEQ ID NO:25); TAGGTCCACTCTCTCTCTTTTCAGCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:166); TAGGTCCACTCTCTCTCTTTTCAGCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:167); CCACTCTCTCTCTTTTCAGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:168) or TAGGTCCACTCTCTCTCTTTTCAGCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:169); a second blocking nucleic acid comprising the sequence: CTTTTCTTTTATTTCTGC(invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO:29); CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:154); CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:155); CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:156) or CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:157); a third blocking nucleic acid comprising the following sequence: GTGCTCAGACACC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO:33); GTGCTCAGACACC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:158); AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:159); AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:160) or AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:161); a fourth blocking nucleic acid comprising the following sequence: CTTCTCGCTTGGTT(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO:37); GTACTTCTCGCTTGGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:162); CTTCTCGCTTGGTT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:163); GTACTTCTCGCTTGGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:164) or GTACTTCTCGCTTGGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:165); and a fifth blocking nucleic acid comprising the following sequence: CAATAGTTTTTTCTGCC(invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO:41); GAATCAATAGTTTTTTCTGCCTC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:170); TCAGAATCAATAGTTTTTTCTG(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:171); GAATCAATAGATTTTACTGCCTC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:172) or AATCAATAGTTTTTCTGCCTC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:173), wherein italic nucleic acids represent locked nucleic acids.

[0027] In some embodiments of any of the above embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the kit further comprises a microcarrier having an identifier corresponding to a positive control and coupled to a probe specific for a positive control gene sequence; and a primer pair specific for a positive control DNA sequence. In some embodiments, the positive control DNA sequence comprises the sequence of a human leukocyte antigen gene. In some embodiments, the primer pair specific for the positive control DNA sequence comprises the sequence TGAGTGTTACTTCTTCCCACACTC (SEQ ID NO: 43) and ATTGCTTTTGCGCAATCCCT (SEQ ID NO: 44). In some embodiments, the probe specific for the positive control gene sequence comprises the sequence TTTTTTTTTTTTGGAGACGGTCTG (SEQ ID NO: 45). In some embodiments, the primer pair specific for the positive control DNA sequence comprises the sequence AATCCCATCACCATCTTCCA (SEQ ID NO: 71) and TGGACTCCACGACGTACTCA (SEQ ID NO: 72). In some embodiments, the probe specific for the positive control gene sequence comprises the sequence CTGTCTTCCACTCACTCC (SEQ ID NO: 73). In some embodiments, the kit further comprises a microcarrier having an identifier corresponding to a negative control and coupled to a probe that does not hybridize with the amplified DNA. In some embodiments, the microcarrier having an identifier corresponding to the negative control comprises a probe comprising the sequence AATATAATATATATA (SEQ ID NO: 46).

[0028] In some embodiments, the identifier of the microcarrier comprises a digital barcode. In some embodiments, each of the microcarriers comprises: (i) a first photopolymer layer; (ii) a second photopolymer layer; and (iii) an intermediate layer between the first layer and the second layer, the intermediate layer having a coded pattern representing an identifier defined thereon, wherein the intermediate layer is partially substantially light-transmissive and partially substantially light-impermeable, representing a code corresponding to the microcarrier, wherein the outermost surface of the microcarrier comprises a photoresist photopolymer, and the photoresist photopolymer is functionalized with a probe specific for a DNA mutation, and wherein the microcarrier has approximately the same density as water. In some embodiments, the identifier of the microcarrier comprises an analog code. In some embodiments, each of the microcarriers comprises: (i) a substantially transparent polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other; (ii) a substantially opaque polymer layer, wherein the substantially opaque polymer layer is affixed to the first surface of the substantially transparent polymer layer and surrounds a central portion of the substantially transparent polymer layer, and wherein the substantially opaque polymer layer comprises a two-dimensional shape representing an analog code, wherein the analog code represents an identifier; and (iii) a probe specific for a DNA mutation, wherein the probe is coupled to at least one of the first surface and the second surface of the substantially transparent polymer layer in at least the central portion of the substantially transparent polymer layer. In some embodiments, each of the microcarriers further comprises: (iv) a second substantially transparent polymer layer aligned with the first substantially transparent polymer layer, the second substantially transparent polymer layer having a central portion aligned with the central portion of the first substantially transparent polymer layer, wherein the second substantially transparent polymer layer is affixed to the second surface of the first substantially transparent polymer layer and does not extend beyond the two-dimensional shape of the first substantially transparent polymer layer; and (v) a substantially opaque magnetic layer surrounding the central portion of the first substantially transparent polymer layer between the substantially opaque polymer layer and the central portion of the substantially transparent polymer layer, wherein the substantially opaque magnetic layer is affixed between the first substantially transparent polymer layer and the second substantially transparent polymer layer. In some embodiments, each of the microcarriers further comprises an orientation indicator for orienting the analog code of the substantially opaque polymer layer. In some embodiments, the two-dimensional shape of the substantially opaque polymer layer comprises a gear shape comprising a plurality of gear teeth, and wherein the simulation code is represented by one or more aspects selected from the group consisting of: a height of one or more gear teeth in the plurality of gear teeth, a width of one or more gear teeth in the plurality of gear teeth, a number of gear teeth in the plurality of gear teeth, and an arrangement of one or more gear teeth within the plurality of gear teeth.In some embodiments, each of the microcarriers further comprises: (vi) one or more pillars protruding from the first surface of the first substantially transparent polymer layer, wherein the one or more pillars are not within the central portion of the first substantially transparent polymer layer; and / or (vii) one or more pillars protruding from the second surface of the first substantially transparent polymer layer or the surface of the second substantially transparent polymer layer that is not fixed to the first substantially transparent polymer layer, wherein the one or more pillars are not within the central portion of the first substantially transparent polymer layer or the second substantially transparent polymer layer. In some embodiments, the substantially transparent polymer of the first substantially transparent polymer layer or the second substantially transparent polymer layer comprises an epoxy polymer. In some embodiments, the epoxy polymer is SU-8. In some embodiments, the kit further comprises instructions for using the kit to detect colon cancer, rectal cancer, colorectal cancer, colon adenoma, rectal adenoma, or colorectal adenoma.

[0029] In another aspect, provided herein are kits and articles of manufacture comprising (a) a plurality of probes including a first probe comprising the sequence TTTTTTTTTTATGGAGCTGATGGCG (SEQ ID NO: 75), a second probe comprising the sequence TTTTTTTTTTTTAAGGAGCTGTTGGTG (SEQ ID NO: 79), a third probe comprising the sequence TTTTTTTTTTGGAGCTAGTGGCGTA (SEQ ID NO: 82), a fourth probe comprising the sequence TTTTTTTTAAAGGTGACGTAGGCAA (SEQ ID NO: 87), a fifth probe comprising the sequence TTTTTTTTTTTATACAGAGAAATCTCGAT (SEQ ID NO: 92), a sixth probe comprising the sequence TTTTTTTTATTCTAGCTACAGAAAAATC (SEQ ID NO: 100), a seventh probe comprising the sequence TTTTTTTTTTTTTAGGAGCTGTGGCAG (SEQ ID NO: 53), a seventh probe comprising the sequence TTTTTTTTTTTGAGCTGTGATAGTGGC (SEQ ID NO: 54), a (SEQ ID NO: 106), an eighth probe containing the sequence of TTTTTTAATACCACTCAGAAAAGGA (SEQ ID NO: 111), a ninth probe containing the sequence of TTTTTTTTTTTATTACCACTCAGAGGA (SEQ ID NO: 116), an eleventh probe containing the sequence of TTTTTTTTTTTTTAGAAATAAAAGATTG (SEQ ID NO: 122), a twelfth probe containing the sequence of TTTTTTTTTATTTGGGTGTCTAAG (SEQ ID NO: 125), a thirteenth probe containing the sequence of TTTTTTTTTTACCAAGTGAGAAGTA (SEQ ID NO: 132), a fourteenth probe containing the sequence of TTTTTTTTACTGCTGAAAAGAGAGAGT (SEQ ID NO: 57), and a fifteenth probe containing the sequence of TTTTTTTTTTTGAGGCAGAAAAAAACTA (SEQ ID NO: 141);(b) a plurality of primer pairs, each comprising a first primer pair comprising a sequence of GTACTGGTGGAGTATTTGATAGTG (SEQ ID NO: 1) and ATCGTCAAGGCACTCTTGCCTAC (SEQ ID NO: 2), a second primer pair comprising a sequence of GGACCCACTCCATCGAGATTT (SEQ ID NO: 8) and CAGATATATTTCTTCATGAAGACCTCACAGTAA (SEQ ID NO: 9), a third primer pair comprising a sequence of GGAATCCATTCTGGTGCCACT (SEQ ID NO: 13) and AGAAAATCCCTGTTCCCACTCATA (SEQ ID NO: 14), a fourth primer pair comprising a sequence of GGTGCCACTACCACAGCTCCT (SEQ ID NO: 18) and TCTCAAAACTGCATTCTGACTTTCA (SEQ ID NO: 19), a fourth primer pair comprising a sequence of TAAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24), and a fifth primer pair comprising a sequence of NO:24), a fifth primer pair comprising the sequence TAGGATGTAATCAGACGACACAGGA (SEQ ID NO:27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO:28), a sixth primer pair comprising the sequence TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO:31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO:32), an eighth primer pair comprising the sequence GCAGAAGTAAAACACCTCCACCA (SEQ ID NO:35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO:36), a ninth primer pair comprising the sequence CAGGAAAATGACAATGGGAATG (SEQ ID NO:39) and ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO:40); and a plurality of blocking nucleic acids comprising the following blocking nucleic acids: a first blocking nucleic acid comprising the sequence TACGCCACCAGCT (invdT); n , wherein n is 1, 2 or 3 (SEQ ID NO: 3); a second blocking nucleic acid comprising the sequence GAGATTTCACTGTAGC (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 10); a third blocking nucleic acid comprising the sequence TGCCACTACCACAG (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 150); a fourth blocking nucleic acid comprising the sequence TCCTTCTCTGAGTGG(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 176); a fifth blocking nucleic acid comprising the sequence AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 161); a sixth blocking nucleic acid comprising the sequence CTTCTCGCTTGGTT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 163); a seventh blocking nucleic acid comprising the sequence CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 157); and an eighth blocking nucleic acid comprising the sequence CCACTCTCTCTCTTTTCAGC(invdT)n. n , wherein n is 1, 2 or 3 (SEQ ID NO:25) and a ninth blocking nucleic acid comprising the sequence TCAGAATCAATAGTTTTTTCTG(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO:171), wherein nucleic acids in italics represent locked nucleic acids.

[0030] In another aspect, provided herein are kits or articles of manufacture comprising (a) a plurality of probes, comprising a first probe comprising the sequence TTTTTTTTTTATGGAGCTGATGGCG (SEQ ID NO: 75), a second probe comprising the sequence TTTTTTTTTATGGAGCTGTAGGTGG (SEQ ID NO: 81), a third probe comprising the sequence TTTTTTTTTTTATGGAGCTAGTGG (SEQ ID NO: 49), a fourth probe comprising the sequence TTTTTTTTTATGGAGCTGGTGACGT (SEQ ID NO: 50), a fifth probe comprising the sequence TTTTTTTTTATACAGAGAAATCTCGAT (SEQ ID NO: 92), a sixth probe comprising the sequence TTTTTTTATGTCTAGCTACAGAAAAAT (SEQ ID NO: 52), a seventh probe comprising the sequence TTTTTTTTTTTAGGAGCTGTGGCAGTG (SEQ ID NO: 101), a seventh probe comprising the sequence TTTTTTTTTTTTTGGAGCTGTGATA (SEQ ID NO: 103); NO: 54), an eighth probe containing the sequence of TTTTTTTTAATACCACTCAGAAAAGGA (SEQ ID NO: 111), a ninth probe containing the sequence of TTTTTTTTTATTACCAATCAGAGGAGG (SEQ ID NO: 119), an eleventh probe containing the sequence of TTTTTTTTTTTTTTTAGAAATAAAAGATTG (SEQ ID NO: 122), a twelfth probe containing the sequence of TTTTTTTTTTTTTGGGTGTCTAAG (SEQ ID NO: 59), a twelfth probe containing the sequence of TTTTTTTTTTACCAAGTGAGAAGTA (SEQ ID NO: 132), a fourteenth probe containing the sequence of TTTTTTTTTTACCCTACTGCTGAAAAG (SEQ ID NO: 135), and a fifteenth probe containing the sequence of TTTTTTTTTTTGAGGCAGAAAAAAACTA (SEQ ID NO: 141);(b) a plurality of primer pairs, each comprising a first primer pair comprising a sequence of GTACTGGTGGAGTATTTGATAGTG (SEQ ID NO: 1) and ATCGTCAAGGCACTCTTGCCTAC (SEQ ID NO: 2), a second primer pair comprising a sequence of GGACCCACTCCATCGAGATTT (SEQ ID NO: 8) and CAGATATATTTCTTCATGAAGACCTCACAGTAA (SEQ ID NO: 9), a third primer pair comprising a sequence of GGAATCCATTCTGGTGCCACT (SEQ ID NO: 13) and AGAAAATCCCTGTTCCCACTCATA (SEQ ID NO: 14), a fourth primer pair comprising a sequence of GGTGCCACTACCACAGCTCCT (SEQ ID NO: 18) and TCTCAAAACTGCATTCTGACTTTCA (SEQ ID NO: 19), a fourth primer pair comprising a sequence of TAAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24), and a fifth primer pair comprising a sequence of NO: 24), a fifth primer pair containing the sequence TAGGATGTAATCAGACGACACAGGA (SEQ ID NO: 27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO: 28), a sixth primer pair containing the sequence TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO: 31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO: 32), respectively; an eighth primer pair containing the sequence GCAGAAGTAAAACACCTCCACCA (SEQ ID NO: 35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO: 36), a ninth primer pair containing the sequence CAGGAAAATGACAATGGGAATG (SEQ ID NO: 39) and ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO: 40), a ninth primer pair containing the sequence TGGCAATCGAACGACTCTCAA (SEQ ID NO: 41), a ninth primer pair containing the sequence TGGCAATCGAACGACTCTCAA (SEQ ID NO: 42), a ninth primer pair containing the sequence TGGCAATCGAACGACTCTCAA (SEQ ID NO: 43), a ninth primer pair containing the sequence TGGCAATCGAACGACTCTCAA (SEQ ID NO: 44), a ninth primer pair containing the sequence TGGCAATCGAACGACTCTCTCAA (SEQ ID NO: NO:40); and (c) a plurality of blocking nucleic acids, the plurality of blocking nucleic acids comprising the following blocking nucleic acids: a first blocking nucleic acid comprising the sequence TTGGAGCTGGTGGCGTA (invdT); n , wherein n is 1, 2 or 3 (SEQ ID NO: 142); a second blocking nucleic acid comprising the sequence GAGATTTCACTGTAGC (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 148); a third blocking nucleic acid comprising the sequence GCCACTACCACAGCT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 15); a fourth blocking nucleic acid comprising the sequence TCCTTCTCTGAGTGG (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 174); a fifth blocking nucleic acid comprising the sequence AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 161); a sixth blocking nucleic acid comprising the sequence CTTCTCGCTTGGTT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 163); a seventh blocking nucleic acid comprising the sequence CTTTTCTTTTATTTCTGC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 29); an eighth blocking nucleic acid comprising the sequence CCACTCTCTCTCTTTTCAGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 168); and a ninth blocking nucleic acid comprising the sequence CAATAGTTTTTTCTGCC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 41), wherein the italicized nucleic acids represent locked nucleic acids.

[0031] In another aspect, provided herein are kits or articles of manufacture comprising (a) a plurality of probes, comprising a first probe comprising the sequence TTTTTTTTTTTTAAGGAGCTGATGG (SEQ ID NO: 47), a second probe comprising the sequence TTTTTTTTTTTTTTAAGGAGCTGTTGG (SEQ ID NO: 48), a third probe comprising the sequence TTTTTTTTTTTTTAAGGAGCTAGTGG (SEQ ID NO: 86), a fourth probe comprising the sequence TTTTTTTTTAAGGAGCTGGTGACGT (SEQ ID NO: 91), a fifth probe comprising the sequence TTTTTTTTTAATTACTAGCTACAGAGAAA (SEQ ID NO: 95), a sixth probe comprising the sequence TTTTTTTATTTCTAGCTACAGAAAAAT (SEQ ID NO: 99), a seventh probe comprising the sequence TTTTTTTTTTTAAGGAGCTGTGGCAG (SEQ ID NO: 104), a seventh probe comprising the sequence TTTTTTTTTTTTTTGGAGCTGTGAT (SEQ ID NO: 106), a sixth probe comprising the sequence NO: 109), an eighth probe containing the sequence of TTTTTTTTATTACCACTCAGAAAAG (SEQ ID NO: 113), a ninth probe containing the sequence of TTTTTTTTTATTACCAATCAGAGGAGG (SEQ ID NO: 119), an eleventh probe containing the sequence of TTTTTTTTTTTTTAGAAATAAAAGATTG (SEQ ID NO: 122), a twelfth probe containing the sequence of TTTTTTTTTATTTGGGTGTCTAAG (SEQ ID NO: 125), a thirteenth probe containing the sequence of TTTTTTTTTTACAAACCAAGTGAGAA (SEQ ID NO: 60), a fourteenth probe containing the sequence of TTTTTTTTACTGCTGAAAAGAGAGAGT (SEQ ID NO: 57), and a fifteenth probe containing the sequence of TTTTTTTTTTAGAGGCAGAAAAAAACT (SEQ ID NO: 61);(b) a plurality of primer pairs, each comprising a first primer pair comprising a sequence of GTACTGGTGGAGTATTTGATAGTG (SEQ ID NO: 1) and ATCGTCAAGGCACTCTTGCCTAC (SEQ ID NO: 2), a second primer pair comprising a sequence of GGACCCACTCCATCGAGATTT (SEQ ID NO: 8) and CAGATATATTTCTTCATGAAGACCTCACAGTAA (SEQ ID NO: 9), a third primer pair comprising a sequence of GGAATCCATTCTGGTGCCACT (SEQ ID NO: 13) and AGAAAATCCCTGTTCCCACTCATA (SEQ ID NO: 14), a fourth primer pair comprising a sequence of GGTGCCACTACCACAGCTCCT (SEQ ID NO: 18) and TCTCAAAACTGCATTCTGACTTTCA (SEQ ID NO: 19), a fourth primer pair comprising a sequence of TAAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24), and a fifth primer pair comprising a sequence of NO:24), a fifth primer pair comprising the sequence TAGGATGTAATCAGACGACACAGGA (SEQ ID NO:27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO:28), a sixth primer pair comprising the sequence TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO:31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO:32), an eighth primer pair comprising the sequence GCAGAAGTAAAACACCTCCACCA (SEQ ID NO:35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO:36), a ninth primer pair comprising the sequence CAGGAAAATGACAATGGGAATG (SEQ ID NO:39) and ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO:40); and (c) a plurality of blocking nucleic acids comprising the following blocking nucleic acids: a first blocking nucleic acid comprising the sequence TACGCCACCAGCT(invdT); n , wherein n is 1, 2 or 3 (SEQ ID NO: 3); a second blocking nucleic acid sequence comprising the sequence GAGATTTCACTGTAGC (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 10); a third blocking nucleic acid sequence comprising the sequence GCCACTACCACAGCT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 15); a fourth blocking nucleic acid sequence comprising the sequence GCTCCTTCTCTGAGT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 20); a fifth blocking nucleic acid sequence comprising the sequence GTGCTCAGACACC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 33); a sixth blocking nucleic acid sequence comprising the sequence CTTCTCGCTTGGTT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 37); a seventh blocking nucleic acid sequence comprising the sequence CTTTTCTTTTATTTCTGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 29); an eighth blocking nucleic acid sequence comprising the sequence CCACTCTCTCTCTTTTCAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 25) and a ninth blocking nucleic acid sequence comprising the sequence CAATAGTTTTTTCTGCC (invdT) n , wherein n is 1, 2, or 3 (SEQ ID NO: 41), wherein italicized nucleic acids represent locked nucleic acids. In some embodiments of any of the aforementioned embodiments, each probe in the plurality of probes is coupled to a microcarrier having a unique identifier corresponding to the probe coupled thereto.

[0032] In another aspect, provided herein are kits or articles of manufacture comprising (1) a primer pair for amplifying a locus of a KRAS mutation (e.g., a KRAS protein encoding or causing a G12D, G12V, G12S, or G13D mutation); and (2) a blocking nucleic acid comprising the sequence TACGCCACCAGCT(invdT). n , wherein n is 1, 2 or 3 (SEQ ID NO: 3); TTGGAGCTGGTGGCGTA (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 142); GCTGGTGGCGTAGGCA (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 143); GCTGGTGGCGTAGGC (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 144) or TTGGAGCTGGTGGCGT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 145), wherein italic nucleic acids represent locked nucleic acids; (3a) a first probe comprising a sequence selected from the group consisting of GGAGCTGATGG (SEQ ID NO: 4), AGCTGATGGCGTA (SEQ ID NO: 178), TGGAGCTGATGGCG (SEQ ID NO: 179), TGGAGCTGATGG (SEQ ID NO: 180), and GCTGATGGCGTA (SEQ ID NO: 181); (3b) a second probe comprising a sequence selected from the group consisting of GGAGCTGTTGG (SEQ ID NO: 5), TGGAGCTGTTGGTGGC (SEQ ID NO: 182), GGAGCTGTTGGTG (SEQ ID NO: 183), TGGAGCTGTTGGT (SEQ ID NO: 184), and TGGAGCTGTAGGTGG (SEQ ID NO: 185). NO: 185); (3c) a third probe comprising a sequence selected from the group consisting of TTGGAGCTAGTGGCGTA (SEQ ID NO: 186), GCTAGTGGCGTAGGC (SEQ ID NO: 187), AGCTAGTGGCGT (SEQ ID NO: 188), GTTGGAGCTAGTGG (SEQ ID NO: 189), and GGAGCTAGTGG (SEQ ID NO: 190); (3d) a fourth probe comprising a sequence selected from the group consisting of GGTGACGTAGGCAA (SEQ ID NO: 191), TGACGTAGGCAAGAG (SEQ ID NO: 192), GCTGGTGACGTAGG (SEQ ID NO: 193), AGCTGGTGACGTAG (SEQ ID NO: 194), and GGAGCTGGTGACGT (SEQ ID NO: 195). NO:195); and wherein each of the four probes is coupled to a microcarrier having a different identifier; (4) a primer pair for amplifying a locus of a BRAF mutation (e.g., a BRAF protein encoding or causing a V600E mutation); and (5) a blocking nucleic acid comprising the following sequence: GAGATTTCACTGTAGC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 10); GAGATTTCACTGTAGC (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 146); GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 147); GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 148) or GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 149), wherein italic nucleic acids represent locked nucleic acids; (6a) a first probe comprising a sequence selected from the group consisting of: TACAGAGAAATCTCGAT (SEQ ID NO: 196); TACAGAGAAATCTC (SEQ ID NO: 197); CTAGCTACAGAGAAAT (SEQ ID NO: 198); CTAGCTACAGAGAAA (SEQ ID NO: 199) and TCTAGCTACAGAG (SEQ ID NO: 200); (6b) a second probe comprising a sequence selected from the group consisting of: GTCTAGCTACAGAAAAATC (SEQ ID NO: 201); GTCTAGCTACAGAAAAAT (SEQ ID NO: 12); TAGCTACAGAAAAA (SEQ ID NO: 202); TCTAGCTACAGAAAAAT (SEQ ID NO: 203) and TCTAGCTACAGAAAAATC (SEQ ID NO: 204); NO:204); (7) a primer pair for amplifying a locus of a CTNNB1 mutation (e.g., a CTNNB1 protein encoding or causing T41A, T41I, S45F, and S45P mutations); (8a) a first blocking nucleic acid comprising the following sequence: GCCACTACCACAGCT(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 15); TGCCACTACCACAG (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 150); CACTACCACAGCTCC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 151); GCCACTACCACAGCT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 152) or GCCACTACCACAGCT (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 153), wherein the italicized nucleic acid represents a locked nucleic acid; (8b) a second blocking nucleic acid comprising the following sequence: GCTCCTTCTCTGAGT(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 20); TCCTTCTCTGAGTGG (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 174); GCTCCTTCTCTGAGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 175); TCCTTCTCTGAGTGG(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 176) or GCTCCTTCTCTGAGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 177), wherein the italic nucleic acids represent locked nucleic acids; (9a) a first probe comprising a sequence selected from the group consisting of: AAATAGCAGAAATAAAAG (SEQ ID NO: 225); GAAATAAAAGATTGGAA (SEQ ID NO: 226); AGAAATAAAAGATTG (SEQ ID NO: 227); GAAATAAATGAATGG (SEQ ID NO: 228) and CAGAAATAAAAGATT (SEQ ID NO: 229); NO:229); (9b) a first probe comprising a sequence selected from the group consisting of TTTGGGTGTCTAAG (SEQ ID NO:230); GGGTGTCTAAGCACCACT (SEQ ID NO:231); CTAAGCACCACTTTT (SEQ ID NO:232); TTTTGGGTGTCTAA (SEQ ID NO:233) and GGTGTCTAAGCACCA (SEQ ID NO:234); (9c) a third probe comprising a sequence selected from the group consisting of AAGTGAGAAGTACCTAA (SEQ ID NO:235); CAAACCAAGTGAGAA (SEQ ID NO:38); TCAAACCAAGTGAG (SEQ ID NO:236); ACCAAGTGAGAAGTA (SEQ ID NO:237) and AGCTCAAACCAAGTGAG (SEQ ID NO:238); (9d) a fourth probe comprising a sequence selected from the group consisting of GCACCTACTGCTGAA (SEQ ID NO:239); NO:239); ACCTACTGCTGAAAAG (SEQ ID NO:240); TGCTGAAAAGAGAGAGT (SEQ ID NO:241); ACTGCTGAAAAGAGAGAGT (SEQ ID NO:26) and CCTACTGCTGAAAAGAGA (SEQ ID NO:242); (9e) a fifth probe comprising a sequence selected from the group consisting of GCAGAAAAAAACTATTG (SEQ ID NO:243);AGAGGCAGAAAAAAACT (SEQ ID NO: 42); CAGAAAAAAACTATTGATT (SEQ ID NO: 244); AGAAAGAGGCAGAAAAAAACT (SEQ ID NO: 245) and GAGGCAGAAAAAAACTA (SEQ ID NO: 246); and wherein each of the five probes is coupled to a microcarrier having a different identifier; (10) a primer pair for amplifying a locus of an APC mutation (e.g., encoding or causing a mutated APC protein as described below); (11a) a first blocking nucleic acid comprising the following sequence: CCACTCTCTCTCTTTTCAGC (invdT); n , wherein n is 1, 2 or 3 (SEQ ID NO: 25); TAGGTCCACTCTCTCTCTTTTCAGCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 166); TAGGTCCACTCTCTCTCTTTTCAGCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 167); CCACTCTCTCTCTTTTCAGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 168) or TAGGTCCACTCTCTCTCTTTTCAGCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 169), wherein italic nucleic acids represent locked nucleic acids; (11b) a second blocking nucleic acid comprising the following sequence: CTTTTCTTTTATTTCTGC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 29); CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 154); CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 155); CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 156) or CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 157), wherein italic nucleic acids represent locked nucleic acids; (11c) a third blocking nucleic acid comprising the following sequence: GTGCTCAGACACC(invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 33); GTGCTCAGACACC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 158); AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 159); AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 160) or AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 161), wherein italic nucleic acids represent locked nucleic acids; (11d) a fourth blocking nucleic acid comprising the following sequence: CTTCTCGCTTGGTT(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 37); GTACTTCTCGCTTGGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 162); CTTCTCGCTTGGTT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 163); GTACTTCTCGCTTGGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 164) or GTACTTCTCGCTTGGT(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 165), wherein italic nucleic acids represent locked nucleic acids; (11e) a fifth blocking nucleic acid comprising the following sequence: CAATAGTTTTTTCTGCC(invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 41); GAATCAATAGTTTTTTCTGCCTC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 170); TCAGAATCAATAGTTTTTTCTG(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 171); GAATCAATAGATTTTACTGCCTC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 172) or AATCAATAGTTTTTCTGCCTC(invdT)n, wherein n is 1, 2 or 3 (SEQ ID NO: 173), wherein italic nucleic acids represent locked nucleic acids; (12a) a first probe comprising a sequence selected from the group consisting of AAATAGCAGAAATAAAAG (SEQ ID NO: 225), GAAATAAAAGATTGGAA (SEQ ID NO: 226), AGAAATAAAAGATTG (SEQ ID NO: 227), NO:227), GAAAATAAATGAATGG (SEQ ID NO:228), and CAGAAATAAAAGATT (SEQ ID NO:229); (12b) a second probe comprising a sequence selected from the group consisting of TTTGGGTGTCTAAG (SEQ ID NO:230), GGGTGTCTAAGCACCACT (SEQ ID NO:231), CTAAGCACCACTTTT (SEQ ID NO:232), TTTTGGGTGTCTAA (SEQ ID NO:233), and GGTGTCTAAGCACCA (SEQ ID NO:234); (12c) a third probe comprising a sequence selected from the group consisting of AAGTGAGAAGTACCTAA (SEQ ID NO:235), CAAACCAAGTGAGAA (SEQ ID NO:38), TCAAACCAAGTGAG (SEQ ID NO:236), ACCAAGTGAGAAGTA (SEQ ID NO:237), and AGCTCAAACCAAGTGAG (SEQ ID NO:238). NO:238); (12d) a fourth probe comprising a sequence selected from the group consisting of GCACCTACTGCTGAA (SEQ ID NO:239), ACCTACTGCTGAAAAG (SEQ ID NO:240), TGCTGAAAAGAGAGAGT (SEQ ID NO:241), ACTGCTGAAAAGAGAGAGT (SEQ ID NO:26), and CCTACTGCTGAAAAGAGA (SEQ ID NO:242);And (12e) a fifth probe comprising a sequence selected from the group consisting of GCAGAAAAAAACTATTG (SEQ ID NO: 243), AGAGGCAGAAAAAAACT (SEQ ID NO: 42), CAGAAAAAAACTATTGATT (SEQ ID NO: 244), AGAAAGAGGCAGAAAAAAACT (SEQ ID NO: 245), and GAGGCAGAAAAAAACTA (SEQ ID NO: 246). In some embodiments, each probe is coupled to a microcarrier having a different identifier. In some embodiments, the primer pair for amplifying the locus of the KRAS mutation comprises the sequence GTACTGGTGGAGTATTTGATAGTG (SEQ ID NO: 1) and ATCGTCAAGGCACTCTTGCCTAC (SEQ ID NO: 2). In some embodiments, the primer pair for amplifying the locus of the BRAF mutation comprises the sequence GGACCCACTCCATCGAGATTT (SEQ ID NO: 8) and CAGATATATTTCTTCATGAAGACCTCACAGTAA (SEQ ID NO: 9). In some embodiments, the primer pair for amplifying the CTNNB1 mutated locus comprises: a first primer pair comprising the sequence GGAATCCATTCTGGTGCCACT (SEQ ID NO: 13) and AGAAAATCCCTGTTCCCACTCATA (SEQ ID NO: 14) and a second primer pair comprising the sequence GGTGCCACTACCACAGCTCCT (SEQ ID NO: 18) and TCTCAAAACTGCATTCTGACTTTCA (SEQ ID NO: 19). In some embodiments, the primer pair for amplifying the locus of the APC mutation comprises: a first primer pair comprising the sequence TAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24); a second primer pair comprising the sequence TAGGATGTAATCAGACGACACAGGA (SEQ ID NO: 27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO: 28); a third primer pair comprising the sequence TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO: 31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO: 32);A fourth primer pair comprising the sequences GCAGAAGTAAAACACCTCCACCA (SEQ ID NO: 35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO: 36), wherein the italicized nucleic acids represent locked nucleic acids; and a fifth primer pair comprising the sequences CAGGAAAATGACAATGGGAATG (SEQ ID NO: 39) and ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO: 40).

[0033] It should be understood that one, some or all of the properties of the various embodiments described herein may be combined to form further embodiments of the invention. These and other aspects of the invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figures 1A and 1B show two views of exemplary microcarriers.

[0036] Figures 1C and ID show exemplary assays for DNA detection using exemplary microcarriers.

[0037] 2A and 2B show two views of exemplary microcarriers.

[0038] Figure 3 An exemplary analog encoding scheme is shown, including multiple shape-varying points for generating a unique analog code.

[0039] Figure 4A Three instances of microcarriers are shown, each with a unique simulation code.

[0040] Figure 4B Shown are examples of microcarriers with unique analog codes according to some embodiments.

[0041] 5A and 5B show two views of exemplary microcarriers.

[0042] 6A and 6B show two views of exemplary microcarriers.

[0043] Figure 6C Dimensions of an exemplary simulation code are shown. Dimensions are based on μm units.

[0044] Figure 7 Exemplary microcarriers are shown.

[0045] Figure 8A An exemplary microcarrier is shown that includes an asymmetric starting position as an orientation indicator.

[0046] Figure 8BAn exemplary analog encoding scheme is shown, including multiple shape-varying points for generating a unique analog code.

[0047] 9A-9C show two views of an exemplary microcarrier (FIG. 9A and FIG. 9B), and a depiction of optional features (FIG. 9C).

[0048] Figure 10 Methods for producing exemplary microcarriers are shown.

[0049] Figure 11A and 11B Methods for producing exemplary microcarriers are shown.

[0050] Figures 12A-12E Methods for producing exemplary microcarriers are shown.

[0051] Figures 13A-13C Methods for producing exemplary microcarriers are shown.

[0052] Figure 14 Shown is a flow chart illustrating an exemplary method for detecting the presence of one or more DNA mutations according to some embodiments.

[0053] Figure 15 and Figure 16 Illustrated are methods for expressing a target DNA mutation relative to a wild-type ( Figure 15 ) loci preferentially amplify and detect mutants ( Figure 16 ) locus. The solid horizontal line represents the amplified DNA sequence, the dashed horizontal line represents the primer / probe / blocking nucleic acid (NA) sequence, and the vertical line represents Watson-Crick base pairing.

[0054] Figure 17 Shown is a flow chart illustrating an exemplary protocol for hybridizing amplified DNA, detecting the presence or absence of hybridization of the amplified DNA to one or more probes coupled to microcarriers, and detecting analog identifiers of the microcarriers, according to some embodiments.

[0055] Figure 18A and Figure 18B Results are shown for detecting the presence of DNA mutations in the KRAS, BRAF, CTNNB1, and APC genes according to some embodiments. The numbers report the fluorescence signal (in arbitrary units (AU)) observed for each pairwise combination of isolated DNA (having mutant or wild-type sequence, as shown in the columns) and probes (as shown in the rows). DETAILED DESCRIPTION

[0056] In one aspect, provided herein is a method for detecting the presence of DNA mutations in KRAS, BRAF, CTNNB1, and APC genes. In some embodiments, the method includes isolating DNA from a sample; using primers specific for the loci of one or more DNA mutations in each of KRAS, BRAF, CTNNB1, and APC genes to amplify the isolated DNA by polymerase chain reaction (PCR); hybridizing the amplified DNA with at least four probes, the at least four probes including one or more DNA mutations in each of KRAS, BRAF, CTNNB1, and APC genes with specific probes, wherein each of the at least four probes is coupled to a microcarrier, and wherein each microcarrier includes an identifier corresponding to the probe coupled thereto; detecting the presence or absence of hybridization of the amplified DNA with the at least four probes, wherein the hybridization between the amplified DNA and one of the probes indicates that the DNA mutation corresponding to the probe exists; and detecting the identifier of the microcarrier, for example, so that each detected identifier is associated with its corresponding probe (and hybridization between the probe and the amplified DNA, or its absence).

[0057] In another aspect, provided herein is a kit or article of manufacture comprising at least four microcarriers, wherein each of the at least four microcarriers comprises: a probe coupled to the microcarrier, wherein the probe is specific for a DNA mutation in the KRAS, BRAF, CTNNB1, and APC genes; and an identifier corresponding to the probe coupled thereto; wherein the kit comprises at least one microcarrier comprising a probe specific for a DNA mutation in the KRAS gene, at least one microcarrier comprising a probe specific for a DNA mutation in the BRAF gene, at least one microcarrier comprising a probe specific for a DNA mutation in the CTNNB1 gene, and at least one microcarrier comprising a probe specific for a DNA mutation in the APC gene.

[0058] In another aspect, provided herein are kits or articles of manufacture comprising (a) a plurality of probes, comprising a first probe comprising the sequence TTTTTTTTTTATGGAGCTGATGGCG (SEQ ID NO: 75), a second probe comprising the sequence TTTTTTTTTTTTAAGGAGCTGTTGGTG (SEQ ID NO: 79), a third probe comprising the sequence TTTTTTTTTTGGAGCTAGTGGCGTA (SEQ ID NO: 82), a fourth probe comprising the sequence TTTTTTTTAAAGGTGACGTAGGCAA (SEQ ID NO: 87), a fifth probe comprising the sequence TTTTTTTTTTTATACAGAGAAATCTCGAT (SEQ ID NO: 92), a sixth probe comprising the sequence TTTTTTTTATTCTAGCTACAGAAAAATC (SEQ ID NO: 100), a seventh probe comprising the sequence TTTTTTTTTTTTTAGGAGCTGTGGCAG (SEQ ID NO: 53), a seventh probe comprising the sequence TTTTTTTTTTTGAGCTGTGATAGTGGC (SEQ ID NO: 54), a the eighth probe containing the sequence of TTTTTTAATACCACTCAGAAAAGGA (SEQ ID NO: 106), the ninth probe containing the sequence of TTTTTTTTAATACCACTCAGAAAAGGA (SEQ ID NO: 111), the tenth probe containing the sequence of TTTTTTTTTTTATTACCACTCAGAGGA (SEQ ID NO: 116), the eleventh probe containing the sequence of TTTTTTTTTTTTTAGAAATAAAAGATTG (SEQ ID NO: 122), the twelfth probe containing the sequence of TTTTTTTTTATTTGGGTGTCTAAG (SEQ ID NO: 125), the thirteenth probe containing the sequence of TTTTTTTTTTACCAAGTGAGAAGTA (SEQ ID NO: 132), the fourteenth probe containing the sequence of TTTTTTTTACTGCTGAAAAGAGAGAGT (SEQ ID NO: 57), and the fifteenth probe containing the sequence of TTTTTTTTTTTGAGGCAGAAAAAAACTA (SEQ ID NO: 141);(b) a plurality of primer pairs, each comprising a first primer pair comprising a sequence of GTACTGGTGGAGTATTTGATAGTG (SEQ ID NO: 1) and ATCGTCAAGGCACTCTTGCCTAC (SEQ ID NO: 2), a second primer pair comprising a sequence of GGACCCACTCCATCGAGATTT (SEQ ID NO: 8) and CAGATATATTTCTTCATGAAGACCTCACAGTAA (SEQ ID NO: 9), a third primer pair comprising a sequence of GGAATCCATTCTGGTGCCACT (SEQ ID NO: 13) and AGAAAATCCCTGTTCCCACTCATA (SEQ ID NO: 14), a fourth primer pair comprising a sequence of GGTGCCACTACCACAGCTCCT (SEQ ID NO: 18) and TCTCAAAACTGCATTCTGACTTTCA (SEQ ID NO: 19), a fourth primer pair comprising a sequence of TAAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24), and a fifth primer pair comprising a sequence of NO:24), a fifth primer pair comprising the sequence TAGGATGTAATCAGACGACACAGGA (SEQ ID NO:27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO:28), a sixth primer pair comprising the sequence TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO:31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO:32), an eighth primer pair comprising the sequence GCAGAAGTAAAACACCTCCACCA (SEQ ID NO:35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO:36), a ninth primer pair comprising the sequence CAGGAAAATGACAATGGGAATG (SEQ ID NO:39) and ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO:40); and a plurality of blocking nucleic acids comprising the following blocking nucleic acids: a first blocking nucleic acid comprising the sequence TACGCCACCAGCT(invdT); n , wherein n is 1, 2 or 3 (SEQ ID NO: 3); a second blocking nucleic acid comprising the sequence GAGATTTCACTGTAGC (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 10); a third blocking nucleic acid comprising the sequence TGCCACTACCACAGinvdTinvdTinvdT (SEQ ID NO: 150); a fourth blocking nucleic acid comprising the sequence TCCTTCTCTGAGTGGinvdTinvdTinvdT (SEQ ID NO: 176); a fifth blocking nucleic acid comprising the sequence AGTGGTGCTCAGACACCCAinvdTinvdTinvdT (SEQ ID NO: 161); a sixth blocking nucleic acid comprising the sequence CTTCTCGCTTGGTTinvdTinvdTinvdT (SEQ ID NO: 163); and a seventh blocking nucleic acid comprising the sequence CTTTTCTTTTATTTCTGCinvdTinvdTinvdT (SEQ ID NO: 157);An eighth blocking nucleic acid comprising the sequence CCACTCTCTCTCTTTTCAGCinvdTinvdTinvdT (SEQ ID NO: 25) and a ninth blocking nucleic acid comprising the sequence TCAGAATCAATAGTTTTTTCTG invdTinvdTinvdT (SEQ ID NO: 171), wherein the italicized nucleic acids represent locked nucleic acids. In another aspect, provided herein are kits or articles of manufacture comprising (a) a plurality of probes, comprising a first probe comprising the sequence TTTTTTTTTTATGGAGCTGATGGCG (SEQ ID NO: 75), a second probe comprising the sequence TTTTTTTTTATGGAGCTGTAGGTGG (SEQ ID NO: 81), a third probe comprising the sequence TTTTTTTTTTTATGGAGCTAGTGG (SEQ ID NO: 49), a fourth probe comprising the sequence TTTTTTTTTATGGAGCTGGTGACGT (SEQ ID NO: 50), a fifth probe comprising the sequence TTTTTTTTTATACAGAGAAATCTCGAT (SEQ ID NO: 92), a sixth probe comprising the sequence TTTTTTTATGTCTAGCTACAGAAAAAT (SEQ ID NO: 52), a seventh probe comprising the sequence TTTTTTTTTTTAGGAGCTGTGGCAGTG (SEQ ID NO: 101), a seventh probe comprising the sequence TTTTTTTTTTTTTGGAGCTGTGATA (SEQ ID NO: 103); NO: 54), an eighth probe containing the sequence of TTTTTTTTAATACCACTCAGAAAAGGA (SEQ ID NO: 111), a ninth probe containing the sequence of TTTTTTTTTATTACCAATCAGAGGAGG (SEQ ID NO: 119), an eleventh probe containing the sequence of TTTTTTTTTTTTTTTAGAAATAAAAGATTG (SEQ ID NO: 122), a twelfth probe containing the sequence of TTTTTTTTTTTTTGGGTGTCTAAG (SEQ ID NO: 59), a thirteenth probe containing the sequence of TTTTTTTTTTACCAAGTGAGAAGTA (SEQ ID NO: 132), a fourteenth probe containing the sequence of TTTTTTTTTTACCCTACTGCTGAAAAG (SEQ ID NO: 135), and a fifteenth probe containing the sequence of TTTTTTTTTTTGAGGCAGAAAAAAACTA (SEQ ID NO: 141);(b) a plurality of primer pairs, each comprising a first primer pair comprising a sequence of GTACTGGTGGAGTATTTGATAGTG (SEQ ID NO: 1) and ATCGTCAAGGCACTCTTGCCTAC (SEQ ID NO: 2), a second primer pair comprising a sequence of GGACCCACTCCATCGAGATTT (SEQ ID NO: 8) and CAGATATATTTCTTCATGAAGACCTCACAGTAA (SEQ ID NO: 9), a third primer pair comprising a sequence of GGAATCCATTCTGGTGCCACT (SEQ ID NO: 13) and AGAAAATCCCTGTTCCCACTCATA (SEQ ID NO: 14), a fourth primer pair comprising a sequence of GGTGCCACTACCACAGCTCCT (SEQ ID NO: 18) and TCTCAAAACTGCATTCTGACTTTCA (SEQ ID NO: 19), a fourth primer pair comprising a sequence of TAAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24), and a fifth primer pair comprising a sequence of NO: 24), a fifth primer pair containing the sequence TAGGATGTAATCAGACGACACAGGA (SEQ ID NO: 27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO: 28), a sixth primer pair containing the sequence TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO: 31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO: 32), an eighth primer pair containing the sequence GCAGAAGTAAAACACCTCCACCA (SEQ ID NO: 35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO: 36), a ninth primer pair containing the sequence CAGGAAAATGACAATGGGAATG (SEQ ID NO: 39) and ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO: 40). NO:40); and (c) and a plurality of blocking nucleic acids, the plurality of blocking nucleic acids comprising the following blocking nucleic acids: a first blocking nucleic acid comprising the sequence TTGGAGCTGGTGGCGTAinvdTinvdTinvdT (SEQ ID NO: 142); a second blocking nucleic acid comprising the sequence GAGATTTCACTGTAGCinvdTinvdTinvdT (SEQ ID NO: 148); and a third blocking nucleic acid comprising the sequence GCCACTACCACAGCT (invdT);n, wherein n is 1, 2 or 3 (SEQ ID NO: 15); a fourth blocking nucleic acid comprising the sequence TCCTTCTCTGAGTGGinvdTinvdTinvdT (SEQ ID NO: 174); a fifth blocking nucleic acid comprising the sequence AGTGGTGCTCAGACACCCAinvdTinvdTinvdT (SEQ ID NO: 161); a sixth blocking nucleic acid comprising the sequence CTTCTCGCTTGGTTinvdTinvdTinvdT (SEQ ID NO: 163); a seventh blocking nucleic acid comprising the sequence CTTTTCTTTTATTTCTGCinvdTinvdTinvdT (SEQ ID NO: 29);An eighth blocking nucleic acid comprising the sequence CCACTCTCTCTCTTTTCAGCinvdTinvdTinvdT (SEQ ID NO: 168) and a ninth blocking nucleic acid comprising the sequence CAATAGTTTTTTCTGCCinvdTinvdTinvdT (SEQ ID NO: 41), wherein nucleic acids in italics represent locked nucleic acids. In another aspect, provided herein are kits or articles of manufacture comprising (a) a plurality of probes, comprising a first probe comprising the sequence TTTTTTTTTTTTAAGGAGCTGATGG (SEQ ID NO: 47), a second probe comprising the sequence TTTTTTTTTTTTTTAAGGAGCTGTTGG (SEQ ID NO: 48), a third probe comprising the sequence TTTTTTTTTTTTTAAGGAGCTAGTGG (SEQ ID NO: 86), a fourth probe comprising the sequence TTTTTTTTTAAGGAGCTGGTGACGT (SEQ ID NO: 91), a fifth probe comprising the sequence TTTTTTTTTAATTACTAGCTACAGAGAAA (SEQ ID NO: 95), a sixth probe comprising the sequence TTTTTTTATTTCTAGCTACAGAAAAAT (SEQ ID NO: 99), a seventh probe comprising the sequence TTTTTTTTTTTAAGGAGCTGTGGCAG (SEQ ID NO: 104), a seventh probe comprising the sequence TTTTTTTTTTTTTTGGAGCTGTGAT (SEQ ID NO: 106), a sixth probe comprising the sequence NO: 109), an eighth probe containing the sequence of TTTTTTTTATTACCACTCAGAAAAG (SEQ ID NO: 113), a ninth probe containing the sequence of TTTTTTTTTATTACCAATCAGAGGAGG (SEQ ID NO: 119), an eleventh probe containing the sequence of TTTTTTTTTTTTTAGAAATAAAAGATTG (SEQ ID NO: 122), a twelfth probe containing the sequence of TTTTTTTTTATTTGGGTGTCTAAG (SEQ ID NO: 125), a thirteenth probe containing the sequence of TTTTTTTTTTACAAACCAAGTGAGAA (SEQ ID NO: 60), a fourteenth probe containing the sequence of TTTTTTTTACTGCTGAAAAGAGAGAGT (SEQ ID NO: 57), and a fifteenth probe containing the sequence of TTTTTTTTTTAGAGGCAGAAAAAAACT (SEQ ID NO: 61);(b) a plurality of primer pairs, each comprising a first primer pair comprising a sequence of GTACTGGTGGAGTATTTGATAGTG (SEQ ID NO: 1) and ATCGTCAAGGCACTCTTGCCTAC (SEQ ID NO: 2), a second primer pair comprising a sequence of GGACCCACTCCATCGAGATTT (SEQ ID NO: 8) and CAGATATATTTCTTCATGAAGACCTCACAGTAA (SEQ ID NO: 9), a third primer pair comprising a sequence of GGAATCCATTCTGGTGCCACT (SEQ ID NO: 13) and AGAAAATCCCTGTTCCCACTCATA (SEQ ID NO: 14), a fourth primer pair comprising a sequence of GGTGCCACTACCACAGCTCCT (SEQ ID NO: 18) and TCTCAAAACTGCATTCTGACTTTCA (SEQ ID NO: 19), a fourth primer pair comprising a sequence of TAAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24), and a fifth primer pair comprising a sequence of NO:24), a fifth primer pair comprising the sequence TAGGATGTAATCAGACGACACAGGA (SEQ ID NO:27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO:28), a sixth primer pair comprising the sequence TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO:31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO:32), an eighth primer pair comprising the sequence GCAGAAGTAAAACACCTCCACCA (SEQ ID NO:35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO:36), and a ninth primer pair comprising the sequence CAGGAAAATGACAATGGGAATG (SEQ ID NO:39) and ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO:40); and (c) a plurality of blocking nucleic acids comprising the following blocking nucleic acids: a first blocking nucleic acid comprising the sequence TACGCCACCAGCT(invdT); n , wherein n is 1, 2 or 3 (SEQ ID NO: 3); a second blocking nucleic acid comprising the sequence GAGATTTCACTGTAGC (invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 10); a third blocking nucleic acid comprising the sequence GCCACTACCACAGCT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 15); a fourth blocking nucleic acid comprising the sequence GCTCCTTCTCTGAGTinvdTinvdTinvdTinvdT (SEQ ID NO: 20); a fifth blocking nucleic acid comprising the sequence GTGCTCAGACACCinvdTinvdTinvdT (SEQ ID NO: 33); a sixth blocking nucleic acid comprising the sequence CTTCTCGCTTGGTTinvdTinvdTinvdT (SEQ ID NO: 37); a seventh blocking nucleic acid comprising the sequence CTTTTCTTTTATTTCTGCinvdTinvdTinvdT (SEQ ID NO: 29); an eighth blocking nucleic acid comprising the sequence CCACTCTCTCTCTTTTCAGCinvdTinvdTinvdT (SEQ ID NO: 25); and a ninth blocking nucleic acid comprising the sequence CAATAGTTTTTTCTGCCinvdTinvdTinvdT (SEQ ID NO: 41), wherein nucleic acids in italics represent locked nucleic acids. In some embodiments, each probe of the plurality of probes is coupled to a microcarrier having a unique identifier corresponding to the probe to which it is coupled.

[0059] I. General Technology

[0060] Unless otherwise indicated, the practice of the techniques described herein will employ conventional techniques in polymer technology, microfabrication, microelectromechanical systems (MEMS) fabrication, photolithography, microfluidics, organic chemistry, biochemistry, oligonucleotide synthesis and modification, bioconjugate chemistry, nucleic acid hybridization, molecular biology, microbiology, genetics, recombinant DNA, and related fields, as are within the capabilities of those skilled in the art. The techniques are described in the references cited herein and are fully explained in the literature.

[0061] For molecular biology and recombinant DNA techniques, see, for example, (Maniatis, T. et al. (1982), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor; Ausubel, F.M. (1987), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F.M. (1989), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Sambrook, J. et al. (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor; Innis, M.A. (1990), PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F.M. (1992), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F.M. (1995), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M.A. et al. (1995), PCR Strategies, Academic Press; Ausubel, F.M.(1999), Short Protocols in Molecular Biology: A Compendium ofMethods from Current Protocols in Molecular Biology, Wiley and annual updates. .

[0062] For DNA synthesis techniques and nucleic acid chemistry, see, for example, Gait, MJ (1990), Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991), Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992), The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994), Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996), Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (1996), Bioconjugate Techniques, Academic Press).

[0063] Regarding microfabrication, see for example (Campbell, SA (1996), The Science and Engineering of Microelectronic Fabrication, Oxford University Press; Zaut, PV (1996), Microarray Fabrication: a Practical Guide to Semiconductor Processing, Semiconductor Services; Madou, MJ (1997), Fundamentals of Microfabrication, CRCPress; Rai-Choudhury, P. (1997). Handbook of Microlithography, Micromachining, & Microfabrication: Microlithography).

[0064] II. Definitions

[0065] Before describing the present invention in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0066] As used herein, the term "microcarrier" may refer to a physical substrate to which a capture agent or probe may be coupled. The microcarriers of the present disclosure may take any suitable geometric form or shape. In some embodiments, the microcarrier may be disc-shaped. Typically, the form or shape of the microcarrier will include at least one about 10 -4 to 10 -7 Dimensionality of level m (hence the prefix "micro").

[0067] As used herein, the term "polymer" may refer to any macromolecular structure comprising repeating monomers. A polymer may be natural (e.g., naturally occurring) or synthetic (e.g., man-made, such as a polymer composed of one or more non-natural monomers and / or polymerized in a configuration or combination not found in nature).

[0068] As used herein, the terms "substantially transparent" and "substantially opaque" can refer to the ability of light (e.g., of a particular wavelength, such as infrared, visible, UV, etc.) to pass through a substance, such as a polymer layer. A substantially transparent polymer can refer to a polymer that is transparent, translucent, and / or translucent to light, while a substantially opaque polymer can refer to a polymer that reflects and / or absorbs light. It should be understood that whether a material is substantially transparent or substantially opaque can depend on the wavelength and / or intensity of the light illuminating the material, as well as the means for detecting the light (or its reduction or absence) that passes through the material. In some embodiments, a substantially opaque material results in a perceptible reduction in transmitted light compared to the surrounding material or image field, for example, as imaged by an optical microscope (e.g., brightfield microscopy, darkfield microscopy, phase contrast microscopy, differential interference contrast (DIC) microscopy, Nomarski interference contrast (NIC) microscopy, Nomarski, Hoffman modulation contrast (HMC) microscopy, or fluorescence microscopy). In some embodiments, the substantially transparent material allows an appreciable amount of transmitted light to pass through the material, for example, for imaging by optical microscopy (e.g., brightfield microscopy, darkfield microscopy, phase contrast microscopy, differential interference contrast (DIC) microscopy, Nomarski interference contrast (NIC) microscopy, Nomarski, Hoffman modulation contrast (HMC) microscopy, or fluorescence microscopy).

[0069] As used herein, the term "analog code" may refer to any code in which the encoded information is represented in a non-quantized and / or non-discrete manner, e.g., in contrast to a digital code. For example, a digital code is sampled at discrete locations for a finite set of values ​​(e.g., 0 / 1 type values), whereas an analog code may be sampled over a larger range of positions (or as a continuous whole) and / or may contain a wider set of values ​​(e.g., shapes). In some embodiments, one or more analog shape recognition techniques may be used to read or decode the analog code.

[0070] As used herein, " sample " refers to a composition containing material (such as molecules) to be detected. In one embodiment, a sample is a " biological sample " (i.e., any material obtained from a living source (e.g., humans, animals, plants, bacteria, fungi, protozoa, viruses)). A biological sample can be in any form, including solid materials (e.g., feces, tissue, cell pellets, and biopsies) and biological fluids (e.g., urine, blood, feces, saliva, lymph, tears, sweat, prostatic fluid, seminal fluid, semen, bile, mucus, amniotic fluid, and mouthwash (containing oral cells)). Solid materials are typically mixed with fluids. Sample can also refer to environmental samples such as water, air, soil, or any other environmental source.

[0071] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and so forth.

[0072] As used herein, the term "about" refers to the usual error range of the corresponding value that is readily known to those skilled in the art. References to "about" values ​​or parameters herein include (and describe) embodiments for the value or parameter itself.

[0073] It is to be understood that aspects and embodiments of the present invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0074] III. Methods for Detecting DNA Mutations

[0075] Certain aspects of the present disclosure relate to methods for detecting the presence of DNA mutations (e.g., one or more mutations in KRAS, BRAF, CTNNB1, and / or APC genes) by using microcarriers (e.g., encoding microcarriers as described herein, or any microcarrier described in International Publication No. WO2016 / 198954). The methods of the present disclosure employ one or more encoding microcarriers having some or all of the microcarrier features and aspects described herein (e.g., in Section IV, Section V, and Section VI). Advantageously, these encoding microcarriers allow for detection of DNA mutations in improved multiplex assays with a large number of potential unique microcarriers and reduced recognition errors compared to conventional multiplex assays. Figure 14-16 A flow chart describing an exemplary method for detecting DNA mutations is provided in The detection methods used herein can be performed in any suitable assay container known in the art, such as a microplate, a petri dish, or many other well-known assay containers.

[0076] In some embodiments, the methods of the present disclosure include isolating DNA from a sample. Standard molecular techniques known in the art allow for the isolation of DNA from a variety of different types of samples. DNA isolation kits suitable for a variety of samples are commercially available. For example, The DNA Stool Mini Kit (QIAGEN; Hilden, Germany) can be used to isolate DNA from stool samples according to the manufacturer's instructions (eg, using an elution volume of 50 μL per 2 g stool sample).

[0077] In some embodiments, the methods of the present disclosure use DNA from a sample at a concentration of about 0.5 ng / μL to 2.5 ng / μL. In some embodiments, the methods of the present disclosure use DNA from a sample at a concentration of at least about 0.5 ng / μL. For example, 20 μL of a 0.5 ng / μL DNA stock solution (a total of 10 ng DNA) or a 2.5 ng / μL DNA stock solution (a total of 50 ng DNA) can be used (e.g., for PCR amplification).

[0078] In some embodiments, the method of the present disclosure is used to detect, monitor, screen or monitor the treatment response of colon cancer, rectal cancer, colorectal cancer, colon adenoma, rectal adenoma or colorectal adenoma of the patient from which the sample is collected. As used herein, colon cancer, rectal cancer and colorectal cancer can refer to various types of cancer, including but not limited to adenocarcinoma, lymphoma, stromal tumor, leiomyosarcoma, carcinoid tumor and melanoma. As DNA mutations accumulate, colorectal cancer is considered to develop from benign tumors such as adenoma (e.g., polyps) to malignant tumors such as cancer and metastatic disease, but even some early adenomas have DNA mutations, such as KRAS activating mutations as described herein. See Fearon, EF and Vogelstein, B. (1990) Cell 61: 759-67. Detection of DNA mutations from fecal samples is particularly meaningful for early detection of colorectal cancer. See, e.g., U.S. Patent No. 7,833,757; Imperiale, TF et al. (2014) N. Engl. J. Med. 370: 1287-97; and Kit (Exact Sciences Corp.). Existing diagnostic techniques, such as sigmoidoscopy and colonoscopy are highly invasive (and therefore can suffer from low patient acceptance) and can miss tumors that are very small and / or in inaccessible locations. In contrast, the methods described herein are believed to be highly accurate, robust, and non-invasive.

[0079] The method of the present disclosure can be used to detect the analyte (for example, DNA mutation) in any suitable solution. In some embodiments, the solution comprises a biological sample. In some embodiments, the solution comprises DNA separated from a biological sample and an optional buffer. Suitable buffers for DNA separation are well known in the art. Examples of biological samples include but are not limited to feces, blood, urine, sputum, bile, cerebrospinal fluid, interstitial fluid of skin or adipose tissue, saliva, tears, bronchoalveolar lavage fluid, oropharyngeal secretions, intestinal fluid, cervical vaginal or uterine secretions and semen. In some embodiments, the sample is a fecal sample. In one embodiment, multiple samples (for example, representing different regions of a sample, such as described in Example 2) are obtained from a single sample and analyzed separately. In other embodiments, a single large fecal sample is analyzed (for example, analyzing whole feces or fecal samples rather than sampling discrete smaller samples). In some embodiments, the biological sample can be from people. In other embodiments, the solution comprises samples that are not biological samples, such as environmental samples, samples prepared in a laboratory (e.g., samples containing one or more analytes that have been prepared, isolated, purified, and / or synthesized), fixed samples (e.g., formalin-fixed, paraffin-embedded, or FFPE samples), and the like.

[0080] In some embodiments, the method of the present disclosure includes amplifying DNA (e.g., DNA isolated from a sample as described above) by polymerase chain reaction (PCR). PCR technology is well known in the art. In brief, using a template DNA chain (e.g., separated and denatured from a sample) and a pair of oligonucleotide primers complementary to the 3' ends of the sense and antisense strands of the DNA template, a thermostable DNA polymerase is used to amplify a copy of the target DNA sequence. DNA polymerase is mixed with two primers, all four deoxynucleotides (dNTPs), a buffer, magnesium ions (e.g., MgCl 2 ) and potassium ions (e.g., KCl) and optional other components in the reaction. The reaction mixture is then subjected to a cycle (e.g., 20-40) of multiple temperature changes, which allows denaturation of the DNA template, annealing of the primers to the denatured single-stranded template, and primer extension by DNA polymerase. Various DNA polymerases with different target properties for PCR have been characterized (e.g., the ability to amplify long templates or repetitive templates, high fidelity, hot start, etc.) and the DNA polymerase is commercially available.

[0081] In some embodiments, the method of the present disclosure includes amplifying (for example, by PCR) one or more DNA mutation loci in one or more specific target genes from isolated DNA.As described herein, " loci " of DNA mutations include enough adjacent sequences on one or both sides of the mutation itself and the mutation, for the hybridization of the PCR amplification and / or probe of the DNA sequence of mutation and the DNA sequence of mutation.As known in the art, the minimum sequence length sufficient for PCR amplification can be affected by several factors, including but not limited to the melting temperature of polymerase, primer, the tendency of primer formation primer dimer, template and primer ratio etc. In some embodiments, the loci of DNA mutations include adjacent sequences of at least about 100 base pairs (that is, including 5' and 3' adjacent sequences of DNA mutations). In some embodiments, the loci of DNA mutations include adjacent sequences of less than or equal to about 200 base pairs (that is, including 5' and 3' adjacent sequences of DNA mutations).As mentioned above, the loci of DNA mutations can use loci as DNA templates, using a pair of primers that have specificity to loci to amplify. Although mutations are described herein as DNA mutations, it will be appreciated that similar techniques can be used to detect RNA mutations, for example using mRNA or cDNA from a sample.

[0082] In some embodiments, multiple PCR reactions can be used, for example, to detect various DNA mutations. In some embodiments, each PCR reaction can include multiple primer pairs, each primer being specific for the target DNA mutation. For example, in some embodiments, as shown in Examples 1 and 2 below, the methods of the present disclosure can include two PCR reactions: a first PCR reaction with reagents for detecting the following mutations: KRAS G12D, KRAS G12V, KRAS G12S, KRAS G13D, BRAFV600E1, BRAF V600E2, CTNNB1 T41A, CTNNB1 T41I, APC E1309, APC Q1367, APC R1450, and APC C1556; and a second PCR reaction with reagents for detecting the following mutations: CTNNB1 S45F, CTNNB1 S45P, and APC S1465. In some embodiments, each PCR reaction includes: 10 μL PCR reaction mixture, 10 μL primer mix, and 20 μL DNA (eg, 20 μL 0.5 ng / μL DNA stock solution, for a total of 10 ng DNA). In some embodiments, multiplex PCR reactions are performed using the same temperature cycling conditions.

[0083] mutation

[0084] In some embodiments, the method of the present disclosure includes amplifying one or more mutated loci in the KRAS gene. As used herein for KRAS, BRAF, CTNNB1 and APC genes, amplifying the loci of DNA mutations includes amplifying mutant loci or corresponding wild-type loci. It should be understood that in most cases, although four or more mutations are screened in multiple assays, any single sample will typically include at most one mutation that is screened. KRAS encodes the KRAS proto-oncogene (a small GTPase that often mutates in human cancers, also known as the Kirsten rag sarcoma viral oncogene homolog), PR310 cK-ras oncogene, c-Ki-ras, c-Kirsten-ras, K-Ras2, K-ras p21, GTPase KRas, cellular c-Ki-ras2 proto-oncogene, cellular transforming proto-oncogene, oncogene KRAS2, transforming protein p21 and v-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homolog. In some embodiments, the KRAS gene is a human KRAS gene. In some embodiments, the human KRAS gene refers to the gene described by NCBI Entrez Gene ID No. 3845, including mutants and variants thereof. In other embodiments, the KRAS gene is from one of the following: mouse (see, e.g., NCBI Entrez Gene ID No. 16653), rat (see, e.g., NCBI Entrez Gene ID No. 24525), cynomolgus monkey (see, e.g., NCBI Entrez Gene ID No. 102131483), fish (see, e.g., NCBI Entrez Gene ID No. 445289), dog (see, e.g., NCBI Entrez Gene ID No. 403871), cow (see, e.g., NCBI Entrez Gene ID No. 541140), horse (see, e.g., NCBI Entrez Gene ID No. 100064473), chicken (see, e.g., NCBI Entrez Gene ID No. 418207), chimpanzee (see, e.g., NCBI Entrez Gene ID No. 473387), rhesus monkey (see, e.g., NCBI Entrez Gene ID No. 707977), or cat (see, e.g., NCBI Entrez Gene ID No. 707977). ID number 751104).

[0085] A variety of KRAS mutations associated with cancer are known and can be appropriately detected by the methods described herein; see, for example, Prior, IA et al. (2012) Cancer Res. 72: 24 57-67. For example, one study found that 30% of colorectal tumors in their cohort had KRAS mutations, primarily at amino acid 12 (Smith, G. et al. (2002) Proc. Natl. Acad. Sci. 99: 9433-8). In some embodiments described herein, KRAS mutations are named based on the resulting amino acid substitution / deletion / frameshift according to the human KRAS protein, for example, as shown in the following sequence: MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHKEKMSKDGKKKKKKSKTKCVIM (SEQ ID NO: 62).An exemplary human KRAS cDNA sequence is shown in the following sequence: TGTGCTCGGAGCTCGATTTTCCTAGGCGGCGGCCGCGGCGGCGGAGGCAGCAGCGGCGGCGGCAGTGGCGGCGGCGAAGGTGGCGGCGGCTCGGCCAGTACTCCCGGCCCCCGCCATTTCGGACTGGGAGCGAGCGCGGCGCAGGCACTGAAGGCGGCGGCGGGGCCAGAGGCTCAGCGGCTCCCAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGATTCCTACAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAGCAGGTCAAGAGGAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGCTTTCTTTGTGTATTTGCCATAAATAATACTAAATCATTTGAAGATATTCACCATTATAGAGAACAAATTAAAAGAGTTAAGGACTCTGAAGATGTACCTATGGTCCTAGTAGGAAATAAATGTGATTTGCCTTCTAGAACAGTAGACACAAAACAGGCTCAGGACTTAGCAAGAAGTTATGGAATTCCTTTTATTGAAACATCAGCAAAGACAAGACAGAGAGTGGAGGATGCTTTTTATACATTGGTGAGAGAG ATCCGACAATACAGATTGAAAAAAATCAGCAAAGAAGAAAAGACTCCTGGCTGTGTGAAAATTAAAAAATGCATTATAATGTAATCTGGGTGTTGATGATGCCTTCTATACATTAGTTCGAGAAATTCGAAAACATAAAGAAAAGATGAGCAAAGATGGTAAAAAGAAGAAAAAG (SEQ ID NO:66). In some embodiments, the DNA mutation results in a mutation of G12 or G13 according to SEQ ID NO:62 or SEQ ID NO:66.For example, in some embodiments, the DNA mutation in the KRAS gene encodes or results in a KRAS protein with a G12D, G12V, G12S, or G13D mutation (encoded according to SEQ ID NO: 62). These DNA mutations can also be described by their nucleotide position (rather than the mutated polypeptide codon) in Table A below. In some embodiments, the DNA mutation in the KRAS gene results in a c.35G>A, c.35G>T, c.34G>A, or c.38G>A mutation in the corresponding cDNA sequence of SEQ ID NO: 66.

[0086] Therefore, as described above, since the sequences of a variety of KRAS genes and corresponding mutations are known, one of ordinary skill in the art can appropriately select primer pairs to amplify the loci of one or more of these DNA mutations. Various factors that affect primer design are known, and tools for identifying primer pairs suitable for amplifying a given DNA template sequence are available (see, for example, www.ncbi.nlm.nih.gov / tools / primer-blast / ). In some embodiments, the primer pairs for amplifying the loci of KRAS mutations (e.g., KRAS proteins encoding or causing G12D, G12V, G12S, or G13D mutations) comprise the sequences GTACTGGTGGAGTATTTGATAGTG (SEQ ID NO: 1) and ATCGTCAAGGCACTCTTGCCTAC (SEQ ID NO: 2), respectively.

[0087] In some embodiments, the methods of the present disclosure include amplifying one or more mutated loci in the BRAF gene. BRAF encodes the BRAF proto-oncogene (a serine / threonine kinase frequently mutated in human cancers, also known as B-Raf, BRAF1, B-RAF1, RAFB1, NS7, 94kDa B-raf protein, p94, murine sarcoma viral (v-raf) oncogene homolog B1, v-raf murine sarcoma viral oncogene homolog B, and v-raf murine sarcoma viral oncogene homolog B1). In some embodiments, the BRAF gene is a human BRAF gene. In some embodiments, the human BRAF gene refers to the gene described by NCBI Entrez Gene ID No. 673, including mutants and variants thereof. In other embodiments, the BRAF gene is from one of the following organisms: mouse (see, e.g., NCBI Entrez Gene ID No. 109880), rat (see, e.g., NCBI Entrez Gene ID No. 114486), cynomolgus monkey (see, e.g., NCBI Entrez Gene ID No. 101866436), fish (see, e.g., NCBI Entrez Gene ID No. 403065), dog (see, e.g., NCBI Entrez Gene ID No. 475526), ​​cow (see, e.g., NCBI Entrez Gene ID No. 536051), horse (see, e.g., NCBI Entrez Gene ID No. 100065760), chicken (see, e.g., NCBI Entrez Gene ID No. 396239), chimpanzee (see, e.g., NCBI Entrez Gene ID No. 463781), rhesus monkey (see, e.g., NCBI Entrez Gene ID No. 693554), or cat (see, e.g., NCBI Entrez Gene ID No. 693555). Entrez Gene ID number 101092346).

[0088]

[0089] Therefore, as described above, since the sequences of a variety of BRAF genes and corresponding mutations are known, one of ordinary skill in the art can appropriately select primer pairs to amplify the loci of one or more of these DNA mutations. Various factors that affect primer design are known, and tools for identifying primer pairs suitable for amplifying a given DNA template sequence are available (see, e.g., www.ncbi.nlm.nih.gov / tools / primer-blast / ). In some embodiments, primer pairs for amplifying the locus of a BRAF mutation (e.g., a BRAF protein encoding or causing a V600E mutation) comprise the sequences GGACCCACTCCATCGAGATTT (SEQ ID NO: 8) and CAGATATATTTCTTCATGAAGACCTCACAGTAA (SEQ ID NO: 9), respectively.

[0090] In some embodiments, the methods of the present disclosure include amplifying one or more mutated loci in the CTNNB1 gene. CTNNB1 encodes CTNNB1β catenin 1 protein, which is a subunit of the cadherin protein complex that transduces Wnt signals and is often mutated in human cancers. It is also known as CTNNB, MRD19, armadillo catenin (cadherin-related protein) β1, and catenin (cadherin-related protein) β1 88kDa. In some embodiments, the CTNNB1 gene is a human CTNNB1 gene. In some embodiments, the human CTNNB1 gene refers to the gene described by NCBI Entrez Gene ID No. 1499, including mutants and variants thereof. In other embodiments, the CTNNB1 gene is from one of the following organisms: mouse (see, e.g., NCBI Entrez Gene ID No. 12387), rat (see, e.g., NCBI Entrez Gene ID No. 84353), cynomolgus monkey (see, e.g., NCBI Entrez Gene ID No. 102146984), fish (see, e.g., NCBI Entrez Gene ID No. 30265), dog (see, e.g., NCBI Entrez Gene ID No. 477032), cow (see, e.g., NCBI Entrez Gene ID No. 539003), horse (see, e.g., NCBI Entrez Gene ID No. 100055241), chicken (see, e.g., NCBI Entrez Gene ID No. 395964), chimpanzee (see, e.g., NCBI Entrez Gene ID No. 450183), rhesus monkey (see, e.g., NCBI Entrez Gene ID No. 574265), or cat (see, e.g., NCBI Entrez Gene ID No. 574265). Gene ID number 101097342).

[0091]

[0092] Since, as described above, the sequences of a variety of CTNNB1 genes and corresponding mutations are known, one of ordinary skill in the art can appropriately select primer pairs to amplify the loci of one or more of these DNA mutations. Various factors that affect primer design are known, and tools for identifying primer pairs suitable for amplifying a given DNA template sequence are available (see, e.g., www.ncbi.nlm.nih.gov / tools / primer-blast / ). In some embodiments, primer pairs for amplifying loci of CTNNB1 mutations (e.g., CTNNB1 proteins encoding or causing T41A, T41I, S45F, and S45P mutations) comprise the sequences GGAATCCATTCTGGTGCCACT (SEQ ID NO: 13) and AGAAAATCCCTGTTCCCACTCATA (SEQ ID NO: 14) (for T41 locus); or GGTGCCACTACCACAGCTCCT (SEQ ID NO: 18) and TCTCAAAACTGCATTCTGACTTTCA (SEQ ID NO: 19) (for S45 locus). In some embodiments, two primer pairs are used for PCR: the first primer pair comprises the sequence GGAATCCATTCTGGTGCCACT (SEQ ID NO: 13) and AGAAAATCCCTGTTCCCACTCATA (SEQ ID NO: 14), and the second primer pair comprises the sequence GGTGCCACTACCACAGCTCCT (SEQ ID NO: 18) and TCTCAAAACTGCATTCTGACTTTCA (SEQ ID NO: 19).

[0093] In some embodiments, the methods of the present disclosure include amplifying one or more mutated loci in the APC gene. APC encodes the APC tumor suppressor, which is a negative regulator of beta-catenin and Wnt signaling, frequently mutated in human cancers, also known as GS, DP2, DP3, BTPS2, DP2.5, PPP1R46, adenomatous polyposis coli protein, WNT signaling pathway negative regulator, adenomatous polyposis coli tumor suppressor (deleted in polyposis coli 2.5), protein phosphatase 1 regulatory subunit 46, and truncated adenomatous polyposis coli. In some embodiments, the APC gene is a human APC gene. In some embodiments, the human APC gene refers to the gene described by NCBI Entrez Gene ID No. 324, including mutants and variants thereof. In other embodiments, the APC gene is from one of the following organisms: mouse (see, e.g., NCBI Entrez Gene ID No. 11789), rat (see, e.g., NCBI Entrez Gene ID No. 24205), cynomolgus monkey (see, e.g., NCBI Entrez Gene ID No. 102126553), fish (see, e.g., NCBI Entrez Gene ID No. 386762), dog (see, e.g., NCBI Entrez Gene ID No. 479139), cow (see, e.g., NCBI Entrez Gene ID No. 533233), horse (see, e.g., NCBI Entrez Gene ID No. 100064431), chicken (see, e.g., NCBI Entrez Gene ID No. 415607), chimpanzee (see, e.g., NCBI Entrez Gene ID No. 461999), rhesus monkey (see, e.g., NCBI Entrez Gene ID No. 693443), or cat (see, e.g., NCBI Entrez Gene ID No. 693443). Gene ID number 101096138).

[0094]

[0095] Since, as described above, the sequences of various APC genes and corresponding mutations are known, one of ordinary skill in the art can appropriately select primer pairs to amplify the loci of one or more of these DNA mutations. Various factors that influence primer design are known, and tools for identifying primer pairs suitable for amplifying a given DNA template sequence are available (see, for example, www.ncbi.nlm.nih.gov / tools / primer-blast / ). In some embodiments, primer pairs for amplifying loci encoding APC mutations (e.g., APC proteins encoding or causing the mutations described above) comprise the sequences TAAAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24) for the S1465 locus, respectively; TAGGATGTAATCAGACGACACAGGA (SEQ ID NO: 27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO: 28) for the E1309 locus, respectively; TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO: 31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO: 32) for the Q1367 locus, respectively; GCAGAAGTAAAACACCTCCACCA (SEQ ID NO: 35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO: 36) for the Q1367 locus, respectively. NO: 36) (for the R1450 locus); or CAGGAAAATGACAATGGGAATG (SEQ ID NO: 39) and ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO: 40) (for the T1556 locus), respectively.In some embodiments, five primer pairs are used for PCR: a first primer pair comprising the sequence TAAAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24); a second primer pair comprising the sequence TAGGATGTAATCAGACGACACAGGA (SEQ ID NO: 27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO: 28); a third primer pair comprising the sequence TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO: 31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO: 32); a fourth primer pair comprising the sequence GCAGAAGTAAAACACCTCCACCA (SEQ ID NO: 35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO: 36); and a fourth primer pair comprising the sequence CAGGAAAATGACAATGGGAATG (SEQ ID NO: 39) and ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO: In some embodiments, the primer pair for amplifying a locus for one or more APC mutations (e.g., an APC protein encoding or causing one or more mutations described above) comprises the sequences GCAGAAGTAAAACACCTCCACCA (SEQ ID NO: 35) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24).

[0096] In some embodiments, the primer pairs of the present disclosure comprise one or more modified nucleotides, such as locked nucleic acids (described in more detail below). For example, in some embodiments, the primer pairs for amplifying the locus of an APC mutation (e.g., encoding or causing the mutated APC protein described above) comprise the sequences GCAGAAGTAAAACACCTCCACCA (SEQ ID NO: 35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO: 36), wherein the italicized nucleic acids represent locked nucleic acids. In some embodiments, the primer pairs for amplifying the locus of an APC mutation (e.g., encoding or causing the mutated APC protein described above) comprise the sequences GCAGAAGTAAAACACCTCCACCA (SEQ ID NO: 35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO: 36), wherein the italicized nucleic acids represent locked nucleic acids. GGTACTTC (SEQ ID NO: 36), wherein the underlined nucleic acid represents a locked nucleic acid. In some embodiments, five primer pairs are used for PCR to amplify the locus of the APC gene: a first primer pair comprising the sequence TAAAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24); a second primer pair comprising the sequence TAGGATGTAATCAGACGACACAGGA (SEQ ID NO: 27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO: 28); a third primer pair comprising the sequence TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO: 31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO: 32); a fourth primer pair comprising the sequence GCAGAAGTAAAACACCTCCACCA (SEQ ID NO: 35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO: 36), wherein italicized nucleic acids represent locked nucleic acids; and a fourth primer pair comprising the sequence CAGGAAAATGACAATGGGAATG (SEQ ID NO: 37). In some embodiments, five primer pairs are used for PCR to amplify the locus of the APC gene: a first primer pair comprising the sequence TAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24); a second primer pair comprising the sequence TAGGATGTAATCAGACGACACAGGA (SEQ ID NO: 27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO: 28); a third primer pair comprising the sequence TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO: 31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO: 32); a third primer pair comprising the sequence GCAGAAGTAAAACACCTCCACCA (SEQ ID NO: 35) and GGTGCTTTATTTTTA (SEQ ID NO: 36). GGa fourth primer pair comprising TACTTC (SEQ ID NO: 36), wherein the underlined nucleic acid represents the locked nucleic acid; and a fifth primer pair comprising the sequence CAGGAAAATGACAATGGGAATG (SEQ ID NO: 39) and ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO: 40).

[0097] Multiplex assays of the present disclosure include detecting a combination of two or more of the above mutations. For example, in some embodiments, the methods of the present disclosure include amplifying one or more mutated loci in the KRAS gene, one or more mutated loci in the BRAF gene, one or more mutated loci in the CTNNB1 gene, and one or more mutated loci in the APC gene. In certain embodiments, the methods of the present disclosure comprise amplifying a mutated locus encoding a KRAS protein with a G12D, G12V, G12S, or G13D mutation; a mutated locus encoding a BRAF protein with a V600E mutation; a mutated locus encoding a CTNNB1 protein with a T41A or T41I mutation and / or a mutated locus encoding a CTNNB1 protein with an S45F or S45P mutation; and one or more of the following loci: a mutated locus encoding an APC protein with a Q1367* mutation, a mutated locus encoding an APC protein with an R1450* mutation, a mutated locus encoding an APC protein with an E1309 out-of-frame mutation, a mutated locus encoding an APC protein with an S1465 out-of-frame mutation, and a mutated locus encoding an APC protein with a T1556 out-of-frame mutation. In certain embodiments, the methods of the present disclosure include amplifying a mutated locus encoding a KRAS protein with G12D, G12V, G12S, and G13D mutations; a mutated locus encoding a BRAF protein with a V600E mutation; a mutated locus encoding a CTNNB1 protein with a T41A or T41I mutation and / or a mutated locus encoding a CTNNB1 protein with an S45F or S45P mutation; and a mutated locus encoding an APC protein with a Q1367* mutation, a mutated locus encoding an APC protein with an R1450* mutation, a mutated locus encoding an APC protein with an E1309 out-of-frame mutation, a mutated locus encoding an APC protein with an S1465 out-of-frame mutation, and a mutated locus encoding an APC protein with a T1556 out-of-frame mutation.

[0098] Blocking nucleic acids

[0099] In some embodiments, the methods of the present disclosure include amplifying the isolated DNA by PCR in the presence of one or more blocking nucleic acids (e.g., a blocking nucleic acid corresponding to each target DNA mutation). Advantageously, the blocking nucleic acids prevent amplification of the wild-type DNA locus, thereby increasing the sensitivity of detecting DNA mutations (see Figure 15 and Figure 16 In some embodiments, the method comprises amplifying the isolated DNA by PCR in the presence of at least four blocking nucleic acids, each of which hybridizes to a wild-type DNA locus corresponding to a DNA mutation in the KRAS, BRAF, CTNNB1, or APC gene.

[0100] In some embodiments, the blocking nucleic acid of the present disclosure comprises: a single-stranded oligonucleotide that hybridizes to a corresponding wild-type DNA locus, and a 3' terminal portion that blocks extension of the single-stranded oligonucleotide, thereby preventing amplification of the wild-type DNA locus. In some embodiments, the 3' terminal portion comprises one or more inverted deoxythymidines (invdT). In some embodiments, the 3' terminal portion comprises three consecutive inverted deoxythymidines.

[0101] In some embodiments, the blocking nucleic acid of the present disclosure comprises one or more modified nucleotides. It is envisioned that oligonucleotides comprising modified nucleotides at some or all sequence positions, and the oligonucleotides may have improved hybridization properties, particularly advantageous for use as blocking nucleic acids during PCR. For example, it is known that oligonucleotides partially or completely synthesized using locked nucleic acids (LNA) have higher thermal stability than corresponding oligonucleotides synthesized using only conventional nucleotides, thereby increasing the melting temperature of the oligonucleotide: LNA duplex and allowing shorter sequences to maintain stable hybridization during thermal cycling. See Koshkin, AA et al. (1998) Tetrahedron 54: 3607-30. A variety of modified nucleotides are known, including but not limited to locked nucleic acids (LNA), peptide nucleic acids (PNA), hexose nucleic acids (HNA), threose nucleic acids (TNA), glycol nucleic acids (GNA) and cyclohexenyl nucleic acids (CeNA). For a more detailed description of exemplary modified nucleotides, see, for example, Schmidt, M. (2010) BioEssays 32: 322-31.

[0102] In some embodiments, a blocking nucleic acid of the present disclosure hybridizes to a wild-type KRAS locus corresponding to a locus encoding one or more DNA mutations of a KRAS protein encoding a G12D, G12V, G12S, or G13D mutation. In some embodiments, a blocking nucleic acid comprises the sequence TACGCCACCAGCT(invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 3); TTGGAGCTGGTGGCGTA (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 142), GCTGGTGGCGTAGGCA (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 143), GCTGGTGGCGTAGGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 144) or TTGGAGCTGGTGGCGT (invdT) n , wherein n is 1, 2, or 3 (SEQ ID NO: 145), wherein the italicized nucleic acids represent locked nucleic acids. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, the blocking nucleic acid comprises the sequence TACGCCACCAGCTinvdTinvdTinvdTinvdT (SEQ ID NO: 3), TTGGAGCTGGTGGCGTAinvdTinvdTinvdT (SEQ ID NO: 142), GCTGGTGGCGTAGGCAinvdTinvdTinvdT (SEQ ID NO: 143), GCTGGTGGCGTAGGCinvdTinvdTinvdT (SEQ ID NO: 144), or TTGGAGCTGGTGGCGTinvdTinvdTinvdT (SEQ ID NO: 145), wherein the italicized nucleic acids represent locked nucleic acids. In certain embodiments, the blocking nucleic acid comprises the sequence TA C G CC A CC A G CTinvdTinvdTinvdT (SEQ ID NO: 3), TT GG A G CT GGTGGC GTAinvdTinvdTinvdT (SEQ ID NO: 142), GCT GG T GG C G TA G G C AinvdTinvdTinvdT(SEQID NO:143)、 GCTGGTGGCGTA GGCinvdTinvdTinvdT (SEQ ID NO:144) or TT GG A G CT GG T GG C GTinvdTinvdTinvdT (SEQ ID NO: 145), wherein the underlined nucleic acid represents a locked nucleic acid. In some embodiments, the blocking nucleic acid comprises the sequence of SEQ ID NO: 3 or 142-145, but optionally includes a different pattern or type of one or more modified nucleotides. In some embodiments, the blocking nucleic acid comprises the sequence of SEQ ID NO: 3 or 142-145, but includes a different 3' terminal portion.

[0103] In some embodiments, the blocking nucleic acid of the present disclosure hybridizes to a wild-type BRAF locus corresponding to a locus encoding one or more DNA mutations of a BRAF protein encoding a V600E mutation. In some embodiments, the blocking nucleic acid comprises the sequence GAGATTTCACTGTAGC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 10); GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 146); GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 147); GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 148) or GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2, or 3 (SEQ ID NO: 149), wherein the italicized nucleic acids represent locked nucleic acids. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, the blocking nucleic acid comprises the sequence GAGATTTCACTGTAGCinvdTinvdTinvdT (SEQ ID NO: 10), GAGATTTCACTGTAGCinvdTinvdTinvdT (SEQ ID NO: 146), GAGATTTCACTGTAGCinvdTinvdTinvdT (SEQ ID NO: 147), GAGATTTCACTGTAGCinvdTinvdTinvdT (SEQ ID NO: 148), or GAGATTTCACTGTAGCinvdTinvdTinvdT (SEQ ID NO: 149), wherein the italicized nucleic acids represent locked nucleic acids. In certain embodiments, the blocking nucleic acid comprises the sequence G AGAT T TCAC T GTAGC invdTinvdTinvdT(SEQ ID NO:10),GA G AT TTCACTGT AGCinvdTinvdTinvdT (SEQ ID NO: 146), G AGA TT TC AC TGTAG CinvdTinvdTinvdT (SEQ ID NO: 147), GAGAT T TCACT G TAGC invdTinvdTinvdT (SEQ ID NO: 148) or G AGA T TT C ACT G T A GC invdTinvdTinvdT (SEQ ID NO: 149), wherein the underlined nucleic acid represents a locked nucleic acid. In some embodiments, the blocking nucleic acid comprises the sequence of SEQ ID NO: 10 or 146-149 but optionally includes a different pattern or type of one or more modified nucleotides. In some embodiments, the blocking nucleic acid comprises the sequence of SEQ ID NO: 10 or 146-149 but includes a different 3' terminal portion.

[0104] In some embodiments, the blocking nucleic acid of the present disclosure hybridizes to a wild-type CTNNB1 locus corresponding to one or more mutant loci of a CTNNB1 protein with a T41A or T41I mutation. In some embodiments, the blocking nucleic acid comprises the sequence GCCACTACCACAGCT(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 15); TGCCACTACCACAG (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 150); CACTACCACAGCTCC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 151); GCCACTACCACAGCT (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 152) or GCCACTACCACAGCT (invdT) n, wherein n is 1, 2, or 3 (SEQ ID NO: 153), wherein the italicized nucleic acids represent locked nucleic acids. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, the blocking nucleic acid comprises the sequence GCCACTACCACAGCTinvdTinvdTinvdTinvdT (SEQ ID NO: 15), TGCCACTACCACAGinvdTinvdTinvdT (SEQ ID NO: 150), CACTACCACAGCTCCinvdTinvdTinvdT (SEQ ID NO: 151), GCCACTACCACAGCTinvdTinvdTinvdT (SEQ ID NO: 152), or GCCACTACCACAGCTinvdTinvdTinvdT (SEQ ID NO: 153), wherein the italicized nucleic acids represent locked nucleic acids. In certain embodiments, the blocking nucleic acid comprises the sequence GC CACTACCACAG CTinvdTinvdTinvdT (SEQ ID NO: 15), T G C CA CT ACCA C AG invdTinvdTinvdT (SEQ ID NO: 150), C AC T ACCA C AGC T C CinvdTinvdTinvdT (SEQ ID NO: 151), G CCACT A CCA C AG C T invdTinvdTinvdT (SEQ ID NO: 152) or GC C ACTA CCA CAG CTinvdTinvdTinvdT (SEQ ID NO: 153), wherein the underlined nucleic acid represents a locked nucleic acid. In some embodiments, the blocking nucleic acid comprises the sequence of SEQ ID NO: 15 or 150-153, but optionally includes a different pattern or type of one or more modified nucleotides. In some embodiments, the blocking nucleic acid comprises the sequence of SEQ ID NO: 15 or 150-153, but includes a different 3' terminal portion.

[0105] In some embodiments, the blocking nucleic acid of the present disclosure hybridizes to a wild-type CTNNB1 locus corresponding to one or more mutant loci encoding a CTNNB1 protein with S45F or S45P mutations. In some embodiments, the blocking nucleic acid comprises the sequence GCTCCTTCTCTGAGT(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 20); TCCTTCTCTGAGTGG (invdT) n , wherein n is 1, 2, or 3 (SEQ ID NO: 174); GCTCCTTCTCTGAGT(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 175); TCCTTCTCTGAGTGG(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 176); or GCTCCTTCTCTGAGT(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 177), wherein italicized nucleic acids represent locked nucleic acids. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, the blocking nucleic acid comprises the sequence GCTCCTTCTCTGAGTinvdTinvdTinvdTinvdT (SEQ ID NO: 20), TCCTTCTCTGAGTGGinvdTinvdTinvdT (SEQ ID NO: 174), GCTCCTTCTCTGAGTinvdTinvdTinvdT (SEQ ID NO: 175), TCCTTCTCTGAGTGGinvdTinvdTinvdT (SEQ ID NO: 176), or GCTCCTTCTCTGAGTinvdTinvdTinvdT (SEQ ID NO: 177), wherein the italicized nucleic acid represents a locked nucleic acid. In certain embodiments, the blocking nucleic acid comprises the sequence GC TCCTTCTCTG AGTinvdTinvdTinvdT (SEQ ID NO: 20), T CC T TCTC T G A G T G GinvdTinvdTinvdT (SEQ ID NO: 174), G C T CC T TC TC TGA GTinvdTinvdTinvdT (SEQ ID NO: 175), T CC TT CT CT GAG T GG invdTinvdTinvdT (SEQ ID NO:176) or G C T CC TT CT C TGAG T invdTinvdTinvdT (SEQ ID NO: 177), wherein the underlined nucleic acid represents a locked nucleic acid. In some embodiments, the blocking nucleic acid comprises the sequence of SEQ ID NO: 20 or 174-177, but optionally includes a different pattern or type of one or more modified nucleotides. In some embodiments, the blocking nucleic acid comprises the sequence of SEQ ID NO: 20 or 174-177, but includes a different 3' terminal portion.

[0106] In some embodiments, the blocking nucleic acid of the present disclosure hybridizes to a wild-type APC locus corresponding to one or more mutant loci encoding an APC protein with a Q1367* mutation. In some embodiments, the blocking nucleic acid comprises the sequence GTGCTCAGACACC(invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 33); GTGCTCAGACACC (invdT) n , wherein n is 1, 2, or 3 (SEQ ID NO: 158); AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 159); AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 160); or AGTGGTGCTCAGACACCCA(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 161), wherein italicized nucleic acids represent locked nucleic acids. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, the blocking nucleic acid comprises the sequence GTGCTCAGACACCinvdTinvdTinvdT (SEQ ID NO: 33), GTGCTCAGACACCinvdTinvdTinvdT (SEQ ID NO: 158), AGTGGTGCTCAGACACCCAinvdTinvdTinvdT (SEQ ID NO: 159), AGTGGTGCTCAGACACCCAinvdTinvdTinvdT (SEQ ID NO: 160), or AGTGGTGCTCAGACACCCAinvdTinvdTinvdT (SEQ ID NO: 161), wherein the italicized nucleic acid represents a locked nucleic acid. In certain embodiments, the blocking nucleic acid comprises the sequence GTG CTCA G ACACCinvdTinvdTinvdT (SEQ ID NO: 33), G TGC TC A G A C ACC invdTinvdTinvdT(SEQID NO:158)、AGTGGTG CTCAGAC ACCCAinvdTinvdTinvdT(SEQ ID NO:159),A GTG GT G C TC AG A C A CCC AinvdTinvdTinvdT (SEQ ID NO:160) or A G T G GTGC TCA G AC A C C C AinvdTinvdTinvdT (SEQ ID NO: 161), wherein the underlined nucleic acid represents a locked nucleic acid. In some embodiments, the blocking nucleic acid comprises the sequence of SEQ ID NO: 33 or 158-161, but optionally includes a different pattern or type of one or more modified nucleotides. In some embodiments, the blocking nucleic acid comprises the sequence of SEQ ID NO: 33 or 158-161, but includes a different 3' terminal portion.

[0107] In some embodiments, the blocking nucleic acid of the present disclosure hybridizes to a wild-type APC locus corresponding to one or more mutations of an APC protein encoding an R1450* mutation. In some embodiments, the blocking nucleic acid comprises the sequence CTTCTCGCTTGGTT(invdT) n, wherein n is 1, 2, or 3 (SEQ ID NO: 37); GTACTTCTCGCTTGGT(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 162); CTTCTCGCTTGGTT(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 163); GTACTTCTCGCTTGGT(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 164) or GTACTTCTCGCTTGGT(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 165), wherein italic nucleic acids represent locked nucleic acids. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, the blocking nucleic acid comprises the sequence CTTCTCGCTTGGTTinvdTinvdTinvdTinvdT (SEQ ID NO: 37), GTACTTCTCGCTTGGTinvdTinvdTinvdT (SEQ ID NO: 162), CTTCTCGCTTGGTTinvdTinvdTinvdT (SEQ ID NO: 163), GTACTTCTCGCTTGGTinvdTinvdTinvdT (SEQ ID NO: 164), or GTACTTCTCGCTTGGTinvdTinvdTinvdT (SEQ ID NO: 165), wherein the italicized nucleic acid represents a locked nucleic acid. In certain embodiments, the blocking nucleic acid comprises the sequence C TTC T CGCT T GGT TinvdTinvdTinvdT (SEQ ID NO: 37), G TAC T T C TCG CT TGG TinvdTinvdTinvdT (SEQ ID NO: 162), C TTC T CGCT T GGT TinvdTinvdTinvdT(SEQ ID NO:163),GT ACT T CTCG CT TGG TinvdTinvdTinvdT (SEQ ID NO: 164) or GT A C TTCTCGC TT GGTinvdTinvdTinvdT (SEQ ID NO: 165), wherein the underlined nucleic acid represents a locked nucleic acid. In some embodiments, the blocking nucleic acid comprises the sequence of SEQ ID NO: 37 or 162-165, but optionally includes a different pattern or type of one or more modified nucleotides. In some embodiments, the blocking nucleic acid comprises the sequence of SEQ ID NO: 37 or 162-165, but includes a different 3' terminal portion.

[0108] In some embodiments, the blocking nucleic acid of the present disclosure hybridizes to a wild-type APC locus corresponding to one or more mutant loci encoding an APC protein with an E1309 in-frame mutation. In some embodiments, the blocking nucleic acid comprises the sequence CTTTTCTTTTATTTCTGC(invdT) n , wherein n is 1, 2, or 3 (SEQ ID NO: 29); CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 154); CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 155); CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 156) or CTTTTCTTTTATTTCTGC(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 157), wherein italic nucleic acids represent locked nucleic acids. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, the blocking nucleic acid comprises the sequence CTTTTCTTTTATTTCTGCinvdTinvdTinvdT (SEQ ID NO: 29), CTTTTCTTTTATTTCTGCinvdTinvdTinvdT (SEQ ID NO: 154), CTTTTCTTTTATTTCTGCinvdTinvdTinvdT (SEQ ID NO: 155), CTTTTCTTTTATTTCTGCinvdTinvdTinvdT (SEQ ID NO: 156), or CTTTTCTTTTATTTCTGCinvdTinvdTinvdT (SEQ ID NO: 157), wherein the italicized nucleic acid represents a locked nucleic acid. In certain embodiments, the blocking nucleic acid comprises the sequence C TTTTCTTTTAT TT C T GC invdTinvdTinvdT (SEQ ID NO: 29), C TTTTC T TTTA T T TC TG C invdTinvdTinvdT (SEQ ID NO: 154), C T TT TC T T T TATTTCTGC invdTinvdTinvdT (SEQ ID NO: 155), C TTT TCT TTT A T T TC T GC invdTinvdTinvdT (SEQ ID NO: 156) or C T TT TCTTTTATTTC TG C invdTinvdTinvdT (SEQ ID NO: 157), wherein the underlined nucleic acid represents a locked nucleic acid. In some embodiments, the blocking nucleic acid comprises the sequence of SEQ ID NO: 29 or 154-157, but optionally includes a different pattern or type of one or more modified nucleotides. In some embodiments, the blocking nucleic acid comprises the sequence of SEQ ID NO: 29 or 154-157, but includes a different 3' terminal portion.

[0109] In some embodiments, the blocking nucleic acid of the present disclosure hybridizes to a wild-type APC locus corresponding to one or more mutant loci encoding an APC protein with an S1465 inframe mutation. In some embodiments, the blocking nucleic acid comprises the sequence CCACTCTCTCTCTTTTCAGC(invdT) n, wherein n is 1, 2, or 3 (SEQ ID NO: 25); TAGGTCCACTCTCTCTCTTTTCAGCA (invdT) n, wherein n is 1, 2, or 3 (SEQ ID NO: 166); TAGGTCCACTCTCTCTCTTTTCAGCA (invdT) n, wherein n is 1, 2, or 3 (SEQ ID NO: 167); CCACTCTCTCTCTTTTCAGC (invdT) n, wherein n is 1, 2, or 3 (SEQ ID NO: 168) or TAGGTCCACTCTCTCTCTTTTCAGCA (invdT) n, wherein n is 1, 2, or 3 (SEQ ID NO: 169), wherein italic nucleic acids represent locked nucleic acids. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, the blocking nucleic acid comprises the sequence CCACTCTCTCTCTTTTCAGCinvdTinvdTinvdTinvdT (SEQ ID NO: 25), TAGGTCCACTCTCTCTCTTTTCAGCAinvdTinvdTinvdT (SEQ ID NO: 166), TAGGTCCACTCTCTCTCTTTTCAGCAinvdTinvdTinvdT (SEQ ID NO: 167), CCACTCTCTCTCTTTTCAGC invdTinvdTinvdT (SEQ ID NO: 168), or TAGGTCCACTCTCTCTCTTTTCAGCA invdTinvdTinvdT (SEQ ID NO: 169), wherein the italicized nucleic acid represents a locked nucleic acid. In certain embodiments, the blocking nucleic acid comprises the sequence CCA C TC TCTCT C T TT T CAGC invdTinvdTinvdT(SEQ ID NO:25),TA GG T CC ACTCTCTCTCT TT T C A GC AinvdTinvdTinvdT (SEQ ID NO: 166), T AGG T CCAC T CTCT C T CTT T TC AG CAinvdTinvdTinvdT (SEQ ID NO: 167), C CA C T C TC T C TC TTTT C A G C invdTinvdTinvdT(SEQ ID NO:168) or TA G GT CC AC T CT C TCT C T T TT C A GC A invdTinvdTinvdT (SEQ ID NO: 169), wherein the underlined nucleic acid represents a locked nucleic acid. In some embodiments, the blocking nucleic acid comprises the sequence of SEQ ID NOs: 25 or 166-169, but optionally includes a different pattern or type of one or more modified nucleotides. In some embodiments, the blocking nucleic acid comprises the sequence of SEQ ID NOs: 25 or 166-169, but includes a different 3' terminal portion.

[0110] In some embodiments, the blocking nucleic acid of the present disclosure hybridizes to a wild-type APC locus corresponding to one or more mutant loci encoding an APC protein with a T1556 inframe mutation. In some embodiments, the blocking nucleic acid comprises the sequence CAATAGTTTTTTCTGCC(invdT) n, wherein n is 1, 2, or 3 (SEQ ID NO: 41); GAATCAATAGTTTTTTCTGCCTC(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 170); TCAGAATCAATAGTTTTTTCTG(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 171); GAATCAATAGATTTTACTGCCTC(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 172); or AATCAATAGTTTTTCTGCCTC(invdT)n, wherein n is 1, 2, or 3 (SEQ ID NO: 173), wherein italicized nucleic acids represent locked nucleic acids. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, the blocking nucleic acid comprises the sequence CAATAGTTTTTTCTGCCinvdTinvdTinvdTinvdT (SEQ ID NO: 41), GAATCAATAGTTTTTTCTGCCTCinvdTinvdTinvdT (SEQ ID NO: 170), TCAGAATCAATAGTTTTTTCTGinvdTinvdTinvdT (SEQ ID NO: 171), GAATCAATAGATTTTACTGCCTCinvdTinvdTinvdT (SEQ ID NO: 172), or AATCAATAGTTTTTCTGCCTCinvdTinvdTinvdT (SEQ ID NO: 173), wherein the italicized nucleic acid represents a locked nucleic acid. In certain embodiments, the blocking nucleic acid comprises the sequence C A ATAG T TTTT T CTGC CinvdTinvdTinvdT (SEQ ID NO: 41), G A A T CAATAG TTTTTT CTGCCT C invdTinvdTinvdT (SEQ ID NO: 170), T CAG A ATC A ATAG TTTTT TCTG invdTinvdTinvdT (SEQ ID NO: 171), G A A T CAATAG ATTTTA CTGCCT CinvdTinvdTinvdT (SEQ ID NO:172) or A A T CAATAGTTTTTTC TGCCT C invdTinvdTinvdT (SEQ ID NO: 173), wherein the underlined nucleic acid represents a locked nucleic acid. In some embodiments, the blocking nucleic acid comprises the sequence of SEQ ID NO: 41 or 170-173, but optionally includes a different pattern or type of one or more modified nucleotides. In some embodiments, the blocking nucleic acid comprises the sequence of SEQ ID NO: 41 or 170-173, but includes a different 3' terminal portion.

[0111] hybridization

[0112] In some embodiments, the methods of the present disclosure include hybridizing the amplified DNA with one or more probes that are specific for the DNA mutations of the present disclosure (e.g., DNA mutations in the KRAS, BRAF, CTNNB1, or APC genes as described above). In some embodiments, the method includes hybridizing the amplified DNA with at least four probes, including one or more probes that are specific for the DNA mutations in each of the KRAS, BRAF, CTNNB1, and APC genes (e.g., one or more probes representing mutations in each gene). As used herein, a probe may refer to an oligonucleotide that is capable of hybridizing to at least a portion of a locus of a target DNA mutation. For example, a probe may include a single-stranded oligonucleotide that is capable of base pairing with most or all base pairs of a single-stranded DNA template comprising a target DNA mutation. For specific detection of DNA mutations, the probe is capable of hybridizing to a locus carrying a DNA mutation, but not to a corresponding wild-type locus (see Figure 15 and 16 Suitable conditions for hybridization of probes to amplified DNA are known in the art (eg, as cited in the materials cited herein) and are exemplified below.

[0113] In some embodiments, the probe of the present disclosure is coupled to an encoded microcarrier of the present disclosure, for example, as described in Section IV. Exemplary methods for coupling polynucleotide probes to microcarrier surfaces are known in the art and are provided in Section IV. For multiple determinations, each type of probe can be coupled to a microcarrier with a specific identifier corresponding to the probe type. Advantageously, this allows the user to associate the signal detected from the probe with the identity of the probe, thereby enabling multiple determinations in which a variety of probes are used. In some embodiments, the probe of the present disclosure comprises a 5' modification, for example, a 5' amino modifier C6.

[0114] In some embodiments, the probe of the present disclosure comprises (1) a sequence that hybridizes to at least a portion of the locus of the target DNA mutation; and (2) one or more additional nucleotides. The one or more additional nucleotides can be used, for example, to couple the probe to a microcarrier surface and / or provide a spacer to reduce steric hindrance between the microcarrier surface and the amplified DNA during hybridization. In some embodiments, the one or more additional nucleotides are located at the 5' end of the probe sequence. In other embodiments, the one or more additional nucleotides are located at the 3' end of the probe sequence. In some embodiments, the one or more additional nucleotides are adenine or thymine nucleotides. Advantageously, this reduces the affinity of non-specific binding. In some embodiments, the probe of the present disclosure comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 or more adenine or thymine nucleotides at the 5' end. In some embodiments, the probe of the present disclosure comprises at least 20, at least 24, at least 25, or at least 30 total nucleotides.

[0115] Exemplary probe sequences are provided below. One of ordinary skill in the art can readily select a variety of probe sequences suitable for hybridizing to an amplified target DNA sequence (eg, a locus comprising a target DNA mutation) using well-known techniques.

[0116] In some embodiments, a probe specific for a DNA mutation in the KRAS gene comprises the sequence GGAGCTGATGG (SEQ ID NO:4), AGCTGATGGCGTA (SEQ ID NO: 178), TGGAGCTGATGGCG (SEQ ID NO: 179), TGGAGCTGATGG (SEQ ID NO: 180), GCTGATGGCGTA (SEQ ID NO: 181), GGAGCTGTTGG (SEQ ID NO: 5), TGGAGCTGTTGGTGGC (SEQ ID NO: 182), GGAGCTGTTGGTG (SEQ ID NO: 183), TGGAGCTGTTGGT (SEQ ID NO: 184), TGGAGCTGTAGGTGG (SEQ ID NO: 185), TGGAGCTAGTGG (SEQ ID NO: 6), TTGGAGCTAGTGGCGTA (SEQ ID NO: 186), GCTAGTGGCGTAGGC (SEQ ID NO: 187), AGCTAGTGGCGT (SEQ ID NO: 188), NO:188), GTTGGAGCTAGTGG (SEQ ID NO:189), GGAGCTAGTGG (SEQ ID NO:190) or TGGAGCTGGTGACGT (SEQ ID NO:7).For example, a probe comprising the following sequence can be used to detect a mutation in a KRAS protein encoding a G12D mutation: GGAGCTGATGG (SEQ ID NO: 4), AGCTGATGGCGTA (SEQ ID NO: 178), TGGAGCTGATGGCG (SEQ ID NO: 179), TGGAGCTGATGG (SEQ ID NO: 180), or GCTGATGGCGTA (SEQ ID NO: 181); a probe comprising the following sequence can be used to detect a mutation in a KRAS protein encoding a G12V mutation: GGAGCTGTTGG (SEQ ID NO: 5), TGGAGCTGTTGGTGGC (SEQ ID NO: 182), GGAGCTGTTGGTG (SEQ ID NO: 183), TGGAGCTGTTGGT (SEQ ID NO: 184), or TGGAGCTGTaGGTGG (SEQ ID NO: 185); a probe comprising the following sequence can be used to detect a mutation in a KRAS protein encoding a G12S mutation: TGGAGCTAGTGG (SEQ ID NO: 186). NO: 6), TTGGAGCTAGTGGCGTA (SEQ ID NO: 186), GCTAGTGGCGTAGGC (SEQ ID NO: 187), AGCTAGTGGCGT (SEQ ID NO: 188), GTTGGAGCTAGTGG (SEQ ID NO: 189), or GGAGCTAGTGG (SEQ ID NO: 190); and / or probes comprising the following sequences can be used to detect mutations in KRAS proteins encoding G13D mutations: TGGAGCTGGTGACGT (SEQ ID NO: 7), GGTGACGTAGGCAA (SEQ ID NO: 191), TGACGTAGGCAAGAG (SEQ ID NO: 192), GCTGGTGACGTAGG (SEQ ID NO: 193), AGCTGGTGACGTAG (SEQ ID NO: 194), or GGAGCTGGTGACGT (SEQ ID NO: 195). As described above, in some embodiments, one or more probes of the present disclosure may comprise 8 or more nucleotides (eg, adenine or thymine) at its 5' end.In certain embodiments, probes comprising the following sequences can be used to detect mutations in KRAS proteins encoding the G12D mutation: TTTTTTTTTTTTAAGGAGCTGATGG (SEQ ID NO:47), TTTTTTTTTTTTAGCTGATGGCGTA (SEQ ID NO:74), TTTTTTTTTTTATGGAGCTGATGGCG (SEQ ID NO:75), TTTTTTTTTTTTATGGAGCTGATGG (SEQ ID NO:76), or TTTTTTTTTTTTTGCTGATGGCGTA (SEQ ID NO:77). In certain embodiments, probes comprising the following sequences can be used to detect mutations in the KRAS protein encoding the G12V mutation: TTTTTTTTTTTTAAGGAGCTGTTGG (SEQ ID NO:48), TTTTTTTTATGGAGCTGTTGGTGGC (SEQ ID NO:78), TTTTTTTTTTTAAGGAGCTGTTGGTG (SEQ ID NO:79), TTTTTTTTTTTATGGAGCTGTTGGT (SEQ ID NO:80), or TTTTTTTTTATGGAGCTGTAGGTGG (SEQ ID NO:81). In certain embodiments, probes comprising the following sequences can be used to detect mutations in KRAS proteins encoding G12S mutations: TTTTTTTTTTTATGGAGCTAGTGG (SEQ ID NO:49), TTTTTTTTTTGGAGCTAGTGGCGTA (SEQ ID NO:82), TTTTTAATTTGCTAGTGGCGTAGGC (SEQ ID NO:83), TTTTTTTTTATTTAGCTAGTGGCGT (SEQ ID NO:84), TTTTTTTTTTTGTTGGAGCTAGTGG (SEQ ID NO:85), or TTTTTTTTTTTTTAAGGAGCTAGTGG (SEQ ID NO:86).In certain embodiments, probes comprising the following sequences can be used to detect mutations in KRAS proteins encoding G13D mutations: TTTTTTTTTATGGAGCTGGTGACGT (SEQ ID NO: 50), TTTTTTTTAAAGGTGACGTAGGCAA (SEQ ID NO: 87), TTTTTTTTTATGACGTAGGCAAGAG (SEQ ID NO: 88), TTTTTTTTTTTGCTGGTGACGTAGG (SEQ ID NO: 89), TTTTTTTTTTAAGCTGGTGACGTAG (SEQ ID NO: 90), or TTTTTTTTTAAGGAGCTGGTGACGT (SEQ ID NO: 91) (probes comprising these sequences that do not contain a 5' adenine and / or thymine are also contemplated).

[0117] In some embodiments, a probe specific for a DNA mutation in the BRAF gene comprises the sequence TCTAGCTACAGAGAAAT (SEQ ID NO: 11) or GTCTAGCTACAGAAAAAT (SEQ ID NO: 12). For example, probes comprising the following sequences can be used to detect mutations in BRAF proteins with V600E mutations (e.g., c.1799T>A DNA mutations): TCTAGCTACAGAGAAAT (SEQ ID NO: 11), TACAGAGAAATCTCGAT (SEQ ID NO: 196), TACAGAGAAATCTC (SEQ ID NO: 197), CTAGCTACAGAGAAAT (SEQ ID NO: 198), CTAGCTACAGAGAAA (SEQ ID NO: 199), or TCTAGCTACAGAG (SEQ ID NO: 200), and / or probes comprising the sequence GTCTAGCTACAGAAAAAT (SEQ ID NO: 12) can be used to detect mutations in BRAF proteins encoding V600E mutations (e.g., c.1799_1800TG>AA DNA mutations). As described above, in some embodiments, one or more probes of the present disclosure may comprise 8 or more nucleotides (e.g., adenine or thymine) at their 5' end and / or comprise at least 24 total nucleotides.In certain embodiments, a probe comprising the following sequence can be used to detect a mutation in a BRAF protein encoding a V600E mutation (e.g., a c.1799T>A DNA mutation): TTTTTTAATTTCTAGCTACAGAGAAAT (SEQ ID NO: 51), TTTTTTTTTATACAGAGAAATCTCGAT (SEQ ID NO: 92), TTTTTTTTTAATTTACAGAGAAATCTC (SEQ ID NO: 93), TTTTTTAATTACTAGCTACAGAGAAAT (SEQ ID NO: 94), TTTTTTTAATTACTAGCTACAGAGAAA (SEQ ID NO: 95), or TTTTTTTTTTAATTTCTAGCTACAGAG (SEQ ID NO: 96), and / or a probe comprising the following sequence can be used to detect a mutation in a BRAF protein encoding a V600E mutation (e.g., a c.1799_1800TG>AA DNA mutation): TTTTTTTATGTCTAGCTACAGAAAAAT (SEQ ID NO: 97). NO: 52), TTTTATGTCTAGCTACAGAAAAATC (SEQ ID NO: 97), TTTTTTTTATTTTTAGCTACAGAAAAA (SEQ ID NO: 98), TTTTTTTATTTCTAGCTACAGAAAAAT (SEQ ID NO: 99), or TTTTTTTTATTCTAGCTACAGAAAAATC (SEQ ID NO: 100) (probes comprising these sequences that do not contain a 5' adenine and / or thymidine are also contemplated).

[0118] In some embodiments, a probe specific for a DNA mutation in the CTNNB1 gene comprises the sequence AGGAGCTGTGGCAG (SEQ ID NO: 16), GGAGCTGTGATA (SEQ ID NO: 17), TTTACCACTCAGAAAAG (SEQ ID NO: 21), TACCACTCAGAGGAG (SEQ ID NO: 22), AGGAGCTGTGGCAGT (SEQ ID NO: 205), AGGAGCTGTGGCAGTG (SEQ ID NO: 206), GCTGTGGCAGTGGC (SEQ ID NO: 207), GCTGTGGCAGTGGCA (SEQ ID NO: 208), AAGGAGCTGTGGCAG (SEQ ID NO: 209), GGAGCTGTGATAGTGG (SEQ ID NO: 210), GAGCTGTGATAGTGGC (SEQ ID NO: 211), AGCTGTGATAGTGGCA (SEQ ID NO: 212), AGAAGGAGCTGTGATA (SEQ ID NO: 213), GGAGCTGTGAT (SEQ ID NO: 214). NO:214), ACTCAGAAAAGGAGCT (SEQ ID NO:215), TACCACTCAGAAAGGA (SEQ ID NO:216), TTTACCACTCAGAAAAGGAG (SEQ ID NO:217), TTACCACTCAGAAAG (SEQ ID NO:218), CAGAAAGGAGCTGTG (SEQ ID NO:219), ACTCAGAGGAGGAGC (SEQ ID NO:214) NO:220), TTACCACTCAGAGGA (SEQ ID NO:221), TTACCACTCAGAGGAGG (SEQ ID NO:222), TTAACACTCAGAGGAG (SEQ ID NO:223) or TTACCAATCAGAGGAGG (SEQ ID NO:224). For example, probes comprising the following sequences can be used to detect mutations in the CTNNB1 protein encoding the T41A mutation: AGGAGCTGTGGCAG (SEQ ID NO: 16), AGGAGCTGTGGCAGT (SEQ ID NO: 205), AGGAGCTGTGGCAGTG (SEQ ID NO: 206), GCTGTGGCAGTGGC (SEQ ID NO: 207), GCTGTGGCAGTGGCA (SEQ ID NO: 208), or AAGGAGCTGTGGCAG (SEQ ID NO: 210).(SEQ ID NO: 209); probes comprising the following sequences can be used to detect mutations in the CTNNB1 protein encoding the T41I mutation: GGAGCTGTGATA (SEQ ID NO: 17), GGAGCTGTGATAGTGG (SEQ ID NO: 210), GAGCTGTGATAGTGGC (SEQ ID NO: 211), AGCTGTGATAGTGGCA (SEQ ID NO: 212), AGAAGGAGCTGTGATA (SEQ ID NO: 213), or GGAGCTGTGAT (SEQ ID NO: 214); probes comprising the following sequences can be used to detect mutations in the CTNNB1 protein encoding the S45F mutation: TTTACCACTCAGAAAAG (SEQ ID NO: 21), ACTCAGAAAAGGAGCT (SEQ ID NO: 215), TACCACTCAGAAAAGGA (SEQ ID NO: 216), TTTACCACTCAGAAAAGGAG (SEQ ID NO: 217), TTACCACTCAGAAAAGGAG (SEQ ID NO: 218). NO: 218) or CAGAAAAGGAGCTGTG (SEQ ID NO: 219; and / or probes comprising the following sequences can be used to detect mutations in CTNNB1 proteins encoding S45P mutations: TACCACTCAGAGGAG (SEQ ID NO: 22), ACTCAGAGGAGGAGC (SEQ ID NO: 220), TTACCACTCAGAGGA (SEQ ID NO: 221), TTACCACTCAGAGGAGG (SEQ ID NO: 222), TTAACACTCAGAGGAG (SEQ ID NO: 223), or TTACCAATCAGAGGAGG (SEQ ID NO: 224). As described above, in some embodiments, one or more probes of the present disclosure can comprise 8 or more nucleotides (e.g., adenine or thymine) at their 5' end and / or comprise at least 24 total nucleotides. In certain embodiments, probes comprising the following sequence can be used to detect mutations in CTNNB1 proteins encoding T41A mutations: TTTTTTTTTTTTAGGAGCTGTGGCAG (SEQ ID NO: 225). NO:53), TTTTTTTTTTTAGGAGCTGTGGCAGTG (SEQ ID NO:101), TTTTTTTTTTTTAGCTGTGGCAGTGGC (SEQ ID NO:102), TTTTTTTTTTTTGCTGTGGCAGTGGCA (SEQ ID NO:103) or TTTTTTTTTTAAGGAGCTGTGGCAG (SEQ ID NO:103)(SEQ ID NO: 104); a probe comprising the following sequence can be used to detect a mutation in a CTNNB1 protein encoding a T41I mutation: TTTTTTTTTTTTTGGAGCTGTGATA (SEQ ID NO: 54), TTTTTTTTTGGAGCTGTGATAGTGG (SEQ ID NO: 105), TTTTTTTTTGAGCTGTGATAGTGGC (SEQ ID NO: 106), TTTTTTTTTAGCTGTGATAGTGGCA (SEQ ID NO: 107), TTTTTTTTTAGAAGGAGCTGTGATA (SEQ ID NO: 108), or TTTTTTTTTTTTTTGGAGCTGTGAT (SEQ ID NO: 109); a probe comprising the following sequence can be used to detect a mutation in a CTNNB1 protein encoding an S45F mutation: TTTTTTTTTTTTACCACTCAGAAAAG (SEQ ID NO: 55), TTTAATTTTACTCAGAAAAGGAGCT (SEQ ID NO: 110), TTTTTTAATACCACTCAGAAAAGGA (SEQ ID NO: 111). NO: 111), TTTTTTTTACCACTCAGAAAAGGAG (SEQ ID NO: 112), TTTTTTTTATTACCACTCAGAAAAG (SEQ ID NO: 113), or TTTTTTTTTCAGAAAAGGAGCTGTG (SEQ ID NO: 114); and / or probes comprising the following sequences can be used to detect mutations in the CTNNB1 protein encoding the S45P mutation: TTTTTTTTTAATACCACTCAGAGGAG (SEQ ID NO: 56), TTTTTTTTTAAAACTCAGAGGAGGAGC (SEQ ID NO: 115), TTTTTTTTTTTATTACCACTCAGAGGA (SEQ ID NO: 116), TTTTTTTTTTTATTACCACTCAGAGGAG (SEQ ID NO: 117), TTTTTTTTTTATTAACACTCAGAGGAG (SEQ ID NO: 118), or TTTTTTTTTATTACCAATCAGAGGAG (SEQ ID NO: 119). NO: 119) (Probes comprising these sequences that do not contain a 5' adenine and / or thymine are also contemplated).

[0119] In some embodiments, a probe specific for a DNA mutation in the APC gene comprises the sequence ACTGCTGAAAAGAGAGAGT (SEQ ID NO:26), GAAATAAAAGATTGG (SEQ ID NO:30), TTTTGGGTGTCTAAG (SEQ ID NO:34), CAAACCAAGTGAGAA (SEQ ID NO:38), AGAGGCAGAAAAAAACT (SEQ ID NO:42), AAATAGCAGAAATAAAAG (SEQ ID NO:225), GAAATAAAAGATTGGAA (SEQ ID NO:226), AGAAATAAAAGATTG (SEQ ID NO:227), GAAATAAATGAATGG (SEQ ID NO:228), CAGAAATAAAAGATT (SEQ ID NO:229), TTTGGGTGTCTAAG (SEQ ID NO:230), GGGTGTCTAAGCACCACT (SEQ ID NO:231), CTAAGCACCACTTTT (SEQ ID NO:232), TTTTGGGTGTCTAA (SEQ ID NO:233). NO:233), GGTGTCTAAGCACCA (SEQ ID NO:234), AAGTGAGAAGTACCTAA (SEQ ID NO:235), TCAAACCAGTGAG (SEQ ID NO:236), ACCAAGTGAGAAGTA (SEQ ID NO:237), AGCTCAAACCAAGTGAG (SEQ ID NO:238), GCACCTACTGCTGAA (SEQ ID NO:239), ACCTACTGCTGAAAAG (SEQ ID NO:240), TGCTGAAAAGAGAGAGT (SEQ ID NO:241), CCTACTGCTGAAAAGAGA (SEQ ID NO:242), GCAGAAAAAAACTATTG (SEQ ID NO:243), CAGAAAAAAACTATTGATT (SEQ ID NO:244), AGAAAGAGGCAGAAAAAAACT (SEQ ID NO:243) NO:245) or GAGGCAGAAAAAAACTA (SEQ ID NO:246). For example, probes comprising the following sequences can be used to detect mutations in the APC protein encoding the S1465 out-of-frame mutation: ACTGCTGAAAAGAGAGAGT (SEQ ID NO: 26), GCACCTACTGCTGAA (SEQ IDNO: 239), ACCTACTGCTGAAAAG (SEQ ID NO: 240), TGCTGAAAAGAGAGAGT (SEQ ID NO: 241), or CCTACTGCTGAAAAGAGA (SEQ ID NO: 242); probes comprising the following sequences can be used to detect mutations in APC proteins encoding E1309 frame-defective mutations: GAAATAAAAGATTGG (SEQ ID NO: 30), AAATAGCAGAAATAAAAG (SEQ ID NO: 225), GAAATAAAAGATTGGAA (SEQ ID NO: 226), AGAAATAAAAGATTG (SEQ ID NO: 227), GAAATAAATGAATGG (SEQ ID NO: 228), or CAGAAATAAAAGATT (SEQ ID NO: 229); probes comprising the following sequences can be used to detect mutations in APC proteins encoding Q1367* mutations: TTTTGGGTGTCTAAG (SEQ ID NO: 34), TTTGGGTGTCTAAG (SEQ ID NO: 35). NO: 230), GGGTGTCTAAGCACCACT (SEQ ID NO: 231), CTAAGCACCACTTTT (SEQ ID NO: 232), TTTTGGGTGTCTAA (SEQ ID NO: 233), or GGTGTCTAAGCACCA (SEQ ID NO: 234); probes comprising the following sequences can be used to detect mutations in APC proteins encoding R1450* mutations: CAAACCAAGTGAGAA (SEQ ID NO: 38), AAGTGAGAAGTACCTAA (SEQ ID NO: 235), TCAAACCAAGTGAG (SEQ ID NO: 236), ACCAAGTGAGAAGTA (SEQ ID NO: 237), or AGCTCAAACCAAGTGAG (SEQ ID NO: 238); and / or probes comprising the following sequences can be used to detect mutations in APC proteins encoding T1556 inframe mutations: GCAGAAAAAAACTATTG (SEQ ID NO: 243), AGAGGCAGAAAAAAACT (SEQ ID NO: 244). NO:42), CAGAAAAAAACTATTGATT (SEQ ID NO:244), AGAAAGAGGCAGAAAAAAACT (SEQ ID NO:245) and GAGGCAGAAAAAAACTA (SEQ IDNO: 246). As described above, in some embodiments, one or more probes of the present disclosure may comprise 8 or more nucleotides (eg, adenine or thymine) at their 5' end and / or comprise at least 24 total nucleotides. In certain embodiments, a probe comprising the following sequence can be used to detect a mutation in an APC protein encoding an S1465 out-of-frame mutation: TTTTTTTTACTGCTGAAAAGAGAGAGT (SEQ ID NO: 57), TTTTTTTTTTGCACCTACTGCTGAA (SEQ ID NO: 134), TTTTTTTTTTACCCTACTGCTGAAAAG (SEQ ID NO: 135), TTTTTTTTTGCTGAAAAGAGAGAGT (SEQ ID NO: 136), or TTTTTTTTTCCTACTGCTGAAAAGAGA (SEQ ID NO: 137); a probe comprising the following sequence can be used to detect a mutation in an APC protein encoding an E1309 out-of-frame mutation: TTTTTTTTTTTTTGAAATAAAAGATTGG (SEQ ID NO: 58), TTTTTTTTTTTTAAATAGCAGAAATAAAAG (SEQ ID NO: 120), TTTTTTTTTTTGAAATAAAAGATTGGAA (SEQ ID NO: 121), TTTTTTTTTTTTTTTGAAATAAAAGATTG (SEQ ID NO: 122). NO: 129); a probe comprising the following sequence can be used to detect a mutation in an APC protein encoding a Q1367* mutation: TTTTTTTTTTTTTGGGTGTCTAAG (SEQ ID NO: 59), TTTTTTTTTATTTGGGTGTCTAAG (SEQ ID NO: 125), TTTTTTGGGTGTCTAAGCACCACT (SEQ ID NO: 126), TTTTTTTTTCTAAGCACCACTTTT (SEQ ID NO: 127), TTTTTTTTTTTTTTGGGTGTCTAA (SEQ ID NO: 128), or TTTTTTTTTGGTGTCTAAGCACCA (SEQ ID NO: 129); a probe comprising the following sequence can be used to detect a mutation in an APC protein encoding a R1450* mutation: TTTTTTTTTTTCAAACCAAGTGAGAA (SEQ ID NO: 51).NO: 60), TTTTTTTTAAGTGAGAAGTACCTAA (SEQ ID NO: 130), TTTTTTTTTTTTCAAACCAAGTGAG (SEQ ID NO: 131), TTTTTTTTTTACCAAGTGAGAAGTA (SEQ ID NO: 132), or TTTTTTTTTAGCTCAAACCAAGTGAG (SEQ ID NO: 133); and / or a probe comprising the following sequence: TTTTTTTTTTAGAGGCAGAAAAAAACT (SEQ ID NO: 61), TTTTTTTTTTGCAGAAAAAAACTATTG (SEQ ID NO: 138), TTTTTTTTTTTCAGAAAAAAACTATTGATT (SEQ ID NO: 139), TTTTTTTTAGAAAGAGGCAGAAAAAAACT (SEQ ID NO: 140), or TTTTTTTTTTTGAGGCAGAAAAAAACTA (SEQ ID NO: 141). NO: 141) (Probes comprising these sequences that do not contain a 5' adenine and / or thymine are also contemplated).

[0120] Detection

[0121] In some embodiments, the methods of the present disclosure include detecting whether the amplified DNA hybridizes with the probes of the present disclosure. Hybridization between the amplified DNA and one of the probes indicates the presence of a DNA mutation corresponding to the probe in the amplified DNA. Exemplary hybridization conditions and detection techniques are described and illustrated herein. In some embodiments, hybridization is performed using 5X SSPE buffer.

[0122] In some embodiments, the DNA amplified by detection reagent labeling is hybridized by, for example, measuring the signal of the detection reagent associated with the microcarrier after a washing step (to reduce or eliminate non-specific binding). In some embodiments, the primer pair of the present disclosure comprises one or two primers coupled with the detection reagent. Therefore, the DNA amplified by detection reagent labeling is amplified after the primer PCR amplification using one or more labels. In some embodiments, the detection reagent can be based on fluorescence, including but not limited to phycoerythrin (PE), blue fluorescent protein, green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein and derivatives thereof.

[0123] In other embodiments, the detection reagent may be based on a radioisotope, including but not limited to 32 P. 33 P. 22 Na, 36 Cl,2 H. 3 H. 35 S and 123 In other embodiments, the detection reagent is light-based, including but not limited to luciferase (e.g., chemiluminescence-based), horseradish peroxidase, alkaline phosphatase, and derivatives thereof. In some embodiments, the amplified DNA can be labeled with a detection reagent prior to contact with the microcarrier composition. In some embodiments, the detection reagent emits a signal when in proximity to the probe, e.g., with Various nucleic acid labeling techniques are known in the art; see, for example, Gibriel, AAY (2012) Briefings in Functional Genomics 11:311-8.

[0124] In some embodiments, detection reagent can be a fluorescence detection reagent. In some embodiments, detection by fluorescence microscopy (e.g., fluorescence microscope or plate reader) detects whether the amplified DNA and probe are hybridized. In some embodiments, detection reagent can be based on colorimetry. In some embodiments, detection reagent can be based on luminescence. In some embodiments, detection by luminescence microscopy (e.g., luminescence microscope or plate reader) detects whether the amplified DNA and probe are hybridized.

[0125] In some embodiments, the detection reagent comprises a label or other portion that can be detected by adding a second reagent that is conjugated to a signal emitting entity (e.g., as described above). For example, in some embodiments, the detection reagent comprises biotin (e.g., a primer such as a reverse or antisense primer can be labeled with biotin at the 5' end). Thus, in some embodiments, detecting the presence or absence of hybridization can include, after hybridization and optional washing, contacting one or more microcarriers with a second reagent that is conjugated to a signal emitting entity, and detecting a signal from the signal emitting entity associated with the one or more microcarriers (e.g., after optional washing). For example, if the detection reagent comprises biotin, the microcarrier can be contacted with streptavidin that is conjugated to a signal emitting entity (such as phycoerythrin (PE)), and the signal from the signal emitting entity can be detected.

[0126] Depending on the specific detection reagent, a variety of techniques can be used to detect hybridization of the amplified DNA to the probe. For example, if the detection reagent comprises a fluorescent detection reagent, detecting the presence or absence of hybridization of the amplified DNA can include imaging the fluorescence of the fluorescent detection reagent.

[0127] In some embodiments, the assay may include one or more washing steps, e.g., to reduce contaminants, remove any substances that non-specifically bind to the probe, DNA, and / or microcarrier surface, etc. In some embodiments, a magnetic separation step may be used to wash microcarriers containing a magnetic layer or material of the present disclosure. In other embodiments, other separation steps known in the art may be used.

[0128] In some embodiments, the methods of the present disclosure comprise detecting the presence of hybridization of the amplified DNA to a total of about 1 to about 1000 microcarriers / probes / assays. In some embodiments, the methods of the present disclosure comprise detecting the presence of hybridization of the amplified DNA to a total of about 1 to about 1000 microcarriers / probes / wells of an assay plate. In some embodiments, the methods of the present disclosure comprise detecting the presence of hybridization of the amplified DNA to at least about 50 microcarriers / probes / assays. In some embodiments, the methods of the present disclosure comprise detecting the presence of hybridization of the amplified DNA to at least about 50 microcarriers / probes / assays. In some embodiments, the microcarriers of the present disclosure comprise probes coupled thereto at a concentration of 1 μM.

[0129] In some embodiments, the method of the present disclosure includes detecting an identifier of an encoded microcarrier. For example, in some embodiments, an image of the identifier of the encoded microcarrier can be obtained and decoded to identify the microcarrier and its corresponding probe. In some embodiments, one or more identifier detection steps can occur after one or more hybridization detection steps. In other embodiments, one or more identifier detection steps can occur before one or more hybridization detection steps. In other embodiments. One or more identifier detection steps can occur simultaneously with one or more hybridization detection steps. In some embodiments, after one or more hybridization detection steps and one or more identifier detection steps, the method of the present disclosure further includes associating the detected identifier of the microcarrier with the presence or absence of hybridization of the detected amplified DNA with the corresponding probe of the microcarrier.

[0130] Various microcarrier encoding schemes are described in Section IV. In some embodiments, detecting the identifier of the coded microcarrier includes imaging the digital barcode of the microcarrier. In one embodiment, the coded microcarrier comprises a body having a series of alternating light-transmitting and opaque parts, a barcode image with relative width (e.g., a series of narrow slits representing "0" codes and wide slits representing "1" codes, or vice versa). When the microcarrier is irradiated with a light beam, the digital barcode 0 or 1 can be determined by a line scan camera, a frame grabber, and a digital signal processor based on the "total intensity" of the transmission peak or the "bandwidth" of the transmission peak from the slit. In one embodiment, a barcode pattern with a series of narrowband and broadband provides clear signals and distinctions for 0 and 1. The position of the slit on the pallet will determine which bit is the least significant bit (LSB) and the most significant bit (MSB). The LSB is placed closer to the edge of the pallet to distinguish it from the MSB at the other longer end.

[0131] In some embodiments, detecting the identifier encoding the microcarrier comprises imaging the identifier of the microcarrier, for example, by bright field imaging of the identifier.

[0132] Various decoding techniques are envisioned. In some embodiments, the analog shape recognition of an identification identifier (e.g., an analog coded identifier) ​​is used to detect the identifier. Conceptually, the decoding may involve, for example, imaging the analog code of each microcarrier (e.g., in a solution or sample), comparing each image with the analog code library, and matching each image with the image from the library, thereby positively identifying the code. Optionally, as described herein, when using a microcarrier comprising an orientation indicator (e.g., asymmetry), decoding may also include rotating each image to align with a specific orientation step (e.g., based in part on an orientation indicator). For example, if the orientation indicator comprises a gap, the image may be rotated until the gap arrives at a predetermined position or orientation (e.g., 0 ° position of image).

[0133] Various shape recognition software, tools and methods are known in the art. Examples of such APIs and tools include, but are not limited to Research FaceSDK, OpenBR, face and scene recognition from ReKognition, Betaface API and various ImageJ plug-ins. In some embodiments, simulated shape recognition can include but is not limited to image processing steps such as foreground extraction, shape detection, thresholding (e.g., automatic or manual image thresholding), etc.

[0134] It will be appreciated by those skilled in the art that the methods and microcarriers described herein are applicable to various imaging devices, including but not limited to microscopes, plate readers, etc. In some embodiments, decoding identifiers (e.g., analog codes) may include passing light through substantially transparent parts (e.g., one or more substantially transparent polymer layers) and / or surrounding solution of the microcarrier. Light may then not pass through or with lower intensity or other perceptible differences pass through substantially opaque parts (e.g., one or more substantially opaque polymer layers) of the microcarrier to generate a simulation coded light pattern corresponding to the identifier. In other words, the pattern of imaging light may correspond to the pattern of the substantially transparent / substantially opaque region of the microcarrier, thereby producing an image of the analog code identifier. The imaging may include steps, including but not limited to capturing an image, performing threshold processing on the image, and expecting any other image processing steps in order to realize more accurate, precise, or robust imaging of the identifier.

[0135] Any type of optical microscope can be used for the methods of the present disclosure, including but not limited to one or more of the following: bright field microscopy, dark field microscopy, phase contrast microscopy, differential interference contrast (DIC) microscopy, Nomarski interference contrast (NIC) microscopy, Nomarski, Hoffman modulation contrast (HMC) microscopy, or fluorescence microscopy. In certain embodiments, the identifier can be decoded using bright field microscopy, and hybridization can be detected using fluorescence microscopy.

[0136] In some embodiments, decoding the identifier may further include matching the image of the simulated code to the simulated code using simulated shape recognition. In some embodiments, the image may be matched to the simulated code (e.g., image files from a library of image files, each of which corresponds to a unique two-dimensional shape / simulated code) within a predetermined threshold, such as to tolerate a predetermined amount of deviation or mismatch between the image and an exemplary simulated code image. Such a threshold may be determined empirically and may naturally be based on the particular type of two-dimensional shape used for the simulated code and the degree of variation between the set of potential two-dimensional shapes.

[0137] In some embodiments, the method of the present disclosure also includes using a microcarrier with an identifier corresponding to a positive or negative control. In some embodiments, the method of the present disclosure includes using a primer pair amplifying a positive control DNA sequence that is specific to the positive control DNA sequence. The positive control DNA sequence can be any sequence that may be present in all samples of a given type, such as a non-mutated or endogenous gene sequence from the organism from which the sample is obtained. The positive control indicates that DNA (e.g., human DNA) is present in the sample at a level sufficient to detect. Like the DNA sequence of the target mutation, the positive control DNA sequence is detected by using a primer pair amplifying a positive control DNA sequence that is specific to the positive control DNA sequence; the amplified positive control gene sequence is hybridized with a probe that is specific to the positive control gene sequence (the probe that is specific to the positive control gene sequence is coupled to a microcarrier with an identifier corresponding to the positive control); whether the amplified positive control DNA sequence is hybridized with a probe that is specific to the positive control gene sequence; and the simulation code of the microcarrier with an identifier corresponding to the positive control is detected.

[0138] In some embodiments, as exemplified below, the positive control DNA sequence comprises the sequence of a human leukocyte antigen (HLA) gene. The sequences of over 150 HLA genes are known, including but not limited to HLA-DRA (see NCBI Entrez Gene ID No. 3122), HLA-DRB1 (see NCBI Entrez Gene ID No. 3123), HLA-A (see NCBI Entrez Gene ID No. 3105), HLA-B (see NCBI Entrez Gene ID No. 3106), HLA-C (see NCBI Entrez Gene ID No. 3107), HLA-DQB1 (see NCBI Entrez Gene ID No. 3119), HLA-DPB1 (see NCBI Entrez Gene ID No. 3115), HLA-E (see NCBI Entrez Gene ID No. 3133), HLA-DQA2 (see NCBI Entrez Gene ID No. 3118), HLA-DPAl (see NCBI Entrez Gene ID No. 3113), HLA-G (see NCBI Entrez Gene ID No. 3114), HLA-H (see NCBI Entrez Gene ID No. 3117), HLA-H (see NCBI Entrez Gene ID No. 3118), HLA-H (see NCBI Entrez Gene ID No. 3119), HLA-H (see NCBI Entrez Gene ID No. 3120), HLA-H HLA-DRB5 (see NCBI Entrez Gene ID No. 3127), HLA-F (see NCBI Entrez Gene ID No. 3134), and HLA-DOA (see NCBI Entrez Gene ID No. 3111). For descriptions of other HLA genes, see Shiina, T. et al. (2009) J. Hum. Genet. 54: 15-39.

[0139] In some embodiments, the primer pair specific for the positive control DNA sequence comprises the sequence TGAGTGTTACTTCTTCCCACACTC (SEQ ID NO: 43) and ATTGCTTTTGCGCAATCCCT (SEQ ID NO: 44). In some embodiments, the probe pair specific for the positive control DNA sequence comprises the sequence TTTTTTTTTTTTGGAGACGGTCTG (SEQ ID NO: 45). In some embodiments, the primer pair specific for the positive control DNA sequence comprises the sequence AATCCCATCACCATCTTCCA (SEQ ID NO: 71) and TGGACTCCACGACGTACTCA (SEQ ID NO: 72). In some embodiments, the probe pair specific for the positive control gene sequence comprises the sequence CTGTCTTCCACTCACTCC (SEQ ID NO: 73).

[0140] In some embodiments, the methods of the present disclosure include detecting the absence of hybridization of the amplified DNA to a microcarrier having an identifier corresponding to a negative control. For example, the microcarrier having an identifier corresponding to the negative control can contain a probe that does not hybridize to the amplified DNA. The use of a microcarrier having an identifier corresponding to a negative control improves the reliability of detecting the presence or absence of hybridization. In some embodiments, the microcarrier contains a probe containing the sequence AATATAATATATTAT (SEQ ID NO: 46).

[0141] Provided below are exemplary and non-limiting descriptions of microcarriers suitable for use in the methods of the present disclosure and optional aspects thereof.

[0142] IV. Encoding Microcarriers

[0143] Provided herein are coded microcarriers suitable for analyte detection, such as multiple analyte detection. This paper contemplates, describes, and illustrates a variety of configurations of coded microcarriers. As used herein, a "coded" microcarrier can refer to a microcarrier having an identifier corresponding to the identity of the probe to which it is coupled. This enables the data of an assay using the microcarrier to be associated with the identity of the probe, allowing the use of multiple microcarriers in a single multiple assay, as the result of any single microcarrier can be associated with the identity of its probe. Exemplary types of identifiers are described below, including digital barcodes and analog codes.

[0144] In some aspects, the methods and kits of the present disclosure utilize digital barcode identifiers. For example, in some embodiments, the encoded microcarrier comprises: a first photopolymer layer; a second photopolymer layer; and an intermediate layer between the first and second layers. In some embodiments, the intermediate layer comprises a coding pattern representing an identifier defined thereon, wherein the intermediate layer is partially substantially light-transmissive and partially substantially light-impermeable, representing a code corresponding to the microcarrier, wherein the outermost surface of the microcarrier comprises a photoresist photopolymer, and the photoresist photopolymer is functionalized with a probe specific for a DNA mutation, and wherein the microcarrier has approximately the same density as water. Exemplary microcarrier descriptions can be found in, for example, U.S. Patent Nos. 7,858,307, 7,871,770, 8,148,139, 8,232,092, and 9,255,922, and US PG Publication Nos. US 2009 / 201504, 2011 / 0007955, and 2012 / 0088691.

[0145] In one embodiment, the digitally encoded microcarriers of the present disclosure comprise a body having a series of alternating light-transmissive and opaque portions with relative positions, widths, and / or spacings similar to a 1D or 2D barcode image (e.g., a series of narrow slits (e.g., having a width of about 1 to 5 microns) representing a "0" code and wide slits (e.g., having a width of about 1 to 10 microns) representing a "1" code forming a binary code, or vice versa). In one embodiment, the dimensions of the microcarrier are designed and configured to be 150×50×10 μm or smaller, and the slit width is about 2.5 μm. Each digital barcode on such a microcarrier can consist of up to 14 slits (or bits), allowing for 16,384 unique codes. In one embodiment, the body of the encoded microcarrier can be configured to have at least two orthogonal cross-sections of different relative geometries and / or sizes. In addition, the cross-sectional geometry can be symmetrical or asymmetrical and / or regular or irregular in shape. In one embodiment, the longest orthogonal axis of the encoded microcarrier is less than 1 mm. In one embodiment, the encoded microcarrier is provided with a reflective film (e.g., electroplated or coated with a metal film or provided with an intermediate layer of a metal film) to improve the contrast and optical efficiency of image recognition for decoding. An alternative embodiment can include the metal layer as a layer sandwiched between two polymer layers by appropriately modifying the above process. Using this embodiment, the surface conditions of the two exposed planar surfaces of the microcarrier can be made identical to provide similar surface coating and immobilization conditions. Another embodiment is to coat the microcarrier with a polymer or functional molecule (such as the probes disclosed herein); thus, the entire microcarrier has the same molecular immobilization conditions.

[0146] In some aspects, the methods and kits of the present disclosure use an analog code identifier. For example, in some embodiments, the methods and kits of the present disclosure use an encoded microcarrier comprising: a substantially transparent polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other; a substantially opaque polymer layer, wherein the substantially opaque polymer layer is fixed to the first surface of the substantially transparent polymer layer and surrounds a central portion of the substantially transparent polymer layer, and wherein the substantially opaque polymer layer comprises a two-dimensional shape representing the analog code; and a probe of the present disclosure that is specific for a DNA mutation, wherein the probe is coupled to at least one of the first surface and the second surface of the substantially transparent polymer layer in at least the central portion of the substantially transparent polymer layer. The analog code represents an identifier. Thus, the microcarrier comprises at least two layers (representing the two-dimensional shape of the analog code identifier): one of which is substantially transparent and the other of which is substantially opaque.

[0147] Advantageously, these microcarriers can adopt a variety of two-dimensional shapes while still maintaining a uniform overall form (e.g., a perimeter of a substantially transparent polymer layer) to achieve uniformity in various aspects, including, for example, overall size, physical properties, and / or behavior in solution. This is advantageous, for example, in allowing greater uniformity between different types of microcarriers (i.e., each of the microcarriers has the same perimeter shape provided by the transparent polymer layer). Examples of this type of microcarrier and aspects thereof are shown in Figures 1A-5B (see, for example, Figure 4B ).

[0148] Figures 1A and 1B show two views of an exemplary microcarrier 100. Microcarrier 100 is a circular disc having a diameter of approximately 50 μm and a thickness of 10 μm. Figure 1A provides a view of microcarrier 100 viewing the circular face of the disc, while Figure 1B shows a side view of microcarrier 100 perpendicular to the surface shown in Figure 1A. Two components of microcarrier 100 are shown. First, a substantially transparent polymer layer 102 provides the bulk of the microcarrier. As described above, layer 102 can be produced, for example, using a polymer such as SU-8.

[0149] Substantially opaque polymer layer 104 is fixed to the surface of layer 102. Although the cross-section of the microcarrier 100 shown in Figure 1B shows the discontinuous view of layer 104, the view shown in Figure 1A shows that the shape of layer 104 is similar to the circular gear with a plurality of teeth. The shape, quantity, size and interval of these gear teeth constitute a two-dimensional shape, and one or more aspects in these aspects of gear teeth can be modified, so that produce a plurality of two-dimensional shapes for simulation coding. Advantageously, the outer edge of the gear teeth of layer 104 is adapted in the periphery of layer 102. This allows various simulation codes, and each simulation code represents the unique identifier of the microcarrier of a kind, keeps uniform overall shape between the microcarriers of a plurality of kinds simultaneously. In other words, each microcarrier kind in a plurality of kinds colonies can have different two-dimensional gear shapes (that is, simulation codes), but each microcarrier will have identical periphery, causes the larger uniformity of physical property (for example, behavior in size, shape, solution etc.). As mentioned above, layer 104 can for example use the polymer such as SU-8 mixed with dye, or uses black matrix resist to produce.

[0150] Layer 104 surrounds a central portion 106 of layer 102. Probes are coupled to at least central portion 106 on one or both surfaces (i.e., upper / lower surfaces) of layer 102. Advantageously, this allows central portion 106 to be imaged without the possibility of interference caused by layer 104.

[0151] Fig. 1 C and 1D show the exemplary mensuration that uses microcarrier 100 to carry out analyte detection.Fig. 1 C shows that microcarrier 100 can be included in the probe 108 that is coupled to one or more surfaces in at least central portion 106.Microcarrier 100 is contacted with the solution containing the DNA 110 of amplification, and the DNA 110 of described amplification has been denatured before contacting microcarrier 100, and hybridized with probe 108.As mentioned above, the DNA 110 of amplification is coupled to detection reagent.In the present example, detection reagent is biotin (for example, by using the primer amplification DNA of biotin labeling to produce).Therefore, the DNA 110 of amplification comprises DNA 110a (for example, comprising the locus of mutation as herein described) and biotin 110b.Fig. 1 C shows single microcarrier kind (that is, microcarrier 100), and it captures the DNA 110 of amplification, but uses multiple microcarrier kind in multiple determination, and each kind has the specific probe of identifying specific DNA mutation.

[0152] Figure 1 D shows the exemplary process for " reading " microcarrier 100.This process comprises two steps that can be completed simultaneously or separately.First, the hybridization of probe 108 and the DNA 110 of amplification is detected.In the example shown in Figure 1 D, the second detection reagent 114 (for example, being conjugated to the streptavidin of PE) is combined with the DNA 110 of amplification by biotin: streptavidin interaction.The DNA of amplification that is not coupled to the probe hybridization of microcarrier 100 can be washed away before detection so that only the DNA that is bound to microcarrier 100 is detected.The PE part of the second detection reagent 114, when excited by light 116 under the wavelength in the excitation spectrum of PE, emits light 118 (such as, photon).Light 118 can be detected by any suitable detection means such as fluorescence microscope, plate reader etc.

[0153] In addition, the unique identifier of the microcarrier 100 is read. In the example shown in Figure 1D, light 112 is used to illuminate the visual field containing the microcarrier 100 (in some embodiments, light 112 can have a wavelength different from that of light 116 and 118). When light 112 illuminates the visual field containing the microcarrier 100, it passes through the substantially transparent polymer layer 102, but is blocked by the substantially opaque polymer layer 104, as shown in Figure 1D. This generates a light pattern that can be imaged, for example, by an optical microscope (for example, using differential interference contrast or DIC microscope). The light pattern is based on the two-dimensional shape of the microcarrier 100 (that is, the analog code). Standard image recognition technology can be used to decode the analog code represented by the image of the microcarrier 100.

[0154] As described in more detail below, analyte detection and identifier imaging steps can occur in any order or simultaneously. Advantageously, the two detection steps shown in Figure 1 D can be completed on an imaging device. As an example, a microscope that can simultaneously carry out fluorescence and light (such as, bright field) microscopy can be used to quantitatively bind to the amount (for example, as detected by detection reagent 114) of the amplified DNA 110 of microcarrier 100 and to image the analog code produced by layers 102 and 104. This allows a more efficient determination process while requiring less equipment.

[0155] In some embodiments, the microcarrier further comprises a substantially opaque magnetic layer affixed to a surface of the substantially transparent polymer layer, the magnetic layer surrounding a central portion of the substantially transparent polymer layer. In some embodiments, the substantially opaque magnetic layer is located between the substantially opaque polymer layer and the central portion of the substantially transparent polymer layer.

[0156] In some embodiments, the microcarrier further comprises a second substantially transparent polymer layer aligned with and fixed to the first substantially transparent polymer layer. In some embodiments, the first substantially transparent polymer layer and the second substantially transparent polymer layer each have a central portion, and the central portions of the first substantially transparent polymer layer and the second substantially transparent polymer layer are aligned. In some embodiments, the microcarrier further comprises a substantially opaque magnetic layer that surrounds the central portions of the first substantially transparent polymer layer and the second substantially transparent polymer layer. In some embodiments, the substantially opaque magnetic layer is fixed between the first substantially transparent polymer layer and the second substantially transparent polymer layer. In some embodiments, the substantially opaque magnetic layer is located between the substantially opaque polymer layer and the central portions of the first substantially transparent polymer layer and the second substantially transparent polymer layer.

[0157] Turning now to Figures 2A and 2B, another exemplary microcarrier 200 is shown. Like microcarrier 100, microcarrier 200 comprises a substantially transparent polymer layer 202 and a substantially opaque polymer layer 204. In addition, microcarrier 200 comprises a magnetic layer 206. As shown in Figure 2A, magnetic layer 206 can be shaped as a ring between a central portion 208 and substantially opaque layer 204.

[0158] FIG2B shows that magnetic layer 206 can be embedded within layer 202. Layer 202 can also include more than one layer, such that magnetic layer 206 is sandwiched between two substantially transparent polymer layers (e.g., as in FIG2B ). Alternatively, magnetic layer 206 can be secured to the same surface of layer 202 as layer 204, or magnetic layer 206 can be secured to the surface of layer 202 opposite layer 204. In some embodiments, magnetic layer 206 can include nickel.

[0159] The magnetic layer 206 imparts magnetic properties to the microcarriers 200, which can be advantageously used in many applications. For example, the microcarriers 200 can be fixed to a surface by magnetic attraction during a washing step, thereby allowing efficient washing without losing or otherwise damaging the microcarriers.

[0160] Except its magnetic property, layer 206 is also substantially opaque.When imaging (for example, using light 112) as shown in Figure 1D, layer 206 will partially or entirely block transmitted light, thereby produces the pattern for imaging.As shown in Figure 2A, layer 206 is also asymmetric-in this example, it comprises gap 210.This asymmetry has produced the directional indicator that can use light 112 imaging, for example, as shown in Figure 1D.Advantageously, can utilize directional indicator to be oriented into uniform orientation by the two-dimensional shape produced by imaging layer 204 during image recognition, so that analog code recognition can be carried out more easily.This allows decoding the microcarrier of imaging in any direction.

[0161] In some embodiments, the substantially opaque magnetic layer has a thickness of about 50 nm to about 10 μm. In some embodiments, the substantially opaque magnetic layer has a thickness of less than about any of the following thicknesses (in nm): 10000, 9500, 9000, 8500, 8000, 7500, 7000, 6500, 6000, 5500, 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, or 100. In some embodiments, the thickness of the substantially opaque magnetic layer is greater than about any of the following thicknesses (in nm): 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 9500. That is, the thickness of the substantially opaque magnetic layer may have an upper limit of 10000, 9500, 9000, 8500, 8000, 7500, 7000, 6500, 6000, 5500, 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150 or 100 and an upper limit of 50, 10 0, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000 or 9500, wherein the lower limit is less than the upper limit.

[0162] In some embodiments, the substantially opaque magnetic layer has a thickness of about 0.1 μm. In some embodiments, the thickness of the substantially opaque magnetic layer is about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm, about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about 4.5 μm, about 5 μm, about 5.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, or about 10 μm. In some embodiments, the thickness of the substantially opaque magnetic layer is about 0.01 μm, about 0.02 μm, about 0.03 μm, about 0.04 μm, about 0.05 μm, about 0.06 μm, about 0.07 μm, about 0.08 μm, about 0.09 μm, about 0.1 μm, about 0.11 μm, about 0.12 μm, about 0.13 μm, about 0.14 μm, about 0.15 μm, about 0.16 μm, about 0.17 μm, about 0.18 μm, about 0.19 μm, about 0.20 μm, about 0.25 μm, about 0.30 μm, about 0.35 μm, about 0.40 μm, about 0.45 μm, or about 0.50 μm.

[0163] In some embodiments, microcarrier also comprises the directional indicator for orienting the simulation code of substantially opaque polymer layer.Can be by imaging (for example, microscopic or other imaging forms as herein described) and / or any characteristic of the microcarrier that is seen and / or detected by image recognition software can be used as directional indicator.Directional indicator can be used as reference point, for example, for image recognition algorithm, to orient the image (that is, the shape of substantially opaque polymer layer) of simulation code with uniform orientation.Advantageously, this simplifies image recognition, because algorithm only needs to compare the image of specific simulation code with the simulation code library on the same orientation, and does not compare with the library of all simulation codes included in all possible orientations.In some embodiments, directional indicator can be independent of substantially opaque polymer layer.For example, it can be formed as a part for magnetic layer and / or substantially transparent polymer layer.In other embodiments, directional indicator can be formed as a part for substantially opaque polymer layer.In some embodiments, directional indicator comprises the asymmetry (for example, as shown in the gap 210 in Fig. 2 A) of substantially opaque magnetic layer.

[0164] In some embodiments, the microcarrier also includes one or more columns protruding from the microcarrier surface (e.g., the top and / or bottom surface of the microcarrier). As used herein, "column" can refer to protruding from the bead surface and does not necessarily represent any regularity in dimension, nor any geometric shape of any cylindrical feature. For example, the outer surface of the column can be parallel or non-parallel to the microcarrier surface. Examples of columnar shapes that can protrude from the microcarrier include but are not limited to rectangular prisms, triangles, pyramids, cubes, cylinders, spheres or hemispheres, cones, etc. In some embodiments, one or more columns are not within the center portion of the first substantially transparent polymer layer and / or the second substantially transparent polymer layer. In some embodiments, one or more columns can protrude from one or more outwardly facing surfaces (e.g., surfaces that are not fixed to another layer) of the first substantially transparent polymer layer and the second substantially transparent polymer layer. In some embodiments, one or more columns are prepared from a substantially opaque magnetic polymer layer. It should be noted that any description of the microcarrier thickness herein does not include one or more columns in the dimensions. That is, the microcarrier thickness as described herein is independent of any optional columns protruding therefrom.

[0165] Turning now to Figures 5A and 5B, another exemplary microcarrier 500 is shown. Like microcarrier 200, microcarrier 500 comprises a substantially transparent polymer layer 502, a substantially opaque polymer layer 504, a magnetic layer 506, and a central portion 508. In addition, microcarrier 500 has four pillars (including pillar 510), which can have any shape extending from the surface of layer 502. As shown in Figure 5A, these pillars can be aligned with magnetic layer 506 to prevent any interference with analyte detection in central portion 508 or the possibility of reading the two-dimensional shape of layer 504 (i.e., the analog code). Figure 5B shows that these pillars can extend from the upper and lower surfaces of microcarrier 500. Pillar 510 can, for example, be made of the same substantially transparent polymer as layer 502 (an exemplary production method is described below). Advantageously, one or more pillars (such as pillar 510) can be used to prevent microcarriers from adhering to each other and / or adhering (e.g., by optical contact bonding) to a container (e.g., the side of a well in a multi-well plate).

[0166] In some embodiments, the height of one or more pillars is about 1 μm to about 10 μm. In some embodiments, the height of one or more pillars is about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about 4.5 μm, about 5 μm, about 5.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, or about 10 μm. In some embodiments, one or more pillars are less than about any of the following heights (in μm): 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5. In some embodiments, one or more pillars are greater than about any of the following heights (in μm): 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5. That is, one or more columns can be any height range within a height range having an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5 and an independently selected lower limit of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5, where the lower limit is less than the upper limit.

[0167] In some embodiments, one or more posts can be cylindrical. In some embodiments, the diameter of one or more posts is from about 1 μm to about 10 μm. In some embodiments, the diameter of one or more posts is from about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about 4.5 μm, about 5 μm, about 5.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, or about 10 μm. In some embodiments, the diameter of one or more posts is less than about any of the following lengths (in μm): 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5. In some embodiments, the diameter of the one or more pillars is greater than about any of the following lengths (in μm): 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5. That is, the one or more pillars can have any diameter range within a diameter range having an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5 and an independently selected lower limit of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5, wherein the lower limit is less than the upper limit. In other embodiments, one or more pillars may have a width approximately the same as any diameter described above, or a width range approximately the same as any diameter range described above, but one or more pillars may take the shape of an ellipsoidal cylinder, a parabolic cylinder, a hyperbolic cylinder, or any other cylindrical or polyhedral shape described herein or known in the art.

[0168] In other aspects, provided herein are coded microcarriers comprising a substantially opaque polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other, wherein the outline of the substantially opaque polymer layer comprises a two-dimensional shape representing an analog code; and a capture agent for capturing an analyte, wherein the capture agent is coupled to at least one of the first surface and the second surface of the substantially opaque polymer layer in a central portion of at least the substantially opaque polymer layer. Thus, the microcarrier is encoded by the shape (e.g., outline) of the microcarrier itself: the two-dimensional shape representing the analog code. Advantageously, these microcarriers can be manufactured efficiently and with high precision, allowing highly accurate decoding and cost-effective production. Examples of this type of microcarrier and aspects thereof are shown in Figures 6A-9C.

[0169] Figures 6A and 6B show an exemplary microcarrier 600 of this type. Microcarrier 600 is a gear-shaped disk having a diameter of approximately 80 μm and a height of 15 μm, which includes an optional post element (similar to post 510 as described above). Microcarrier 600 is made of a single opaque polymer layer 602, rather than being made of a separate transparent polymer layer and an opaque polymer layer. Microcarrier 600 can be imaged as shown in Figure 1D, but its simulation code is based on imaging of the entire microcarrier shape (such as, the periphery of the opaque polymer layer). One or both surfaces of microcarrier 600 can be used to couple capture agents as described above, and the central portion or the entire surface can be used.

[0170] Figure 6C The dimensions of the gear teeth 604 of the microcarrier 600 are shown. As shown, in this embodiment, the gear teeth 604 are 4 μm wide and are spaced 4 μm apart from adjacent gear teeth 606. Because the two-dimensional shape of the microcarrier 600 is analog-coded, the perimeter between adjacent gear teeth can be varied, allowing for a variety of gear tooth shapes. For example, the height of the gear tooth 604 can extend 4 μm or 6.5 μm relative to the adjacent perimeter segments immediately to the left or right, respectively.

[0171] Figure 7 Another embodiment of this type of microcarrier, microcarrier 700, is shown. Like microcarrier 600, microcarrier 700 is made of an opaque polymer layer 702. In addition, the microcarrier comprises a magnetic layer 704. The magnetic layer 704 can be fixed to one of the surfaces of the microcarrier 700, or it can be embedded within the microcarrier 700 (e.g., between two opaque polymer layers). For example, the magnetic layer 704 can be generated by depositing nickel. As described above, the magnetic layer allows for additional functionality, such as the option to wash the microcarrier 700 while magnetically attached to another surface.

[0172] In some embodiments, the microcarriers further comprise one or more pillars protruding from the surface of the substantially opaque polymer layer. As described in more detail above, a "pillar" can refer to any geometric shape protruding from the surface of the microcarrier and does not necessarily represent any regularity in one or more columnar dimensions. Any of the exemplary columnar shapes described above can be used.

[0173] In some embodiments, the height of one or more pillars is about 1 μm to about 10 μm. In some embodiments, the height of one or more pillars is about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about 4.5 μm, about 5 μm, about 5.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, or about 10 μm. In some embodiments, one or more pillars are less than about any of the following heights (in μm): 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5. In some embodiments, one or more pillars are greater than about any of the following heights (in μm): 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5. That is, one or more columns can be any height range within a height range having an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5 and an independently selected lower limit of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5, where the lower limit is less than the upper limit.

[0174] In some embodiments, one or more posts can be cylindrical. In some embodiments, the diameter of one or more posts is from about 1 μm to about 10 μm. In some embodiments, the diameter of one or more posts is from about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about 4.5 μm, about 5 μm, about 5.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, or about 10 μm. In some embodiments, the diameter of one or more posts is less than about any of the following lengths (in μm): 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5. In some embodiments, the diameter of the one or more pillars is greater than about any of the following lengths (in μm): 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5. That is, the one or more pillars can have any diameter range within a diameter range having an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5 and an independently selected lower limit of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5, wherein the lower limit is less than the upper limit. In other embodiments, one or more pillars may have a width approximately the same as any diameter described above, or a width range approximately the same as any diameter range described above, but one or more pillars may take the shape of an ellipsoidal cylinder, a parabolic cylinder, a hyperbolic cylinder, or any other cylindrical or polyhedral shape described herein or known in the art.

[0175] In some embodiments, the microcarrier further comprises a magnetic layer comprising a magnetic material affixed to the surface of the substantially opaque polymer layer. In some embodiments, the magnetic layer does not extend beyond the two-dimensional shape of the substantially opaque polymer layer. That is, if the contours of the substantially opaque polymer layer are to be imaged, the resulting image is not altered by the presence or absence of the magnetic layer. In some embodiments, the magnetic layer may comprise one or more of the aforementioned pillars. That is, the one or more pillars may be made of the magnetic material described herein.

[0176] In some embodiments, the microcarrier also comprises an orientation indicator for orienting the analog code of the substantially opaque polymer layer. Any characteristic of the microcarrier that can be seen and / or detected by imaging (e.g., microscopic or other imaging forms as described herein) and / or by image recognition software can be used as an orientation indicator. The orientation indicator can be used as a reference point, for example, for an image recognition algorithm to orient the image of the analog code (i.e., the shape of the substantially opaque polymer layer) in a uniform orientation. Advantageously, this simplifies image recognition because the algorithm only needs to compare the image of a particular analog code with a library of analog codes at the same orientation, rather than with a library of all analog codes included in all possible orientations. In some embodiments, the orientation indicator comprises an asymmetry of the profile of the substantially opaque polymer layer. For example, the orientation indicator may comprise a visible characteristic of the microcarrier profile, such as an asymmetry (e.g., as described below) Figure 8A and starting positions 804 and 904 in 9A).

[0177] Now go to Figure 8A , another exemplary microcarrier 800 is shown. Like microcarrier 700, microcarrier 800 comprises an opaque polymer layer 802 (and optionally, a magnetic layer such as layer 704). In addition, microcarrier 800 comprises a starting position 804 having a shape that is different from the rest of the perimeter of microcarrier 800. Starting position 804 can be used as an orientation indicator for image recognition, as described above with respect to gap 210 shown in FIG. 2A .

[0178] Figure 8B The encoding schemes that can be used are shown. Figure 8B Microcarrier 810 is shown, which like microcarrier 800 comprises an opaque polymer layer 812 and a starting position 814 (and optionally a magnetic layer such as layer 704). In this scheme, potential shape change points around the gear are marked, for example, at positions 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, and 844. Figure 8B As shown, even though only two potential shapes can be used for positions 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, and 844, this embodiment allows for up to two 13 Additionally, as discussed above, the use of analog coding allows for the use of analog codes at any or all indicated locations around the perimeter (e.g., at Figure 8B Using more than two potential shapes (at each shape change point marked in ) greatly expands this number.

[0179] Figures 9A-9C illustrate another possible embodiment, microcarrier 900. Like microcarrier 800, microcarrier 900 is a gear-shaped microcarrier comprising an opaque polymer layer 902 and a starting position 904 (and optionally, a magnetic layer such as layer 704). In addition, microcarrier 900 may have one or more posts (e.g., posts 906) affixed to one or both surfaces of microcarrier 900. As shown in the cross-section in Figure 9B, posts 906 extend from the surface of layer 902. Advantageously, posts 906 help reduce the likelihood of optical contact bonding (as described above for posts 510).

[0180] FIG9C shows the dimensions of post 906. In this embodiment, post 906 is a cylinder having a height of 3 μm and a diameter of 3 μm, however, as described above, such posts are by no means limited to cylindrical shapes. In some embodiments, post 906 is made of a magnetic material such as nickel. This allows post 906 to additionally serve as a magnetic element for magnetic manipulation of microcarriers 900, as described above.

[0181] Any of the microcarriers described herein may include one or more of the characteristics, elements or aspects described below. Additionally, depending on the embodiment of the microcarrier, one or more of the characteristics, elements or aspects described below may adopt different features, for example, as described above.

[0182] In some embodiments, the substantially transparent polymers of the present disclosure include epoxy polymers. Suitable epoxy polymers for making the compositions described herein include, but are not limited to, EPON supplied by Hexion Specialty Chemicals, Inc. (Columbus, OH). TMThe epoxy resin of the family and any numbered epoxy resin provided by The Dow Chemical Company (Midland, MI). Many examples of suitable polymers are well known in the art, including but not limited to SU-8, EPON 1002F, EPON 165 / 154 and poly (methyl methacrylate) / poly (acrylic acid) block copolymers (PMMA-co-PAA). For other polymers, see, for example, Warad, IC Packaging: Package Construction Analysis in Ultra Small IC Packaging, LAP LAMBERT Academic Publishing (2010); The Electronic Packaging Handbook, CRC Press (Blackwell, ed.), (2000); and Pecht et al., Electronic Packaging Materials and Their Properties, CCRPress, 1st edition, (1998). These types of materials have the advantage of not swelling in aqueous environments, which ensures that uniform microcarrier size and shape are maintained within the microcarrier population. In some embodiments, the substantially transparent polymer is a photoresist polymer. In some embodiments, the epoxy-based polymer is an epoxy-based negative near-UV photoresist. In some embodiments, the epoxy-based polymer is SU-8.

[0183] In some embodiments, substantially opaque polymer is a polymer as described herein (for example, SU-8) mixed with one or more opaque or colored dyes. In other embodiments, substantially opaque polymer is a black matrix resist. Any black matrix resist known in the art can be used; About exemplary black matrix resist and method related thereto, see, for example, U.S. Patent No. 8,610,848. In some embodiments, black matrix resist can be a photoresist colored with black pigment, for example, as patterned as a part of black matrix on the color filter of LCD. Black matrix resist can include but is not limited to those sold by Toppan Printing Co. (Tokyo), Tokyo OHKAKogyo (Kawasaki) and Daxin Materials Corp.

[0184] In some embodiments, the center of one or more polymer layers can be referenced. The center of the present disclosure can adopt any shape. In some embodiments, the shape of the center can reflect or correspond to the shape (for example, profile) of the corresponding polymer layer. In other embodiments, the shape of the center can be irrelevant to the shape (for example, profile) of the corresponding polymer layer. For example, the center of a circular microcarrier surface can be circular in some embodiments, and can be square in other embodiments. In some embodiments, the center of a square microcarrier surface can be square, and can be circular in other embodiments.

[0185] In some embodiments, the central portion of a polymeric layer of the present disclosure is about 5%, about 7%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% of the surface area of ​​the polymeric layer. In some embodiments, the central portion of a polymeric layer of the present disclosure is less than about any of the following fractions (in %) of the substantially transparent polymeric layer: 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 7. In some embodiments, the central portion of the polymeric layer of the present disclosure is greater than about any of the following fractions (%) of the substantially transparent polymeric layer: 5, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85. That is, the fraction of the polymeric layer surface area contained in the central portion can be any percentage range having an upper limit of 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 7 and an independently selected lower limit of 5, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85, wherein the lower limit is less than the upper limit. In some embodiments, the central portion of the polymeric layer comprises about 25% of the surface area of ​​the polymeric layer. In some embodiments, the central portion of the microcarrier surface comprises the entire surface minus the outline of the microcarrier.

[0186] As described above, the microcarriers of the present disclosure may also include a magnetic layer, which may take various shapes as described herein. In some embodiments, the magnetic layer may be a substantially opaque layer. In some embodiments, the magnetic layer may include a magnetic material. The magnetic layer of the present disclosure may be made of any suitable magnetic material (such as a material with paramagnetism, ferromagnetism or ferrimagnetism). Examples of magnetic materials include, but are not limited to, iron, nickel, cobalt and some rare earth metals (e.g., gadolinium, dysprosium, neodymium, etc.), and alloys thereof. In some embodiments, the magnetic material comprises nickel, including but not limited to elemental nickel and magnetic nickel alloys, such as aluminum nickel cobalt and permalloy. Including a magnetic layer in the microcarriers of the present disclosure may be advantageous, for example, in promoting magnetic separation, and the magnetic layer may be used for washing, collecting and otherwise manipulating one or more microcarriers.

[0187] As described above, in some embodiments, the magnetic layer can be fixed to the surface of the substantially transparent polymer layer and surround the central portion of the substantially transparent polymer layer. In other embodiments, as described above, the magnetic layer can include one or more pillars; that is, the one or more pillars can be made of the magnetic materials described herein.

[0188] In some aspects, provided herein are encoded microcarriers comprising: a substantially transparent polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other; a substantially opaque magnetic polymer layer affixed to the first surface of the substantially transparent polymer layer and surrounding a central portion of the substantially transparent polymer layer; and a probe specific for a DNA mutation in the KRAS, BRAF, CTNNB1, or APC gene, wherein the probe is coupled to at least one of the first and second surfaces of the substantially transparent polymer layer in at least the central portion of the substantially transparent polymer layer. In some embodiments, the substantially opaque magnetic polymer layer comprises at least a portion of a two-dimensional shape representing an analog code. For example, a portion of the two-dimensional shape may be made of a substantially opaque non-magnetic polymer layer, and a portion of the two-dimensional shape may be made of a substantially opaque non-magnetic polymer layer (e.g., SU-8 as described herein). In other embodiments, the substantially opaque magnetic polymer layer comprises all of the two-dimensional shape representing the analog code. Thus, the microcarrier comprises at least two layers: one of which is substantially transparent, and the other of which is a substantially opaque magnetic two-dimensional shape representing the analog code.

[0189] Advantageously, these microcarriers can adopt a variety of two-dimensional shapes while still maintaining a uniform overall form (e.g., a substantially transparent polymer layer around the periphery) to achieve uniformity in various aspects, including, for example, overall size, physical properties, and / or behavior in solution. Various optional aspects and elements of this type of microcarrier are shown in Figures 1A-5B (see, e.g., Figure 4BThe magnetic layer advantageously combines a two-dimensional shape host for simulating the encoding while also providing magnetic functionality allowing, for example, to facilitate magnetic separation, which can be used to wash, collect and otherwise manipulate one or more microcarriers.

[0190] In some embodiments, the analog code of the present disclosure is made of the two-dimensional shape of a substantially opaque magnetic polymer layer. Advantageously, this provides microcarriers that can be efficiently produced on a large scale (e.g., using the technology described herein). In addition, using a substantially opaque magnetic polymer layer to generate analog codes facilitates multiple determinations by allowing a variety of different microcarrier types (each having a unique analog code) while also providing simple operation through the magnetic properties of the resulting microcarriers.

[0191] In some embodiments, the substantially opaque magnetic layer has a thickness of about 50 nm to about 10 μm. In some embodiments, the substantially opaque magnetic layer has a thickness of less than about any of the following thicknesses (in nm): 10000, 9500, 9000, 8500, 8000, 7500, 7000, 6500, 6000, 5500, 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, or 100. In some embodiments, the thickness of the substantially opaque magnetic layer is greater than about any of the following thicknesses (in nm): 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 9500. That is, the thickness of the substantially opaque magnetic layer may have an upper limit of 10000, 9500, 9000, 8500, 8000, 7500, 7000, 6500, 6000, 5500, 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150 or 100 and an upper limit of 50, 10 0, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000 or 9500, wherein the lower limit is less than the upper limit.

[0192] In some embodiments, the substantially opaque magnetic layer has a thickness of about 0.1 μm. In some embodiments, the thickness of the substantially opaque magnetic layer is about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm, about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about 4.5 μm, about 5 μm, about 5.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, or about 10 μm. In some embodiments, the thickness of the substantially opaque magnetic layer is about 0.01 μm, about 0.02 μm, about 0.03 μm, about 0.04 μm, about 0.05 μm, about 0.06 μm, about 0.07 μm, about 0.08 μm, about 0.09 μm, about 0.1 μm, about 0.11 μm, about 0.12 μm, about 0.13 μm, about 0.14 μm, about 0.15 μm, about 0.16 μm, about 0.17 μm, about 0.18 μm, about 0.19 μm, about 0.20 μm, about 0.25 μm, about 0.30 μm, about 0.35 μm, about 0.40 μm, about 0.45 μm, or about 0.50 μm.

[0193] In some embodiments, microcarriers of the present disclosure can be encoded with the substantially opaque layer constituting a two-dimensional shape. For example, as mentioned above, the two-dimensional shape can constitute the shape of a substantially opaque layer that contrasts with the substantially transparent layer of the microcarrier, or it can constitute the shape (for example, periphery) of the microcarrier itself. Any two-dimensional shape that can comprise multiple resolvable and distinguishable variation types can be used. In some embodiments, the two-dimensional shape comprises one or more in linear, circular, elliptical, rectangular, quadrilateral or higher polygonal outward appearance, element and / or shape. In certain embodiments, the substantially opaque magnetic layer of the present disclosure can be used to produce a two-dimensional shape.

[0194] Figure 4A Shown Figure 3 Three exemplary embodiments of the coding scheme are shown in FIG: microcarriers 400, 402, and 404. The unique codes of microcarriers 400, 402, and 404 are generated using Figure 3Importantly, as described above, more complex encoding schemes can be obtained using analog image recognition, thereby greatly expanding the number of potential unique codes. In some embodiments, the two-dimensional shape of the substantially opaque polymeric layer includes one or more rings surrounding a central portion of the substantially transparent polymeric layer. In some embodiments, at least one of the one or more rings includes a discontinuity. Exemplary and non-limiting two-dimensional shapes formed using one or more rings (e.g., two rings) having different numbers and configurations of discontinuities are shown in FIG. Figure 4B middle. Figure 4B Ten exemplary embodiments of the code are shown, particularly with respect to the number of shapes (e.g., two different shapes in code ZN_3 as compared to seven different shapes in code ZN_10) and / or the size of the shapes (e.g., large, small, and medium-sized shapes in code ZN_2).

[0195] In some embodiments, the two-dimensional shape of the substantially opaque polymer layer includes a gear shape. The gear shape used herein may refer to a plurality of shapes (e.g., gear teeth) arranged on the periphery of a substantially circular, elliptical, or circular body, wherein at least two of the plurality of shapes are spatially separated. In some embodiments, the gear shape comprises a plurality of gear teeth. In some embodiments, the simulation code is represented by one or more aspects selected from the height of one or more gear teeth in the plurality of gear teeth, the width of one or more gear teeth in the plurality of gear teeth, the number of gear teeth in the plurality of gear teeth, and the arrangement of one or more gear teeth in the plurality of gear teeth. Advantageously, the gear shape includes a plurality of aspects, including the height of the gear teeth, the width of the gear teeth, the number of gear teeth, and the arrangement of the gear teeth, which can be varied to generate a wide variety of potentially unique two-dimensional shapes. However, it should be understood that since the gear shape of the present disclosure is used for encoding and does not need to be physically meshed with another gear (e.g., like a mechanical gear that transmits torque), the gear teeth of the present disclosure are not subject to the need for identical or intermeshing shapes, whether within a gear shape or between multiple gear shapes. Therefore, it can be considered that the diversity of the shapes of the gear teeth of the present disclosure is significantly greater than that of a mechanical gear.

[0196] Figure 3 A large number of potential simulation codes are shown that are possible using the gear shapes shown in Figures 1A-2B. Figure 3 An exemplary coding scheme is shown, where, for example, multiple shape change points are marked at positions 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, and 328 on an exemplary microcarrier 300. Even using a simple "fill or not fill" scheme, there may be up to 214 This scheme is convenient for manufacturing and generating two-dimensional shapes that are easily distinguishable for image recognition analysis. However, due to the use of analog coding, more complex schemes with more than 2 possibilities (e.g., in Figure 3 1A-3 facilitates a wide range of unique analog codes while providing a large central portion (e.g., central portions 106 and 208) for analyte detection.

[0197] In some embodiments, the plurality of gear teeth includes one or more gear teeth having a width of about 1 μm to about 10 μm. In some embodiments, the plurality of gear teeth includes one or more gear teeth having a width of about 1 μm, a width of about 1.5 μm, a width of about 2 μm, a width of about 2.5 μm, a width of about 3 μm, a width of about 3.5 μm, a width of about 4 μm, a width of about 4.5 μm, a width of about 5 μm, a width of about 5.5 μm, a width of about 6 μm, a width of about 6.5 μm, a width of about 7 μm, a width of about 7.5 μm, a width of about 8 μm, a width of about 8.5 μm, a width of about 9 μm, a width of about 9.5 μm, or a width of about 10 μm. In some embodiments, the plurality of gear teeth includes one or more gear teeth having a width less than any of the following widths (in μm): 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5. In some embodiments, the plurality of gear teeth includes one or more gear teeth having a width greater than any of the following widths (in μm): 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5. That is, the plurality of gear teeth may include one or more gear teeth that may be any width range within a width range having an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5 and an independently selected lower limit of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5, where the lower limit is less than the upper limit.

[0198] In some embodiments, the plurality of gear teeth includes one or more gear teeth having a height of about 1 μm to about 10 μm. In some embodiments, the plurality of gear teeth includes one or more gear teeth having a height of about 1 μm, a height of about 1.5 μm, a height of about 2 μm, a height of about 2.5 μm, a height of about 3 μm, a height of about 3.5 μm, a height of about 4 μm, a height of about 4.5 μm, a height of about 5 μm, a height of about 5.5 μm, a height of about 6 μm, a height of about 6.5 μm, a height of about 7 μm, a height of about 7.5 μm, a height of about 8 μm, a height of about 8.5 μm, a height of about 9 μm, a height of about 9.5 μm, or a height of about 10 μm. In some embodiments, the plurality of gear teeth includes one or more gear teeth having a width less than about any of the following heights (in μm): 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5. In some embodiments, the plurality of gear teeth includes one or more gear teeth having a height greater than about any of the following heights (in μm): 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5. That is, the plurality of gear teeth may include one or more gear teeth that may be any height range within a height range having an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5 and an independently selected lower limit of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5, where the lower limit is less than the upper limit. It should be understood that if the adjacent peripheral section from which the gear teeth extend is non-uniform, the gear teeth may have different measurable heights depending on the reference point (see, for example, Figure 6C The gear teeth 602 in FIG. 5 may have a height of 4 or 6.5 μm depending on the reference point).

[0199] In some embodiments, the plurality of gear teeth includes one or more gear teeth spaced about 1 μm to about 10 μm apart. In some embodiments, the plurality of gear teeth includes one or more gear teeth spaced about 1 μm apart, about 1.5 μm apart, about 2 μm apart, about 2.5 μm apart, about 3 μm apart, about 3.5 μm apart, about 4 μm apart, about 4.5 μm apart, about 5 μm apart, about 5.5 μm apart, about 6 μm apart, about 6.5 μm apart, about 7 μm apart, about 7.5 μm apart, about 8 μm apart, about 8.5 μm apart, about 9 μm apart, about 9.5 μm apart, or about 10 μm apart. In some embodiments, the plurality of gear teeth includes one or more gear teeth having a spacing less than about any of the following widths (in μm): 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5. In some embodiments, the plurality of gear teeth includes one or more gear teeth having a spacing greater than about any of the following widths (in μm): 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5. That is, the plurality of gear teeth may include one or more gear teeth that may be spaced apart having any range of width spacings within a range of width spacings having an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5 and an independently selected lower limit of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5, where the lower limit is less than the upper limit.

[0200] In some embodiments, the microcarriers of the present disclosure are substantially circular disks. As used herein, a substantially circular shape may refer to any shape having approximately the same distance between all points on the perimeter of the shape and the geometric center of the shape. In some embodiments, a shape is considered substantially circular if the variation between any potential radius connecting the geometric center and a given point on the perimeter exhibits a length variation of 10% or less. As used herein, a substantially circular disk may refer to any substantially circular shape in which the thickness of the shape is significantly less than its diameter. For example, in some embodiments, the thickness of a substantially circular disk may be less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% of its diameter. In certain embodiments, the thickness of a substantially circular disk may be about 20% of its diameter. It should be understood that a microcarrier of the present disclosure having a gear-shaped profile may also be considered a substantially circular disk; for example, the shape of a microcarrier other than one or more gear teeth may comprise a substantially circular disk.

[0201] In some embodiments, the diameter of the microcarrier is less than about 200 μm. For example, in some embodiments, the diameter of the microcarrier is less than about 200 μm, less than about 180 μm, less than about 160 μm, less than about 140 μm, less than about 120 μm, less than about 100 μm, less than about 80 μm, less than about 60 μm, less than about 40 μm, or less than about 20 μm.

[0202] In some embodiments, the diameter of the microcarrier is about 180 μm, about 160 μm, about 140 μm, about 120 μm, about 100 μm, about 90 μm, about 80 μm, about 70 μm, about 60 μm, about 50 μm, about 40 μm, about 30 μm, about 20 μm or about 10 μm. In certain embodiments, the diameter of the microcarrier is about 60 μm.

[0203] In some embodiments, the thickness of the microcarrier is less than about 50 μm. For example, in some embodiments, the thickness of the microcarrier is less than about 70 μm, about 60 μm, about 50 μm, about 40 μm, about 30 μm, less than about 25 μm, less than about 20 μm, less than about 15 μm, less than about 10 μm, or less than about 5 μm. In some embodiments, the thickness of the microcarrier is less than about any of the following thicknesses (in μm): 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2. In some embodiments, the thickness of the microcarrier is greater than about any of the following thicknesses (in μm): 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65. That is, the thickness of the microcarriers can be any range of thicknesses in μm having an upper limit of 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 and an independently selected lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65, wherein the lower limit is less than the upper limit.

[0204] In some embodiments, the thickness of the microcarrier is about 50 μm, about 45 μm, about 40 μm, about 35 μm, about 30 μm, about 25 μm, about 20 μm, about 19 μm, about 18 μm, about 17 μm, about 16 μm, about 15 μm, about 14 μm, about 13 μm, about 12 μm, about 11 μm, about 10 μm, about 9 μm, about 8 μm, about 7 μm, about 6 μm, about 5 μm, about 4 μm, about 3 μm, about 2 μm, or about 1 μm. In certain embodiments, the thickness of the microcarrier is about 10 μm.

[0205] The probe of the present disclosure can be coupled to the encoded microcarrier using a variety of techniques. In one embodiment, the probe is coupled to the surface of the microcarrier (in some embodiments, at least the center portion of the microcarrier surface). In some embodiments, the probe can be coupled to one or both of the first surface or the second surface of the polymer layer. In some embodiments, the polymer comprises an epoxy polymer or otherwise contains epoxy groups.

[0206] In some embodiments, the probe can be chemically attached to the microcarrier. In other embodiments, the probe can be physically adsorbed to the surface of the microcarrier. In some embodiments, the connection linkage between the capture agent and the bead surface can be a covalent bond. In other embodiments, the connection linkage between the probe and the microcarrier surface can be a non-covalent bond, including but not limited to salt bridges or other ionic bonds, one or more hydrogen bonds, hydrophobic interactions, van der Waals forces, London dispersion forces, mechanical bonds, one or more halogen bonds, aurophilic effects, embedding or stacking.

[0207] In some embodiments, coupling the probe comprises reacting the polymer with a photoacid generator and light to generate a cross-linked polymer. In some embodiments, the light has a wavelength that activates the photoacid generator, such as UV or near-UV light. Photoacid generators are commercially available from Sigma-Aldrich (St. Louis) and BASF (Ludwigshafen). Any suitable photoacid generator known in the art can be used, including but not limited to triphenyl or triarylsulfonium hexafluoroantimonate; triarylsulfonium hexafluorophosphate; triphenylsulfonium perfluoro-1-butanesulfonate; triphenylsulfonium trifluoromethanesulfonate; tri(4-tert-butylphenyl)sulfonium perfluoro-1-butanesulfonate or trifluoromethanesulfonate; photoacid generators containing bis(4-tert-butylphenyl)iodonium, such as bis(4-tert-butylphenyl)iodonium perfluoro-1-butanesulfonate, p-toluenesulfonate and trifluoromethanesulfonic acid. salt; Boc-methoxyphenyldiphenylsulfonium trifluoromethanesulfonate; (tert-butoxycarbonylmethoxynaphthyl)-diphenylsulfonium trifluoromethanesulfonate; (4-tert-butylphenyl)diphenylsulfonium trifluoromethanesulfonate; diphenyliodonium hexafluorophosphate, nitrate, perfluoro-1-butanesulfonate, trifluoromethanesulfonate or p-toluenesulfonate; (4-fluorophenyl)diphenylsulfonium trifluoromethanesulfonate; N-hydroxynaphthalimide trifluoromethanesulfonate; N-hydroxy-5-norbornene-2,3-dicarboximide perfluoro- 1-butanesulfonate; (4-iodophenyl)diphenylsulfonium trifluoromethanesulfonate; (4-methoxyphenyl)diphenylsulfonium trifluoromethanesulfonate; 2-(4-methoxyphenyl)-4,6-bis(trichloromethyl)-1,3,5-triazine; (4-methylphenyl)diphenylsulfonium trifluoromethanesulfonate; (4-methylthiophenyl)methylphenylsulfonium trifluoromethanesulfonate; (4-phenoxyphenyl)diphenylsulfonium trifluoromethanesulfonate; (4-phenylthiophenyl)diphenylsulfonium trifluoromethanesulfonate; or any of the photoacid generators described at product-finder.basf.com / group / corporate / product-finder / de / literature-document: / Brand+Irgacure-Brochure--Photoacid+Generator+Selection+Guide-English.pdf. In some embodiments, the photoacid generator is a sulfonium-containing photoacid generator.

[0208] In some embodiments, coupling the probe comprises reacting an epoxide of a cross-linked polymer with a functional group such as an amine, carboxyl, sulfhydryl, or the like. Alternatively, the epoxy groups on the surface can be oxidized to hydroxyl groups, which are then used as initiation sites for graft polymerization of a water-soluble polymer such as poly(acrylic acid). The carboxyl groups in the poly(acrylic acid) are then used to form a covalent bond with an amino or hydroxyl group in the capture agent. For example, in certain embodiments, the carboxyl groups in the poly(acrylic acid) are used to form a covalent bond with an amino group in the probe.

[0209] In some embodiments, the coupling probe includes reacting the epoxide of the cross-linked polymer with a compound containing an amine and a carboxyl group. In some embodiments, the amine of the compound reacts with the epoxide to form a cross-linked polymer of the compound coupling. Without being bound by theory, it is believed that the probe can be coupled to the polymer before the polymer is cross-linked; however, this can reduce the uniformity of the resulting surface. Any compound with a primary amine and a carboxyl group can be used. The compound may include but is not limited to glycine, aminoundecanoic acid, aminocaproic acid, acrylic acid, 2-carboxyethyl acrylic acid, 4-vinylbenzoic acid, 3-acrylamido-3-methyl-1-butyric acid, glycidyl methacrylate, etc. In some embodiments, the carboxyl group of the cross-linked polymer of the compound coupling reacts with the amine (e.g., primary amine) of the probe to couple the capture agent to a substantially transparent polymer.

[0210] V. Kit

[0211] Also provided herein are kits or articles comprising a plurality of microcarriers of the present disclosure. These kits or articles are particularly useful for performing multiple assays, such as the exemplary multiple assays described herein (see, e.g., Section III above). Any microcarrier described herein (see, e.g., Section IV) can be used for kits or articles of the present disclosure.

[0212] In some embodiments, the kit or article of the present disclosure includes at least four coding microcarriers. In some embodiments, each of the four coding microcarriers includes (i) a probe coupled to a microcarrier that has specificity for a DNA mutation in a KRAS, BRAF, CTNNB1 or APC gene; and (ii) an identifier corresponding to a probe. In some embodiments, the kit includes at least one microcarrier comprising a probe specific for a DNA mutation in a KRAS gene, at least one microcarrier comprising a probe specific for a DNA mutation in a BRAF gene, at least one microcarrier comprising a probe specific for a DNA mutation in a CTNNB1 gene, and at least one microcarrier comprising a probe specific for a DNA mutation in a APC gene. That is, each of the KRAS, BRAF, CTNNB1, and APC genes is presented in the kit by a microcarrier having a probe specific for a mutation in a gene. Exemplary KRAS, BRAF, CTNNB1, and APC genes and mutations are described above. In some embodiments, KRAS, BRAF, CTNNB1, and APC genes are human genes. In some embodiments, the kit comprises microcarriers with probes suitable for detecting each of the following mutations (e.g., there is at least one microcarrier + probe species for each mutation in the kit): DNA mutations encoding G12D, G12V, G12S, and G13D mutations in KRAS protein; one or more DNA mutations encoding V600E mutation in BRAF protein; DNA mutations encoding T41A and T41I mutations in CTNNB1 protein; DNA mutations encoding S45F and S45P mutations in CTNNB1 protein; and DNA mutations encoding Q1367*, R1450*, E1309 frameshift, S1465 frameshift, and T1556 frameshift mutations in APC protein.

[0213] In some embodiments, the kit further comprises at least four blocking nucleic acids. In some embodiments, the at least four blocking nucleic acids hybridize to wild-type DNA loci corresponding to DNA mutations in each of the KRAS, BRAF, CTNNB1, and APC genes. Exemplary descriptions of blocking nucleic acids are provided in Section III. In some embodiments, the kit comprises a blocking nucleic acid comprising the following sequence: TACGCCACCAGCT(invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO:3); TTGGAGCTGGTGGCGTAinvdTinvdTinvdTinvdT (SEQ ID NO:142); GCTGGTGGCGTAGGCAinvdTinvdTinvdT (SEQ ID NO:143); GCTGGTGGCGTAGGCinvdTinvdTinvdT (SEQ ID NO:144) or TTGGAGCTGGTGGCGTinvdTinvdTinvdT (SEQ ID NO:145); a blocking nucleic acid comprising the sequence: GAGATTTCACTGTAGC (invdT) n , wherein n is 1, 2 or 3 (SEQ ID NO: 10); GAGATTTCACTGTAGCinvdTinvdTinvdT (SEQ ID NO: 146); GAGATTTCACTGTAGCinvdTinvdTinvdT (SEQ ID NO: 147); GAGATTTCACTGTAGCinvdTinvdTinvdT (SEQ ID NO: 148) or GAGATTTCACTGTAGCinvdTinvdTinvdT (SEQ ID NO: 149); a blocking nucleic acid comprising the sequence GCCACTACCACAGCT(invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 15); TGCCACTACCACAGinvdTinvdTinvdTinvdT (SEQ ID NO: 150); CACTACCACAGCTCCinvdTinvdTinvdT (SEQ ID NO: 151); GCCACTACCACAGCTinvdTinvdTinvdT (SEQ ID NO: 152) or GCCACTACCACAGCTinvdTinvdTinvdT (SEQ ID NO: 153) and / or a blocking nucleic acid comprising the following sequence: GCTCCTTCTCTGAGTinvdTinvdTinvdT (SEQ ID NO: 20); TCCTTCTCTGAGTGGinvdTinvdTinvdT (SEQ ID NO: 174); GCTCCTTCTCTGAGTinvdTinvdTinvdT (SEQ ID NO: 176). NO: 175); TCCTTCTCTGAGTGGinvdTinvdTinvdT (SEQ ID NO: 176) or GCTCCTTCTCTGAGTinvdTinvdTinvdT (SEQ ID NO: 177); and one or more of the following sequences: a blocking nucleic acid comprising the following sequence: GTGCTCAGACACCinvdTinvdTinvdT (SEQ ID NO: 33), GTGCTCAGACACCinvdTinvdTinvdT (SEQ ID NO: 158), AGTGGTGCTCAGACACCCAinvdTinvdTinvdT (SEQ ID NO: 159), AGTGGTGCTCAGACACCCAinvdTinvdTinvdT (SEQ ID NO: 160) or AGTGGTGCTCAGACACCCAinvdTinvdTinvdT (SEQ ID NO: 161). ID NO: 161); a blocking nucleic acid comprising the following sequence: CTTCTCGCTTGGTTinvdTinvdTinvdTinvdT (SEQ ID NO: 37), GTACTTCTCGCTTGGTinvdTinvdTinvdT (SEQ ID NO: 162), CTTCTCGCTTGGTTinvdTinvdTinvdT (SEQ ID NO: 163), GTACTTCTCGCTTGGTinvdTinvdTinvdT (SEQ ID NO: 164), or GTACTTCTCGCTTGGTinvdTinvdTinvdT (SEQ ID NO: 165);Blocking nucleic acids comprising the following sequences: CTTTTCTTTTATTTCTGCinvdTinvdTinvdT (SEQ ID NO: 29), CTTTTCTTTTATTTCTGCinvdTinvdTinvdT (SEQ ID NO: 154), CTTTTCTTTTATTTCTGCinvdTinvdTinvdT (SEQ ID NO: 155), CTTTTCTTTTATTTCTGCinvdTinvdTinvdT (SEQ ID NO: 156), or CTTTTCTTTTATTTCTGCinvdTinvdTinvdT (SEQ ID NO: 157); blocking nucleic acids comprising the following sequences: CCACTCTCTCTCTTTTCAGCinvdTinvdTinvdT (SEQ ID NO: 25), TAGGTCCACTCTCTCTCTTTTCAGCAinvdTinvdTinvdT (SEQ ID NO: 166), TAGGTCCACTCTCTCTCTTTTCAGCA invdTinvdTinvdT (SEQ ID NO: 167), CCACTCTCTCTCTTTTCAGC invdTinvdTinvdT (SEQ ID NO: 168), or TAGGTCCACTCTCTCTCTTTTCAGCA invdTinvdTinvdT (SEQ ID NO: 169); and blocking nucleic acids comprising the following sequences: CAATAGTTTTTTCTGCCinvdTinvdTinvdTinvdT (SEQ ID NO: 41), GAATCAATAGTTTTTTCTGCCTCinvdTinvdTinvdT (SEQ ID NO: 170), TCAGAATCAATAGTTTTTTCTGinvdTinvdTinvdT (SEQ ID NO: 171), GAATCAATAGATTTTACTGCCTC invdTinvdTinvdT (SEQ ID NO: 172), or AATCAATAGTTTTTTTCTGCCTC invdTinvdTinvdT (SEQ ID NO: 173), (nucleic acids in italics represent locked nucleic acids). ;

[0214] In some embodiments, the kit further comprises one or more primer pairs, for example, for amplifying a locus of a target DNA mutation. In some embodiments, the kit comprises primer pairs specific for one or more DNA mutation loci in each of the KRAS, BRAF, CTNNB1, and APC genes, for example, at least four primer pairs. In some embodiments, the kit comprises a primer pair comprising the sequence GTACTGGTGGAGTATTTGATAGTG (SEQ ID NO: 1) and ATCGTCAAGGCACTCTTGCCTAC (SEQ ID NO: 2) for amplifying a locus comprising a KRAS mutation (e.g., one or more KRAS mutations encoding a KRAS protein with G12D, G12V, G12S, and G13D mutations); a primer pair comprising the sequence GGACCCACTCCATCGAGATTT (SEQ ID NO: 8) and CAGATATATTTCTTCATGAAGACCTCACAGTAA (SEQ ID NO: 9) for amplifying a locus comprising a BRAF mutation (e.g., one, two, or more BRAF mutations encoding a BRAF protein with a V600E mutation); a primer pair comprising the sequence GGAATCCATTCTGGTGCCACT (SEQ ID NO: 13) and AGAAAATCCCTGTTCCCACTCATA (SEQ ID NO: 14) and / or a primer pair comprising the sequence GGTGCCACTACCACAGCTCCT (SEQ ID NO: 15). NO: 18) and TCTCAAAACTGCATTCTGACTTTCA (SEQ ID NO: 19) to amplify a locus comprising a CTNNB1 mutation (e.g., one or more CTNNB1 mutations encoding a CTNNB1 protein with a T41A, T41I, S45F, or S45P mutation); and one or more of the following primer pairs: a first primer pair comprising the sequence TAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24); a second primer pair comprising the sequence TAGGATGTAATCAGACGACACAGGA (SEQ ID NO: 27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO: 28); and a third primer pair comprising the sequence TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO: 31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO: 32);A fourth primer pair comprising the sequences GCAGAAGTAAAACACCTCCACCA (SEQ ID NO: 35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO: 36), wherein italicized nucleic acids represent locked nucleic acids; and a fifth primer pair comprising the sequences CAGGAAAATGACAATGGGAATG (SEQ ID NO: 39) and ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO: 40) for amplifying a locus comprising an APC mutation (e.g., one or more APC mutations encoding an APC protein with a Q1367*, R1450*, E1309 frameshift, S1465 frameshift, or T1556 frameout mutation).

[0215] Any of the probes described herein (eg, in Part III) can be included in the kit, eg, coupled to an encoding microcarrier. For example, suitable probes for detecting DNA mutations in the KRAS gene can comprise the sequences GGAGCTGATGG (SEQ ID NO:4), AGCTGATGGCGTA (SEQ ID NO: 178), TGGAGCTGATGGCG (SEQ ID NO: 179), TGGAGCTGATGG (SEQ ID NO: 180), GCTGATGGCGTA (SEQ ID NO: 181), GGAGCTGTTGG (SEQ ID NO: 5), TGGAGCTGTTGGTGGC (SEQ ID NO: 182), GGAGCTGTTGGTG (SEQ ID NO: 183), TGGAGCTGTTGGT (SEQ ID NO: 184), TGGAGCTGTaGGTGG (SEQ ID NO: 185), TGGAGCTAGTGG (SEQ ID NO: 6), TTGGAGCTAGTGGCGTA (SEQ ID NO: 186), GCTAGTGGCGTAGGC (SEQ ID NO: 187), AGCTAGTGGCGT (SEQ ID NO: 188), NO: 188), GTTGGAGCTAGTGG (SEQ ID NO: 189), GGAGCTAGTGG (SEQ ID NO: 190), TGGAGCTGGTGACGT (SEQ ID NO: 7), GGTGACGTAGGCAA (SEQ ID NO: 191), TGACGTAGGCAAGAG (SEQ ID NO: 192), GCTGGTGACGTAGG (SEQ ID NO:193), AGCTGGTGACGTAG (SEQ ID NO:194), GGAGCTGGTGACGT (SEQ ID NO:195), TTTTTTTTTTTTAAGGAGCTGATGG (SEQ ID NO:47), TTTTTTTTTTTTAGCTGATGGCGTA (SEQ ID NO:74), TTTTTTTTTTATGGAGCTGATGGCG (SEQ ID NO:74) NO:75),TTTTTTTTTTTTATGGAGCTGATGG(SEQ ID NO:76),TTTTTTTTTTTTTGCTGATGGCGTA(SEQ ID NO: 77), TTTTTTTTTTTTAAGGAGCTGTTGG (SEQ ID NO: 48), TTTTTTTTTATGGAGCTGTTGGTGGC (SEQ IDNO: 78), TTTTTTTTTTAAGGAGCTGTTGGTG (SEQ ID NO: 79), TTTTTTTTTTTATGGAGCTGTTGGT (SEQ ID NO: 80), TTTTTTTTTATGGAGCTGTAGGTGG (SEQ ID NO: 81), TTTTTTTTTTTATGGAGCTAGTGG (SEQ ID NO: 49), TTTTTTTTTTGGAGCTAGTGGCGTA (SEQ ID NO: 81) NO: 82), TTTTTAATTTGCTAGTGGCGTAGGC (SEQ ID NO: 83), TTTTTTTTTATTTAGCTAGTGGCGT (SEQ ID NO: 84), TTTTTTTTTTTGTTGGAGCTAGTGG (SEQ ID NO: 85), TTTTTTTTTTTTAAGGAGCTAGTGG (SEQ ID NO: 86), TTTTTTTTTATGGAGCTGGTGACGT (SEQ ID NO: 85) IDNO:50),TTTTTTTTAAAGGTGACGTAGGCAA(SEQ ID NO: 87), TTTTTTTTTATGACGTAGGCAAGAG (SEQ ID NO: 88), TTTTTTTTTTTGCTGGTGACGTAGG (SEQ ID NO: 89), TTTTTTTTTTAAGCTGGTGACGTAG (SEQ ID NO: 90), and TTTTTTTTTAAGGAGCTGGTGACGT (SEQ ID NO: 91). In some embodiments, the probes of the present disclosure may comprise 8 or more nucleotides (e.g., adenine or thymine) at their 5' end. Suitable probes for detecting DNA mutations in the BRAF gene may comprise the sequences TCTAGCTACAGAGAAAT (SEQ ID NO: 11), GTCTAGCTACAGAAAAAT (SEQ ID NO: 12), TACAGAGAAATCTCGAT (SEQ ID NO: 196), TACAGAGAAATCTC (SEQ ID NO: 197), CTAGCTACAGAGAAAT (SEQ ID NO: 198), CTAGCTACAGAGAAA (SEQ ID NO: 199), TCTAGCTACAGAG (SEQ ID NO: 200), TTTTTTAATTTCTAGCTACAGAGAAAT (SEQ ID NO: 51), TTTTTTTTTATACAGAGAAATCTCGAT (SEQ ID NO: 52).NO: 92), TTTTTTTTTTAATTTACAGAGAAATCTC (SEQ ID NO: 93), TTTTTTAATTACTAGCTACAGAGAAAT (SEQ ID NO: 94), TTTTTTTTAATTACTAGCTACAGAGAAA (SEQ ID NO: 95), TTTTTTTTTTTAATTTCTAGCTACAGAG (SEQ ID NO: 96), TTTTTTTATGTCTAGCTACAGAAAAAT (SEQ ID NO: 52), TTTATGTCTAGCTACAGAAAAATC (SEQ ID NO: 97), TTTTTTTTATTTTTAGCTACAGAAAAA (SEQ ID NO: 98), TTTTTTTATTTCTAGCTACAGAAAAAT (SEQ ID NO: 99) and / or TTTTTTTTTATTCTAGCTACAGAAAAATC (SEQ ID NO: 100). Suitable probes for detecting DNA mutations in the CTNNB1 gene may comprise the sequences AGGAGCTGTGGCAG (SEQ ID NO: 16), GGAGCTGTGATA (SEQ ID NO: 17), TTTACCACTCAGAAAAG (SEQ ID NO: 21), TACCACTCAGAGGAG (SEQ ID NO: 22), TTTTTTTTTTTTAGGAGCTGTGGCAG (SEQ ID NO: 53), TTTTTTTTTTTTAGGAGCTGTGGCAGTG (SEQ ID NO: 101), TTTTTTTTTTTTAGCTGTGGCAGTGGC (SEQ ID NO: 102), TTTTTTTTTTTTGCTGTGGCAGTGGCA (SEQ ID NO: 103), TTTTTTTTTTAAGGAGCTGTGGCAG (SEQ ID NO: 104), TTTTTTTTTTTTTTGGAGCTGTGATA (SEQ ID NO: 54), TTTTTTTTGGAGCTGTGATAGTGG (SEQ ID NO: 105), TTTTTTTTTTGAGCTGTGATAGTGGC (SEQ ID NO: 106), TTTTTTTTTTAGCTGTGATAGTGGCA (SEQ ID NO: 107), TTTTTTTTTTAGAAGGAGCTGTGATA (SEQ ID NO: 108), TTTTTTTTTTTTTTTGGAGCTGTGAT (SEQ IDNO:109), TTTTTTTTTTACCACTCAGAAAAG (SEQ ID NO:55), TTTAATTTTACTCAGAAAAGGAGCT (SEQ ID NO:110), TTTTTTTAATACCACTCAGAAAAGGA (SEQ ID NO:111), TTTTTTTTACCACTCAGAAAAGGAG (SEQ ID NO:112), TTTTTTTTATTACCACTCAGAAAAG (SEQ ID NO:113), TTTTTTTTTCAGAAAAGGAGCTGTG (SEQ ID NO:114), TTTTTTTTTAATACCACTCAGAGGAG (SEQ ID NO:56), TTTTTTTTAAAACTCAGAGGAGGAGC (SEQ ID NO:115), TTTTTTTTTTATTACCACTCAGAGGA (SEQ ID NO:NO:120),TTTTTTTTTTTGAAATAAAGATTGGAA(SEQID NO:121),TTTTTTTTTTTTTAGAAATAAAAGATTG(SEQ ID NO:122),TTTTTTTTTTTTTGAAATAAATGAATGG(SEQ ID NO:123),TTTTTTTTTTTTTCAGAAATAAAAGATT(SEQID NO: 124), TTTTTTTTTTTGGGTGTCTAAG (SEQ ID NO: 59), TTTTTTTTTATTTGGGTGTCTAAG (SEQ ID NO: 125), TTTTTTGGGTGTCTAAGCACCACT (SEQ ID NO: 126), TTTTTTTTTCTAAGCACCACTTTT (SEQ ID NO: 127), TTTTTTTTTTTTTTGGGTGTCTAA (SEQ ID NO: 124) NO:128),TTTTTTTTTGGTGTCTAAGCACCA(SEQ ID NO:129),TTTTTTTTTACAAACCAAGTGAGAA(SEQ NO: 139), TTTTTTTTTAAGTGAGAAGTACCTAA (SEQ ID NO: 140), TTTTTTTTTTTCAAACCAAGTGAG (SEQ ID NO: 141), TTTTTTTTTTACCAAGTGAGAAGTA (SEQ ID NO: 142), TTTTTTTTAGCTCAAACCAAGTGAG (SEQ ID NO: 143), TTTTTTTTTTAGAGGCAGAAAAAAACT (SEQ ID NO: 61), TTTTTTTTTTGCAGAAAAAAACTATTG (SEQ ID NO: 144), TTTTTTTTTTTCAGAAAAAAACTATTGATT (SEQ ID NO: 145), TTTTTTTTTTTGAGGCAGAAAAAAACT (SEQ ID NO: 146), and / or TTTTTTTTTTTGAGGCAGAAAAAAACTA (SEQ ID NO: 147) (probes comprising these sequences that do not contain a 5' adenine and / or thymidine are also contemplated). In some embodiments, each probe is coupled to a microcarrier of the disclosure that has a unique identifier.

[0216] The kits or articles of manufacture of the present disclosure suitable for detecting mutations in the KRAS gene (e.g., KRAS mutations encoding KRAS proteins with G12D, G12V, G12S, and G13D mutations) may optionally include: four probes of the present disclosure specific for one or more mutations in the KRAS gene, wherein each of the four probes is coupled to a microcarrier with a different identifier; a primer pair comprising the sequences GTACTGGTGGAGTATTTGATAGTG (SEQ ID NO: 1) and ATCGTCAAGGCACTCTTGCCTAC (SEQ ID NO: 2); and / or a blocking nucleic acid comprising the sequence: TACGCCACCAGCT(invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO:3); TTGGAGCTGGTGGCGTAinvdTinvdTinvdTinvdT (SEQ ID NO:142); GCTGGTGGCGTAGGCAinvdTinvdTinvdT (SEQ ID NO:143); GCTGGTGGCGTAGGCinvdTinvdTinvdT (SEQ ID NO:144) or TTGGAGCTGGTGGCGTinvdTinvdTinvdT (SEQ ID NO:145), wherein italicized nucleic acids represent locked nucleic acids. In some embodiments, the kit comprises: four probes each comprising the following sequences: GGAGCTGATGG (SEQ ID NO:4), GGAGCTGTTGG (SEQ ID NO:5), TGGAGCTAGTGG (SEQ ID NO:6), and TGGAGCTGGTGACGT (SEQ ID NO:7); four probes each comprising the following probes: a first probe comprising a sequence selected from the group consisting of: GGAGCTGATGG (SEQ ID NO:4), AGCTGATGGCGTA (SEQ ID NO:178), TGGAGCTGATGGCG (SEQ ID NO:179), TGGAGCTGATGG (SEQ ID NO:180), or GCTGATGGCGTA (SEQ ID NO:181); a second probe comprising a sequence selected from the group consisting of: GGAGCTGTTGG (SEQ ID NO:5), TGGAGCTGTTGGTGGC (SEQ ID NO:182), GGAGCTGTTGGTG (SEQ ID NO:183), TGGAGCTGTTGGT (SEQ ID NO:184), or GCTGATGGCGTA (SEQ ID NO:185). NO: 184) or TGGAGCTGTaGGTGG (SEQ ID NO: 185); a third probe comprising a sequence selected from the group consisting of TTGGAGCTAGTGGCGTA (SEQ ID NO: 186), GCTAGTGGCGTAGGC (SEQ ID NO: 187), AGCTAGTGGCGT (SEQ ID NO: 188), GTTGGAGCTAGTGG (SEQ ID NO: 189), or GGAGCTAGTGG (SEQ ID NO: 190);and a fourth probe comprising a sequence selected from the group consisting of GGTGACGTAGGCAA (SEQ ID NO: 191), TGACGTAGGCAAGAG (SEQ ID NO: 192), GCTGGTGACGTAGG (SEQ ID NO: 193), AGCTGGTGACGTAG (SEQ ID NO: 194), or GGAGCTGGTGACGT (SEQ ID NO: 195); or four probes each comprising the following probes: a first probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTAAGGAGCTGATGG (SEQ ID NO: 47), TTTTTTTTTTTTAGCTGATGGCGTA (SEQ ID NO: 74), TTTTTTTTTTTATGGAGCTGATGGCG (SEQ ID NO: 75), TTTTTTTTTTTTATGGAGCTGATGG (SEQ ID NO: 76), and TTTTTTTTTTTTTGCTGATGGCGTA (SEQ ID NO: 77). ID NO:77); a second probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTAAGGAGCTGTTGG (SEQ ID NO:48), TTTTTTTTATGGAGCTGTTGGTGGC (SEQ ID NO:78), TTTTTTTTTTAAGGAGCTGTTGGTG (SEQ ID NO:79), TTTTTTTTTTTATGGAGCTGTTGGT (SEQ ID NO:80), and TTTTTTTTTATGGAGCTGTAGGTGG (SEQ ID NO:81); a third probe comprising a sequence selected from the group consisting of TTTTTTTTTTTATGGAGCTAGTGG (SEQ ID NO:49), TTTTTTTTTTGGAGCTAGTGGCGTA (SEQ ID NO:82), TTTTTAATTTGCTAGTGGCGTAGGC (SEQ ID NO:83), TTTTTTTTTATTTAGCTAGTGGCGT (SEQ ID NO:84), TTTTTTTTTTTGTTGGAGCTAGTGG (SEQ ID NO:85). NO:85) and TTTTTTTTTTTTAAGGAGCTAGTGG (SEQ ID NO:86);and TTTTTTTTTATGGAGCTGGTGACGT (SEQ ID NO: 50) (probes comprising these sequences that do not contain 5' adenine and / or thymine are also contemplated). The kit may optionally include any of the elements described below for detecting mutations in the BRAF, CTNNB1, and / or APC genes. In some embodiments, each probe is coupled to a microcarrier of the present disclosure having a unique identifier. ;

[0217] A kit or article of manufacture of the present disclosure suitable for detecting mutations in the BRAF gene (e.g., two or more BRAF mutations encoding a BRAF protein with a V600E mutation) may optionally include: two probes of the present disclosure specific for one or more mutations in the BRAF gene, wherein each of the two probes is coupled to a microcarrier having a different identifier; a primer pair comprising the sequences GGACCCACTCCATCGAGATTT (SEQ ID NO: 8) and CAGATATATTTCTTCATGAAGACCTCACAGTAA (SEQ ID NO: 9); and / or a blocking nucleic acid comprising the sequence: GAGATTTCACTGTAGC(invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 10); GAGATTTCACTGTAGCinvdTinvdTinvdT (SEQ ID NO: 146); GAGATTTCACTGTAGC invdTinvdTinvdT (SEQ ID NO: 147); GAGATTTCACTGTAGCinvdTinvdTinvdT (SEQ ID NO: 148) or GAGATTTCACTGTAGCinvdTinvdTinvdT (SEQ ID NO: 149), wherein italicized nucleic acids represent locked nucleic acids. In some embodiments, the kit comprises: two probes comprising the sequence TCTAGCTACAGAGAAAT (SEQ ID NO: 11) and GTCTAGCTACAGAAAAAT (SEQ ID NO: 12), respectively; two probes comprising the sequence TTTTTTAATTTCTAGCTACAGAGAAAT (SEQ ID NO: 51) and TTTTTTTATGTCTAGCTACAGAAAAAT (SEQ ID NO: 52), respectively; (1) a first probe comprising a sequence selected from the group consisting of TACAGAGAAATCTCGAT (SEQ ID NO: 196), TACAGAGAAATCTC (SEQ ID NO: 197), CTAGCTACAGAGAAAT (SEQ ID NO: 198), CTAGCTACAGAGAAA (SEQ ID NO: 199), and TCTAGCTACAGAG (SEQ ID NO: 200); and (2) a second probe comprising a sequence selected from the group consisting of GTCTAGCTACAGAAAAATC (SEQ ID NO: 201), GTCTAGCTACAGAAAAAT (SEQ ID NO: 202). NO: 12), TAGCTACAGAAAAA (SEQ ID NO: 202), TCTAGCTACAGAAAAAT (SEQ ID NO: 203), and TCTAGCTACAGAAAAATC (SEQ ID NO: 204);or (1) a first probe comprising a sequence selected from the group consisting of TTTTTTAATTTCTAGCTACAGAGAAAT (SEQ ID NO:51), TTTTTTTTTATACAGAGAAATCTCGAT (SEQ ID NO:92), TTTTTTTTTAATTTACAGAGAAATCTC (SEQ ID NO:93), TTTTTTAATTACTAGCTACAGAGAAAT (SEQ ID NO:94), TTTTTTTAATTACTAGCTACAGAGAAA (SEQ ID NO:95), and TTTTTTTTTTAATTTCTAGCTACAGAG (SEQ ID NO:96); and (2) a second probe comprising a sequence selected from the group consisting of TTTTTTTATGTCTAGCTACAGAAAAAT (SEQ ID NO:52), TTTTATGTCTAGCTACAGAAAAATC (SEQ ID NO:97), TTTTTTTTATTTTTAGCTACAGAAAAA (SEQ ID NO:98), TTTTTTTATTTCTAGCTACAGAAAAAT (SEQ ID NO:99). NO:99) and TTTTTTTTATTCTAGCTACAGAAAAATC (SEQ ID NO:100) (probes comprising these sequences that do not contain 5' adenine and / or thymine are also contemplated). The kit may optionally include any of the elements described below for detecting mutations in the KRAS gene, and / or any of the elements described below for detecting mutations in the CTNNB1 and / or APC genes. In some embodiments, each probe is coupled to a microcarrier of the present disclosure having a unique identifier. ;

[0218] The kits or articles of manufacture of the present disclosure suitable for detecting mutations in the CTNNB1 gene (e.g., CTNNB1 mutations encoding CTNNB1 proteins with T41A, T41I, S45F, and S45P mutations) may optionally comprise: four probes of the present disclosure specific for one or more mutations in the CTNNB1 gene, wherein each of the four probes is coupled to a microcarrier with a different identifier; a first primer pair comprising the sequence GGAATCCATTCTGGTGCCACT (SEQ ID NO: 13) and AGAAAATCCCTGTTCCCACTCATA (SEQ ID NO: 14), and a second primer pair comprising the sequence GGTGCCACTACCACAGCTCCT (SEQ ID NO: 18) and TCTCAAAACTGCATTCTGACTTTCA (SEQ ID NO: 19); and / or a first blocking nucleic acid comprising the following sequence: GCCACTACCACAGCT(invdT) n, wherein n is 1, 2 or 3 (SEQ ID NO: 15); TGCCACTACCACAGinvdTinvdTinvdTinvdT (SEQ ID NO: 150); CACTACCACAGCTCCinvdTinvdTinvdT (SEQ ID NO: 151); GCCACTACCACAGCTinvdTinvdTinvdT (SEQ ID NO: 152) or GCCACTACCACAGCTinvdTinvdTinvdT (SEQ ID NO: 153), and a second blocking nucleic acid comprising the following sequence: GCTCCTTCTCTGAGTinvdTinvdTinvdT (SEQ ID NO: 20), TCCTTCTCTGAGTGGinvdTinvdTinvdT (SEQ ID NO: 174), GCTCCTTCTCTGAGTinvdTinvdTinvdT (SEQ ID NO: 175), NO: 175), TCCTTCTCTGAGTGGinvdTinvdTinvdT (SEQ ID NO: 176) and GCTCCTTCTCTGAGTinvdTinvdTinvdT (SEQ ID NO: 177), wherein the italicized nucleic acids represent locked nucleic acids. In some embodiments, the kit comprises: four probes comprising the following sequences, respectively: AGGAGCTGTGGCAG (SEQ ID NO: 16), GGAGCTGTGATA (SEQ ID NO: 17), TTTACCACTCAGAAAAG (SEQ ID NO: 21), and TACCACTCAGAGGAG (SEQ ID NO: 22); four probes comprising the following probes: a first probe comprising a sequence selected from the group consisting of AGGAGCTGTGGCAGT (SEQ ID NO: 205), AGGAGCTGTGGCAGTG (SEQ ID NO: 206), GCTGTGGCAGTGGC (SEQ ID NO: 207), GCTGTGGCAGTGGCA (SEQ ID NO: 208), and AAGGAGCTGTGGCAG (SEQ ID NO: 209); a second probe comprising a sequence selected from the group consisting of GGAGCTGTGATAGTGG (SEQ ID NO: 210), GAGCTGTGATAGTGGC (SEQ ID NO: 211), AGCTGTGATAGTGGCA (SEQ ID NO: 212). NO: 212), AGAAGGAGCTGTGATA (SEQ ID NO: 213), and GGAGCTGTGAT (SEQ ID NO: 214);a third probe comprising a sequence selected from the group consisting of ACTCAGAAAAGGAGCT (SEQ ID NO: 215), TACCACTCAGAAAAGGA (SEQ ID NO: 216), TTTACCACTCAGAAAAGGAG (SEQ ID NO: 217), TTACCACTCAGAAAAG (SEQ ID NO: 218), and CAGAAAAGGAGCTGTG (SEQ ID NO: 219); and (4) a fourth probe comprising a sequence selected from the group consisting of ACTCAGAGGAGGAGC (SEQ ID NO: 220), TTACCACTCAGAGGA (SEQ ID NO: 221), TTACCACTCAGAGGAGG (SEQ ID NO: 222), TTAACACTCAGAGGAG (SEQ ID NO: 223), and TTACCAATCAGAGGAGG (SEQ ID NO: 224); or four probes comprising the following probes: a first probe comprising a sequence selected from the group consisting of TTTTTTTTTTTAGGAGCTGTGGCAG (SEQ ID NO: 225). NO: 53), TTTTTTTTTTTTAGGAGCTGTGGCAGTG (SEQ ID NO: 101), TTTTTTTTTTTAGCTGTGGCAGTGGC (SEQ ID NO: 102), TTTTTTTTTTTGCTGTGGCAGTGGCA (SEQ ID NO: 103), and TTTTTTTTTTTAAGGAGCTGTGGCAG (SEQ ID NO: 104); a second probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTTGGAGCTGTGATA (SEQ ID NO: 54), TTTTTTTTTGGAGCTGTGATAGTGG (SEQ ID NO: 105), TTTTTTTTTGAGCTGTGATAGTGGC (SEQ ID NO: 106), TTTTTTTTTAGCTGTGATAGTGGCA (SEQ ID NO: 107), TTTTTTTTTAGAAGGAGCTGTGATA (SEQ ID NO: 108), and TTTTTTTTTTTTTTGGAGCTGTGAT (SEQ ID NO: 109);(SEQ ID NO: 114); and (5) a fourth probe comprising a sequence selected from the group consisting of: TTTTTTTTTTTTACCACTCAGAGGAG (SEQ ID NO: 56), TTTTTTTTTTAAAACTCAGAAGGAGCT (SEQ ID NO: 115), TTTTTTTTTTTAATACCACTCAGAAAAGGA (SEQ ID NO: 116), TTTTTTTTTATTACCACTCAGAAAAGGAG (SEQ ID NO: 117). NO: 117), TTTTTTTTTTATTAACACTCAGAGGAG (SEQ ID NO: 118), and TTTTTTTTTATTACCAATCAGAGGAGG (SEQ ID NO: 119), wherein each of the four probes is coupled to a microcarrier having a different identifier (probes comprising these sequences that do not contain 5' adenine and / or thymine are also contemplated). The kit may optionally include any of the elements described above for detecting mutations in the KRAS and / or BRAF genes and / or any of the elements described above for detecting mutations in the APC gene. In some embodiments, each probe is coupled to a microcarrier of the present disclosure having a unique identifier. ;

[0219] A kit or article of manufacture of the present invention suitable for detecting mutations in the APC gene (e.g., APC mutations encoding APC proteins with Q1367*, R1450*, E1309 frameshift, S1465 frameshift, and T1556 frameout mutations) may optionally include: five probes of the present invention specific for one or more mutations in the APC gene, wherein each of the five probes is coupled to a microcarrier having a different identifier; a first primer pair comprising the sequence TAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24); a second primer pair comprising the sequence TAGGATGTAATCAGACGACACAGGA (SEQ ID NO: 27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO: 28); a second primer pair comprising the sequence TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO: 31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO: 32); NO: 32); a third primer pair comprising the sequence GCAGAAGTAAAACACCTCCACCA (SEQ ID NO: 35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO: 36), wherein the italicized nucleic acids represent locked nucleic acids; and a fifth primer pair comprising the sequence CAGGAAAATGACAATGGGAATG (SEQ ID NO: 39) and ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO: 40); and / or a first blocking nucleic acid comprising the following sequences: CCACTCTCTCTCTTTTCAGCinvdTinvdTinvdT (SEQ ID NO: 25), TAGGTCCACTCTCTCTCTTTTCAGCAinvdTinvdTinvdT (SEQ ID NO: 166), TAGGTCCACTCTCTCTCTTTTCAGCAinvdTinvdTinvdT (SEQ ID NO: 167), CCACTCTCTCTCTTTTCAGC invdTinvdTinvdT (SEQ ID NO: 168) or TAGGTCCACTCTTCCTCTTTTCAGCA invdTinvdTinvdT (SEQ ID NO: 169);a second blocking nucleic acid comprising the following sequence: CTTTTCTTTTATTTCTGCinvdTinvdTinvdT (SEQ ID NO: 29), CTTTTCTTTTATTTCTGCinvdTinvdTinvdT (SEQ ID NO: 154), CTTTTCTTTTATTTCTGCinvdTinvdTinvdT (SEQ ID NO: 155), CTTTTCTTTTATTTCTGCinvdTinvdTinvdT (SEQ ID NO: 156), or CTTTTCTTTTATTTCTGCinvdTinvdTinvdT (SEQ ID NO: 157); a third blocking nucleic acid comprising the following sequence: GTGCTCAGACACCinvdTinvdTinvdT (SEQ ID NO: 33), GTGCTCAGACACCinvdTinvdTinvdT (SEQ ID NO: 158), AGTGGTGCTCAGACACCCAinvdTinvdTinvdT (SEQ ID NO: 159). NO:159), AGTGGTGCTCAGACACCCAinvdTinvdTinvdT (SEQ ID NO:160) or AGTGGTGCTCAGACACCCAinvdTinvdTinvdT (SEQ ID NO:161);a fourth blocking nucleic acid comprising the following sequence: CTTCTCGCTTGGTTinvdTinvdTinvdTinvdT (SEQ ID NO: 37), GTACTTCTCGCTTGGTinvdTinvdTinvdT (SEQ ID NO: 162), CTTCTCGCTTGGTTinvdTinvdTinvdT (SEQ ID NO: 163), GTACTTCTCGCTTGGTinvdTinvdTinvdT (SEQ ID NO: 164), or GTACTTCTCGCTTGGTinvdTinvdTinvdT (SEQ ID NO: 165); and a fifth blocking nucleic acid comprising the following sequence: CAATAGTTTTTTCTGCCinvdTinvdTinvdT (SEQ ID NO: 41), GAATCAATAGTTTTTTCTGCCTC invdTinvdTinvdT (SEQ ID NO: 42). NO: 170), TCAGAATCAATAGTTTTTTCTGinvdTinvdTinvdTinvdT (SEQ ID NO: 171), GAATCAATAGATTTTACTGCCTCinvdTinvdTinvdT (SEQ ID NO: 172) or AATCAATAGTTTTTCTGCCTCinvdTinvdTinvdT (SEQ ID NO: 173), wherein the italicized nucleic acids represent locked nucleic acids. In some embodiments, the kit comprises: five probes each comprising the following sequences: ACTGCTGAAAAGAGAGAGT (SEQ ID NO: 26), GAAATAAAAGATTGG (SEQ ID NO: 30), TTTTGGGTGTCTAAG (SEQ ID NO: 34), CAAACCAAGTGAGAA (SEQ ID NO: 38), and AGAGGCAGAAAAAAACT (SEQ ID NO: 42); five probes each comprising the following probes: (1) a first probe comprising a sequence selected from the group consisting of AAATAGCAGAAATAAAAG (SEQ ID NO: 225), GAAATAAAAGATTGGAA (SEQ ID NO: 226), AGAAATAAAAGATTG (SEQ ID NO: 227), GAAATAAATGAATGG (SEQ ID NO: 228), and CAGAAATAAAAGATT (SEQ ID NO: 229);(2) a second probe comprising a sequence selected from the group consisting of TTTGGGTGTCTAAG (SEQ ID NO: 230), GGGTGTCTAAGCACCACT (SEQ ID NO: 231), CTAAGCACCACTTTT (SEQ ID NO: 232), TTTTGGGTGTCTAA (SEQ ID NO: 233), and GGTGTCTAAGCACCA (SEQ ID NO: 234); (3) a third probe comprising a sequence selected from the group consisting of AAGTGAGAAGTACCTAA (SEQ ID NO: 235), CAAACCAAGTGAGAA (SEQ ID NO: 38), TCAAACCAAGTGAG (SEQ ID NO: 236), ACCAAGTGAGAAGTA (SEQ ID NO: 237), and AGCTCAAACCAAGTGAG (SEQ ID NO: 238); (4) a fourth probe comprising a sequence selected from the group consisting of GCACCTACTGCTGAA (SEQ ID NO: 239), ACCTACTGCTGAAAAG (SEQ ID NO: 240). NO:240), TGCTGAAAAGAGAGAGT (SEQ ID NO:241), ACTGCTGAAAAGAGAGAGT (SEQ ID NO:26), and CCTACTGCTGAAAAGAGA (SEQ ID NO:242); and (5) a fifth probe comprising a sequence selected from the group consisting of GCAGAAAAAAACTATTG (SEQ ID NO:243), AGAGGCAGAAAAAAACT (SEQ ID NO:42), CAGAAAAAAACTATTGATT (SEQ ID NO:244), AGAAAGAGGCAGAAAAAAACT (SEQ ID NO:245), and GAGGCAGAAAAAAACTA (SEQ ID NO:246);or five probes each comprising the following probes: (1) a first probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTTGAAATAAAAGATTGG (SEQ ID NO:58), TTTTTTTTTTAAATAGCAGAAATAAAAG (SEQ ID NO:120), TTTTTTTTTTTGAAATAAAAGATTGGAA (SEQ ID NO:121), TTTTTTTTTTTTTAGAAATAAAAGATTG (SEQ ID NO:122), TTTTTTTTTTTTTGAAATAAATGAATGG (SEQ ID NO:123), and TTTTTTTTTTTTTCAGAAATAAAAGATT (SEQ ID NO:124); (2) a second probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTTGGGTGTCTAAG (SEQ ID NO:59), TTTTTTTTTATTTGGGTGTCTAAG (SEQ ID NO:125), TTTTTTGGGTGTCTAAGCACCACT (SEQ ID NO:126). NO: 126), TTTTTTTTTCTAAGCACCACTTTT...

Claims

1. Preparation of reagents for detection and Use of a kit in a method for detecting the presence of a DNA mutation in a gene, wherein the reagent comprises and The method comprises: (a) isolating DNA from a sample; (b) using the method for treating a human and amplifying the isolated DNA from (a) by polymerase chain reaction (PCR), wherein each of the at least four blocking nucleic acids is amplified by a primer pair specific for the locus of one or more DNA mutations in each of the genes, wherein each of the at least four blocking nucleic acids is amplified by a primer pair specific for the locus of one or more DNA mutations in each of the genes or wherein each of the at least four blocking nucleic acids comprises a nucleic acid sequence that is specific for the wild-type DNA locus of one of the DNA mutations in the gene and prevents amplification of the wild-type DNA locus; wherein each of the at least four blocking nucleic acids comprises a nucleic acid sequence that is specific for the wild-type DNA locus of the gene corresponding to the or a single-stranded oligonucleotide that hybridizes to the wild-type DNA locus of one of the DNA mutations in the gene, a 3' terminal portion that blocks extension of the single-stranded oligonucleotide, and one or more locked nucleic acids (LNAs); (c) hybridizing the amplified DNA with the at least four probes, wherein the at least four probes include one or more probes specific for the and a probe specific for a DNA mutation in each of the at least four genes, wherein each of the at least four probes is coupled to a microcarrier, and wherein each of the microcarriers comprises an identifier corresponding to the probe coupled thereto; (d) detecting the presence or absence of hybridization of the amplified DNA to the at least four probes, wherein hybridization between the amplified DNA and one of the probes indicates the presence of the DNA mutation corresponding to the probe; (e) detecting an identifier of the microcarrier; and (f) correlating the detected identifier of the microcarrier with the detected presence or absence of hybridization of the amplified DNA to the corresponding probe of the microcarrier; in: (1) The one or more DNA mutations in the gene include one or more DNA mutations encoding a KRAS protein that is a G12D, G12V, G12S, or G13D mutation, wherein step (b) comprises using a primer pair comprising the sequence GTACTGGTGGAGTATTTGATAGTG (SEQ ID NO: 1) and ATCGTCAAGGCACTCTTGCCTAC (SEQ ID NO: 2) and a primer pair comprising The isolated DNA is amplified by PCR in the presence of a blocking nucleic acid of a sequence, wherein the italicized nucleic acid represents a locked nucleic acid, and the at least four probes comprise one or more probes comprising a sequence selected from the group consisting of: TTTTTTTTTTTTAAGGAGCTGATGG (SEQ ID NO: 47), TTTTTTTTTTTTAAGGAGCTGTTGG (SEQ ID NO: 48), TTTTTTTTTTTATGGAGCTAGTGG (SEQ ID NO: 49), TTTTTTTTTTTTAAGGAGCTAGTGG (SEQ ID NO:86), TTTTTTTTTATGGAGCTGGTGACGT (SEQ ID NO:50) and TTTTTTTTAAGGAGCTGGTGACGT (SEQ ID NO:91); (2) One or more DNA mutations in a gene including one or more genes encoding a BRAF protein with a V600E mutation gene, wherein step (b) comprises using a primer pair comprising the sequences GGACCCACTCCATCGAGATTT (SEQ ID NO: 8) and CAGATATATTTCTTCATGAAGACCTCACAGTAA (SEQ ID NO: 9) and (SEQ ID NO: 10), wherein the italicized nucleic acid represents a locked nucleic acid, amplifying the isolated DNA by PCR, and the at least four probes comprise one or more probes comprising a sequence selected from the group consisting of: TTTTTTAATTTCTAGCTACAGAGAAAT(SEQ ID NO:51)、 TTTTTTTAATTACTAGCTACAGAGAAA(SEQ ID NO:95)、 TTTTTTTATGTCTAGCTACAGAAAAAT (SEQ ID NO: 52) and TTTTTTTATTTCTAGCTACAGAAAAAT(SEQ ID NO:99); (3) One or more DNA mutations in the gene encoding the CTNNB1 protein including one or more of the T41A, T41I, S45F, and S45P mutations mutation, wherein step (b) comprises using a primer pair comprising the sequence GGAATCCATTCTGGTGCCACT (SEQ ID NO: 13) and AGAAAATCCCTGTTCCCACTCATA (SEQ ID NO: 14) and in the presence of (SEQ ID NO: 15), wherein the italicized nucleic acid represents a locked nucleic acid, or using a primer pair comprising the sequences GGTGCCACTACCACAGCTCCT (SEQ ID NO: 18) and TCTCAAAACTGCATTCTGACTTTCA (SEQ ID NO: 19) and in the presence of a blocking nucleic acid comprising (SEQ ID NO: 20) The isolated DNA is amplified by PCR in the presence of PCR products, and the at least four probes comprise one or more probes comprising a sequence selected from the group consisting of: TTTTTTTTTTAGGAGCTGTGGCAG (SEQ ID NO: 53), TTTTTTTTTTAAGGAGCTGTGGCAG (SEQ ID NO: 104), TTTTTTTTTTTTTGGAGCTGTGATA (SEQ ID NO: 54), TTTTTTTTTTTTTTGGAGCTGTGAT (SEQ ID NO: 109), TTTTTTTTTTTACCACTCAGAAAG(SEQ ID NO:55)、 TTTTTTTTATTACCACTCAGAAAG(SEQ ID NO:113) TTTTTTTTTAATACCACTCAGAGGAG (SEQ ID NO:56) and TTTTTTTTTATTACCAATCAGAGGAGG (SEQ ID NO: 119); and (3) One or more DNA mutations in the gene encoding the APC protein include one or more of Q1367*, R1450*, E1309 frameshift, S1465 frameshift, and T1556 frameshift mutations mutation, wherein step (b) comprises using a primer pair comprising the sequence TAAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24) and (SEQ ID NO: 25), wherein the italicized nucleic acid represents a locked nucleic acid, or using a primer pair comprising the sequences TAGGATGTAATCAGACGACACAGGA (SEQ ID NO: 27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO: 28) and in the presence of a blocking nucleic acid of the sequence (SEQ ID NO: 29), wherein the italicized nucleic acid represents the locked nucleic acid, or using a primer pair comprising the sequences TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO: 31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO: 32) and in the presence of a blocking nucleic acid of the sequence (SEQ ID NO: 33), wherein the italicized nucleic acid represents a locked nucleic acid, or using a primer pair comprising the sequences GCAGAAGTAAAACACCTCCACCA (SEQ ID NO: 35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO: 36) and in the presence of a blocking nucleic acid comprising (SEQ ID NO: 37), wherein the italicized nucleic acid represents a locked nucleic acid, or using a primer pair comprising the sequence CAGGAAAATGACAATGGGAATG (SEQ ID NO: 39) and ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO: 40) and in the presence of a blocking nucleic acid comprising (SEQ ID NO: 41), wherein the italicized nucleic acid represents a locked nucleic acid, amplifying the isolated DNA by PCR, and the at least four probes comprise one or more probes comprising a sequence selected from the group consisting of: TTTTTTTTTTTGAAAATAAAAGATTGG (SEQ ID NO: 58), TTTTTTTTTTTTAGAAATAAAAGATTG (SEQ ID NO: 122), TTTTTTTTTTTTTGGGTGTCTAAG (SEQ ID NO: 59), TTTTTTGGGTGTCTAAGCACCACT(SEQ ID NO:126)、 TTTTTTTTTACAAACCAAGTGAGAA (SEQ ID NO: 60), TTTTTTTTACTGCTGAAAAGAGAGAGT (SEQ ID NO:57) and TTTTTTTTTTAGAGGCAGAAAAAAACT (SEQ ID NO: 61).

2. The method according to claim 1, wherein the 3' terminal portion comprises one or more inverted deoxythymidines.

3. The method of claim 1, wherein the One or more DNA mutations in the gene include at least one encoding a T41A or T41I mutation Protein first mutation.

4. The method of claim 1, wherein the One or more DNA mutations in the gene include at least the first CTNNB1 protein encoding the S45F or S45P mutation. mutation.

5. The method of claim 1, wherein the One or more DNA mutations in the gene include at least the first APC protein encoding the Q1367* mutation mutation.

6. The method of claim 1, wherein the One or more DNA mutations in the gene include at least the first APC protein encoding the R1450* mutation. mutation.

7. The method of claim 1, wherein the One or more DNA mutations in the gene include at least the first APC protein encoding the E1309 frameshift mutation mutation.

8. The method of claim 1, wherein the One or more DNA mutations in the gene include at least the first APC protein encoding the S1465 frameshift mutation mutation.

9. The method of claim 1, wherein the One or more DNA mutations in the gene include at least the first APC protein encoding the T1556 frameshift mutation mutation.

10. The use of claim 1, wherein each of the primer pairs comprises a primer coupled to a detection agent.

11. The use of claim 10, wherein the detection reagent comprises a fluorescent detection reagent, and wherein detecting the presence or absence of hybridization of the amplified DNA to the at least four probes in step (d) comprises fluorescent imaging of the fluorescent detection reagent.

12. The method of claim 10, wherein the detection reagent comprises biotin, and wherein in step (d), detecting the presence or absence of hybridization of the amplified DNA to the at least four probes comprises: (1) after hybridization in step (c), contacting the microcarrier with streptavidin conjugated to a signal emitting entity; and (2) Detecting a signal from the signal emitting entity associated with the microcarrier.

13. The use according to claim 12, wherein the signal emitting entity comprises phycoerythrin.

14. The use of claim 1, wherein detecting the identifier of the microcarrier in step (e) comprises bright field imaging of the identifier.

15. The method according to claim 1, wherein said One or more DNA mutations in the gene encoding the KRAS protein including G12D, G12V, G12S, and G13D mutations mutation.

16. The method according to claim 15, wherein the probe comprises: (1) a first probe comprising the sequence TTTTTTTTTTTTTAAGGAGCTGATGG (SEQ ID NO: 47); (2) a second probe comprising the sequence TTTTTTTTTTTTTAAGGAGCTGTTGG (SEQ ID NO: 48); (3) a third probe comprising a sequence selected from the group consisting of TTTTTTTTTTTATGGAGCTAGTGG (SEQ ID NO: 49) and TTTTTTTTTTTTAAGGAGCTAGTGG (SEQ ID NO: 86); and (4) a fourth probe comprising a sequence selected from the group consisting of TTTTTTTTTATGGAGCTGGTGACGT (SEQ ID NO: 50) and TTTTTTTTTAAGGAGCTGGTGACGT (SEQ ID NO: 91); and wherein each of the four probes is coupled to a microcarrier having a different identifier.

17. The method of claim 1, wherein the One or more DNA mutations in the gene including two or more BRAF proteins encoding the V600E mutation mutation.

18. The method according to claim 17, wherein the probe comprises: (1) a first probe comprising a sequence selected from the group consisting of TTTTTTAATTTCTAGCTACAGAGAAAT (SEQ ID NO: 51) and TTTTTTTTAATTACTAGCTACAGAGAAA (SEQ ID NO: 95); and (2) a second probe comprising a sequence selected from the group consisting of: TTTTTTTATGTCTAGCTACAGAAAAAT (SEQ ID NO: 52) and TTTTTTTATTTCTAGCTACAGAAAAAT (SEQ ID NO: 99); and wherein each of the two probes is coupled to a microcarrier having a different identifier.

19. The method of claim 1, wherein said One or more DNA mutations in the gene encoding the CTNNB1 protein including T41A, T41I, S45F and S45P mutations mutation.

20. The use according to claim 19, wherein the probe comprises: (1) a first probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTAGGAGCTGTGGCAG (SEQ ID NO: 53) and TTTTTTTTTTAAGGAGCTGTGGCAG (SEQ ID NO: 104); (2) a second probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTTGGAGCTGTGATA (SEQ ID NO: 54) and TTTTTTTTTTTTTTGGAGCTGTGAT (SEQ ID NO: 109); (3) a third probe comprising a sequence selected from the group consisting of TTTTTTTTTTTACCACTCAGAAAAG (SEQ ID NO: 55) and TTTTTTTTATTACCACTCAGAAAAG (SEQ ID NO: 113); and (4) A fourth probe comprising a sequence selected from the group consisting of TTTTTTTTTAATACCACTCAGAGGAG (SEQ ID NO: 56) and TTTTTTTTTATTACCAATCAGAGGAGG (SEQ ID NO: 119), and wherein each of the four probes is coupled to a microcarrier having a different identifier.

21. The use of claim 19, wherein step (b) comprises amplifying the isolated DNA by PCR using a first primer pair comprising the sequence GGAATCCATTCTGGTGCCACT (SEQ ID NO: 13) and AGAAAATCCCTGTTCCCACTCATA (SEQ ID NO: 14) and a second primer pair comprising the sequence GGTGCCACTACCACAGCTCCT (SEQ ID NO: 18) and TCTCAAAACTGCATTCTGACTTTCA (SEQ ID NO: 19).

22. The method of claim 19, wherein step (b) comprises amplifying the isolated DNA by PCR in the presence of a blocking nucleic acid comprising the sequence (SEQ ID NO: 15), and a first blocking nucleic acid comprising the sequence (SEQ ID NO: 20), wherein the italicized nucleic acid represents a locked nucleic acid.

23. The method of claim 1, wherein said One or more DNA mutations in the gene encoding the APC protein include Q1367*, R1450*, E1309 frameshift, S1465 frameshift, and T1556 frameshift mutations. mutation.

24. The method of claim 23, wherein the probe comprises: (1) a first probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTTGAAATAAAAGATTGG (SEQ ID NO: 58) and TTTTTTTTTTTTTTTAGAAATAAAAGATTG (SEQ ID NO: 122); (2) a second probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTTGGGTGTCTAAG (SEQ ID NO: 59) and TTTTTTTGGGTGTCTAAGCACCACT (SEQ ID NO: 126); (3) a third probe comprising the sequence TTTTTTTTTTACAAACCAAGTGAGAA (SEQ ID NO: 60); (4) a fourth probe comprising the sequence TTTTTTTTACTGCTGAAAAGAGAGAGT (SEQ ID NO: 57); and (5) a fifth probe comprising the sequence TTTTTTTTTTAGAGGCAGAAAAAAACT (SEQ ID NO: 61); and wherein each of the five probes is coupled to a microcarrier having a different identifier.

25. The method of claim 23, wherein step (b) comprises amplifying the isolated DNA by PCR using the following primer pairs: a first primer pair comprising the sequence TAAAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24); a second primer pair comprising the sequence TAGGATGTAATCAGACGACACAGGA (SEQ ID NO: 27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO: 28); a third primer pair comprising the sequence TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO: 31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO: 32); a third primer pair comprising the sequence GCAGAAGTAAAACACCTCCACCA (SEQ ID NO: 35) and (SEQ ID NO: 36), wherein the italicized nucleic acid represents the locked nucleic acid; and a fifth primer pair comprising the sequence CAGGAAAATGACAATGGGAATG (SEQ ID NO: 39) and ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO: 40).

26. The method of claim 23, wherein step (b) comprises amplifying the isolated DNA by PCR in the presence of a blocking nucleic acid comprising the sequence (SEQ ID NO: 25); comprising the sequence (SEQ ID NO: 29); comprising the sequence (SEQ ID NO: 33) a third blocking nucleic acid comprising the sequence (SEQ ID NO: 37); and a fourth blocking nucleic acid comprising the sequence (SEQ ID NO: 41), wherein the italicized nucleic acid represents a locked nucleic acid.

27. The method of claim 1, wherein the method further comprises: amplifying the positive control DNA sequence using a primer pair specific for the positive control DNA sequence; hybridizing the amplified positive control DNA sequence with a probe specific for the positive control DNA sequence, wherein the probe specific for the positive control DNA sequence is coupled to a microcarrier having an identifier corresponding to the positive control; detecting the presence or absence of hybridization of the amplified positive control DNA sequence to the probe specific for the positive control DNA sequence; and detecting an identifier corresponding to the positive control.

28. The use according to claim 27, wherein the positive control DNA sequence comprises the sequence of a human leukocyte antigen gene.

29. The use according to claim 28, wherein the primer pair specific for the positive control DNA sequence comprises the sequences TGAGTGTTACTTCTTCCCACACTC (SEQ ID NO: 43) and ATTGCTTTTGCGCAATCCCT (SEQ ID NO: 44).

30. The use of claim 28, wherein the probe specific for the positive control DNA sequence comprises the sequence TTTTTTTTTTTTGGAGACGGTCTG (SEQ ID NO: 45).

31. The use of claim 27, wherein the positive control DNA sequence comprises the sequence of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene.

32. The use of claim 31, wherein the primer pair specific for the positive control DNA sequence comprises the sequences AATCCCATCACCATCTTCCA (SEQ ID NO: 71) and TGGACTCCACGACGTACTCA (SEQ ID NO: 72).

33. The use of claim 31, wherein the probe specific for the positive control DNA sequence comprises the sequence CTGTCTTCCACTCACTCC (SEQ ID NO: 73).

34. The method of claim 1, wherein the method further comprises: detecting the absence of hybridization of the amplified DNA to a microcarrier having an identifier corresponding to a negative control, wherein the microcarrier having an identifier corresponding to the negative control comprises a probe that does not hybridize to the amplified DNA; and detecting the identifier corresponding to the negative control.

35. The use of claim 34, wherein the microcarrier having an identifier corresponding to the negative control comprises a probe comprising the sequence AATATATATATATTAT (SEQ ID NO: 46).

36. The use of claim 1, wherein the identifier of the microcarrier comprises a digital barcode.

37. The method of claim 36, wherein each of the microcarriers comprises: (i) a first photopolymer layer; (ii) a second photopolymer layer; and (iii) an intermediate layer between the first layer and the second layer, the intermediate layer having a coding pattern representing an identifier defined thereon, wherein the intermediate layer is partially light-transmissive and partially light-opaque, representing a code corresponding to the microcarrier, wherein the outermost surface of the microcarrier comprises a photoresist photopolymer, and the photoresist photopolymer is functionalized with the probe specific for the DNA mutation, and wherein the microcarrier has the same density as water.

38. The use of claim 1, wherein the identifier of the microcarrier comprises an analog code.

39. The method of claim 38, wherein each of the microcarriers comprises: (i) a transparent polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other; (ii) an opaque polymer layer, wherein the opaque polymer layer is secured to the first surface of the transparent polymer layer and surrounds a central portion of the transparent polymer layer, and wherein the opaque polymer layer comprises a two-dimensional shape representing an analog code, wherein the analog code represents an identifier; and (iii) the probe specific for the DNA mutation, wherein the probe is coupled to at least one of the first surface and the second surface of the transparent polymer layer in at least a central portion of the transparent polymer layer.

40. The method of claim 39, wherein each of the microcarriers further comprises: (iv) a second transparent polymer layer aligned with the first transparent polymer layer, the second transparent polymer layer having a central portion aligned with the central portion of the first transparent polymer layer, wherein the second transparent polymer layer is secured to the second surface of the first transparent polymer layer and does not extend beyond the two-dimensional shape of the first transparent polymer layer; and (v) an opaque magnetic layer surrounding a central portion of the first transparent polymer layer between the opaque polymer layer and a central portion of the transparent polymer layer, wherein the opaque magnetic layer is secured between the first transparent polymer layer and the second transparent polymer layer.

41. The use of claim 39, wherein each of the microcarriers further comprises an orientation indicator for orienting the simulated code of the opaque polymer layer.

42. The use of claim 39, wherein the two-dimensional shape of the opaque polymer layer comprises a gear shape comprising a plurality of gear teeth, and wherein the simulation code is represented by one or more aspects selected from the group consisting of: a height of one or more gear teeth in the plurality of gear teeth, a width of one or more gear teeth in the plurality of gear teeth, a number of gear teeth in the plurality of gear teeth, and an arrangement of one or more gear teeth within the plurality of gear teeth.

43. The method of claim 39, wherein each of the microcarriers further comprises: (vi) one or more pillars protruding from the first surface of the first transparent polymeric layer, wherein the one or more pillars are not within a central portion of the first transparent polymeric layer; and / or (vii) one or more pillars protruding from the second surface of the first transparent polymer layer or a surface of the second transparent polymer layer that is not fixed to the first transparent polymer layer, wherein the one or more pillars are not within a central portion of the first transparent polymer layer or the second transparent polymer layer.

44. The use of claim 39, wherein the transparent polymer of the first transparent polymer layer or the second transparent polymer layer comprises an epoxy-based polymer.

45. The use according to claim 44, wherein the epoxy polymer is SU-8.

46. ​​The use of claim 1, wherein the sample is a stool sample.

47. The use of claim 1, wherein the method is used to detect colon cancer, rectal cancer, colorectal cancer, colon adenoma, rectal adenoma or colorectal adenoma.

48. A kit comprising at least four microcarriers, wherein each of the at least four microcarriers comprises: (i) a probe coupled to the microcarrier, wherein the probe is sensitive to human or DNA mutations in genes are specific; and (ii) an identifier corresponding to the probe to which it is coupled; The kit comprises at least one A microcarrier comprising a probe specific for a DNA mutation in a gene, at least one probe comprising a probe specific for the A microcarrier comprising a probe specific for a DNA mutation in a gene, at least one probe comprising a probe specific for the A microcarrier comprising a probe specific for a DNA mutation in a gene and at least one probe comprising a probe specific for the A microcarrier having a probe specific for a DNA mutation in a gene, the A DNA mutation in a gene encoding a G12D, G12V, G12S, or G13D mutant KRAS protein, wherein DNA mutations in the gene encoding the V600E mutated BRAF protein, the The DNA mutation in the gene encodes a CTNNB1 protein with T41A, T41I, S45F or S45P mutation, wherein DNA mutations in the gene encoding the APC protein with Q1367*, R1450*, E1309 frameshift, S1465 frameshift, and T1556 frameshift mutations; and The kit further comprises at least four blocking nucleic acids, wherein each of the at least four blocking nucleic acids corresponds to or hybridizes to a wild-type DNA locus of one of the DNA mutations in a gene, wherein each of the at least four blocking nucleic acids comprises a single-stranded oligonucleotide that hybridizes to the corresponding wild-type DNA locus, a 3' terminal portion that blocks extension of the single-stranded oligonucleotide, and one or more locked nucleic acids (LNAs); Which corresponds to the The blocking nucleic acid that hybridizes to the wild-type DNA locus of one of the DNA mutations in the gene comprises the sequence (SEQ ID NO: 3), corresponding to the The blocking nucleic acid that hybridizes to the wild-type DNA locus of one of the DNA mutations in the gene comprises the sequence (SEQ ID NO: 10), corresponding to the The blocking nucleic acid that hybridizes to the wild-type DNA locus of one of the DNA mutations in the gene comprises the sequence (SEQ ID NO: 15) or (SEQ ID NO: 20), and corresponding to the The blocking nucleic acid that hybridizes to the wild-type DNA locus of one of the DNA mutations in the gene comprises a sequence selected from the group consisting of: (SEQ ID NO: 25), (SEQ ID NO: 29), (SEQ ID NO: 33), (SEQ ID NO: 37) and (SEQ ID NO:41), wherein the italicized nucleic acid represents a blocking nucleic acid; and Among them The probe specific for a DNA mutation in a gene comprises a sequence selected from the group consisting of TTTTTTTTTTTTAAGGAGCTGATGG (SEQ ID NO: 47), TTTTTTTTTTTTAAGGAGCTGTTGG (SEQ ID NO: 48), TTTTTTTTTTTATGGAGCTAGTGG (SEQ ID NO: 49), TTTTTTTTTTTTTAAGGAGCTAGTGG (SEQ ID NO: 86), TTTTTTTTTATGGAGCTGGTGACGT (SEQ ID NO: 50), and TTTTTTTTTAAGGAGCTGGTGACGT (SEQ ID NO: 91); The probe specific for a DNA mutation in a gene comprises a sequence selected from the group consisting of TTTTTTAATTTCTAGCTACAGAGAAAT (SEQ ID NO: 51), TTTTTTTAATTACTAGCTACAGAGAAA (SEQ ID NO: 95), TTTTTTTATGTCTAGCTACAGAAAAAT (SEQ ID NO: 52), and TTTTTTTATTTCTAGCTACAGAAAAAT (SEQ ID NO: 99); The probe having specificity for a DNA mutation in a gene comprises a sequence selected from the group consisting of TTTTTTTTTTTAGGAGCTGTGGCAG (SEQ ID NO: 53), TTTTTTTTTTAAGGAGCTGTGGCAG (SEQ ID NO: 104), TTTTTTTTTTTTTGGAGCTGTGATA (SEQ ID NO: 54), TTTTTTTTTTTTTTGGAGCTGTGAT (SEQ ID NO: 109), TTTTTTTTTTTTACCACTCAGAAAAG (SEQ ID NO: 55), TTTTTTTTATTACCACTCAGAAAAG (SEQ ID NO: 113), TTTTTTTTTAATACCACTCAGAGGAG (SEQ ID NO: 56), and TTTTTTTTTATTACCAATCAGAGGAGG (SEQ ID NO: 119); and for human The probe specific for a DNA mutation in the gene comprises a sequence selected from the group consisting of TTTTTTTTTTTTTGAAATAAAAGATTGG (SEQ ID NO:58), TTTTTTTTTTTTTAGAAATAAAAGATTG (SEQ ID NO:122), TTTTTTTTTTTTTGGGTGTCTAAG (SEQ ID NO:59), TTTTTTGGGTGTCTAAGCACCACT (SEQ ID NO:126), TTTTTTTTTTACAAACCAAGTGAGAA (SEQ ID NO:60), TTTTTTTTACTGCTGAAAAGAGAGAGT (SEQ ID NO:57), and TTTTTTTTTTAGAGGCAGAAAAAAACT (SEQ ID NO:61).

49. The kit of claim 48, wherein the DNA mutations in the gene include at least the first CTNNB1 protein encoding the T41A or T41I mutation. mutation.

50. The kit of claim 48, wherein the DNA mutations in the gene include at least the first CTNNB1 protein encoding the S45F or S45P mutation. mutation.

51. The kit of claim 48, wherein the DNA mutations in the gene encoding at least the first APC protein with the Q1367* mutation mutation.

52. The kit of claim 48, wherein the DNA mutations in the gene include at least the first APC protein encoding the R1450* mutation. mutation.

53. The kit of claim 48, wherein the DNA mutations in the gene include at least the first APC protein encoding the E1309 frameshift mutation. mutation.

54. The kit of claim 48, wherein the DNA mutations in the gene include at least the first APC protein encoding the S1465 frameshift mutation. mutation.

55. The kit of claim 48, wherein the DNA mutations in the gene include at least the first APC protein encoding the T1556 frameshift mutation. mutation.

56. The kit of claim 48, further comprising at least four primer pairs, wherein the kit comprises at least four primer pairs. and Each of the genes has a specific primer pair for the locus of one or more DNA mutations.

57. The kit of claim 56, wherein each of the at least four primer pairs comprises a primer coupled to a detection reagent.

58. The kit of claim 57, wherein the detection reagent comprises a fluorescent detection reagent.

59. The kit of claim 57, wherein the detection reagent comprises biotin, and wherein the kit further comprises streptavidin conjugated to a signaling entity.

60. The kit of claim 59, wherein the signaling entity comprises phycoerythrin.

61. The kit of claim 48, wherein the DNA mutations in the gene encoding the KRAS protein include G12D, G12V, G12S, and G13D mutations. mutation.

62. The kit of claim 61, wherein the kit comprises: (1) a first probe comprising the sequence TTTTTTTTTTTTTAAGGAGCTGATGG (SEQ ID NO: 47); (2) a second probe comprising the sequence TTTTTTTTTT15 TTAAGGAGCTGTTGG (SEQ ID NO: 48); (3) a third probe comprising a sequence selected from the group consisting of TTTTTTTTTTTATGGAGCTAGTGG (SEQ ID NO: 49) and TTTTTTTTTTTTAAGGAGCTAGTGG (SEQ ID NO: 86); and (4) a fourth probe comprising a sequence selected from the group consisting of: TTTTTTTTTATGGAGCTGGTGACGT (SEQ ID NO: 50) and TTTTTTTTTAAGGAGCTGGTGACGT (SEQ ID NO: 91); and wherein each of the four probes is coupled to a microcarrier having a different identifier.

63. The kit of claim 61, further comprising a primer pair comprising the sequences GTACTGGTGGAGTATTTGATAGTG (SEQ ID NO: 1) and ATCGTCAAGGCACTCTTGCCTAC (SEQ ID NO: 2).

64. The kit of claim 48, wherein the DNA mutations in the gene include two or more copies of the BRAF protein encoding the V600E mutation. mutation.

65. The kit of claim 64, wherein the kit comprises: (1) a first probe comprising a sequence selected from the group consisting of TTTTTTAATTTCTAGCTACAGAGAAAT (SEQ ID NO: 51) and TTTTTTTTAATTACTAGCTACAGAGAAA (SEQ ID NO: 95); and (2) a second probe comprising a sequence selected from the group consisting of: TTTTTTTATGTCTAGCTACAGAAAAAT (SEQ ID NO: 52), TTTTATGTCTAGCTACAGAAAAATC (SEQ ID NO: 97), and TTTTTTTATTTCTAGCTACAGAAAAAT (SEQ ID NO: 99); and wherein each of the two probes is coupled to a microcarrier having a different identifier.

66. The kit of claim 64, further comprising a primer pair comprising the sequences GGACCCACTCCATCGAGATTT (SEQ ID NO: 8) and CAGATATATTTCTTCATGAAGACCTCACAGTAA (SEQ ID NO: 9).

67. The kit of claim 48, wherein the DNA mutations in the gene encoding the CTNNB1 protein include T41A, T41I, S45F, and S45P mutations. mutation.

68. The kit of claim 67, wherein the kit comprises: (1) a first probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTAGGAGCTGTGGCAG (SEQ ID NO: 53) and TTTTTTTTTTAAGGAGCTGTGGCAG (SEQ ID NO: 104); (2) a second probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTTGGAGCTGTGATA (SEQ ID NO: 54) and TTTTTTTTTTTTTTGGAGCTGTGAT (SEQ ID NO: 109); (3) a third probe comprising a sequence selected from the group consisting of TTTTTTTTTTTACCACTCAGAAAAG (SEQ ID NO: 55) and TTTTTTTTATTACCACTCAGAAAAG (SEQ ID NO: 113); and (4) A fourth probe comprising a sequence selected from the group consisting of TTTTTTTTTAATACCACTCAGAGGAG (SEQ ID NO: 56) and TTTTTTTTTATTACCAATCAGAGGAGG (SEQ ID NO: 119), and wherein each of the four probes is coupled to a microcarrier having a different identifier.

69. The kit of claim 67, further comprising a first primer pair comprising the sequence GGAATCCATTCTGGTGCCACT (SEQ ID NO: 13) and AGAAAATCCCTGTTCCCACTCATA (SEQ ID NO: 14), and a second primer pair comprising the sequence GGTGCCACTACCACAGCTCCT (SEQ ID NO: 18) and TCTCAAAACTGCATTCTGACTTTCA (SEQ ID NO: 19).

70. The kit of claim 48, wherein the DNA mutations in the gene include APC mutations encoding the APC protein with Q1367*, R1450*, E1309 frameshift, S1465 frameshift, and T1556 frameshift mutations.

71. The kit of any one of claims 70, wherein the kit comprises: (1) a first probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTTGAAATAAAAGATTGG (SEQ ID NO: 58) and TTTTTTTTTTTTTTTAGAAATAAAAGATTG (SEQ ID NO: 122); (2) a second probe comprising a sequence selected from the group consisting of TTTTTTTTTTTTTGGGTGTCTAAG (SEQ ID NO: 59) and TTTTTTTGGGTGTCTAAGCACCACT (SEQ ID NO: 126); (3) a third probe comprising the sequence TTTTTTTTTTACAAACCAAGTGAGAA (SEQ ID NO: 60); (4) a fourth probe comprising the sequence TTTTTTTTACTGCTGAAAAGAGAGAGT (SEQ ID NO: 57); and (5) a fifth probe comprising the sequence TTTTTTTTTTAGAGGCAGAAAAAAACT (SEQ ID NO: 61); and wherein each of the five probes is coupled to a microcarrier having a different identifier.

72. The kit of claim 70, further comprising the following primer pairs: a first primer pair comprising the sequence TAAAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24); a second primer pair comprising the sequence TAGGATGTAATCAGACGACACAGGA (SEQ ID NO: 27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO: 28); a third primer pair comprising the sequence TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO: 31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO: 32); a fourth primer pair comprising the sequence GCAGAAGTAAAACACCTCCACCA (SEQ ID NO: 35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO: 36), wherein italicized nucleic acids represent locked nucleic acids; and a fourth primer pair comprising the sequence CAGGAAAATGACAATGGGAATG (SEQ ID NO: The fifth primer pair is ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO: 39) and ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO: 40).

73. The kit of claim 48, further comprising: a microcarrier having an identifier corresponding to a positive control and coupled to a probe specific for a positive control gene sequence; and a primer pair specific for the positive control DNA sequence.

74. The kit of claim 73, wherein the positive control DNA sequence comprises the sequence of a human leukocyte antigen gene.

75. The kit of claim 74, wherein the primer pair specific for the positive control DNA sequence comprises the sequences TGAGTGTTACTTCTTCCCACACTC (SEQ ID NO: 43) and ATTGCTTTTGCGCAATCCCT (SEQ ID NO: 44).

76. The kit of claim 74, wherein the probe specific for the positive control gene sequence comprises the sequence TTTTTTTTTTTTGGAGACGGTCTG (SEQ ID NO: 45).

77. The kit of claim 73, wherein the positive control DNA sequence comprises the sequence of the human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene.

78. The kit of claim 77, wherein the primer pair specific for the positive control DNA sequence comprises the sequences AATCCCATCACCATCTTCCA (SEQ ID NO: 71) and TGGACTCCACGACGTACTCA (SEQ ID NO: 72).

79. The kit of claim 77, wherein the probe specific for the positive control gene sequence comprises the sequence CTGTCTTCCACTCACTCC (SEQ ID NO: 73).

80. The kit of claim 48, further comprising a microcarrier having an identifier corresponding to a negative control and coupled to a probe that does not hybridize to the amplified DNA.

81. The kit of claim 80, wherein the microcarrier having an identifier corresponding to the negative control comprises a probe comprising the sequence AATATATATATATTAT (SEQ ID NO: 46).

82. The kit of claim 48, wherein the identifier of the microcarrier comprises a digital barcode.

83. The kit of claim 82, wherein each of the microcarriers comprises: (i) a first photopolymer layer; (ii) a second photopolymer layer; and (iii) an intermediate layer between the first layer and the second layer, the intermediate layer having a coding pattern representing the identifier defined thereon, wherein the intermediate layer is partially light-transmissive and partially light-opaque, representing a code corresponding to the microcarrier, wherein the outermost surface of the microcarrier comprises a photoresist photopolymer, and the photoresist photopolymer is functionalized with the probe specific for the DNA mutation, and wherein the microcarrier has the same density as water.

84. The kit of claim 48, wherein the identifier of the microcarrier comprises an analog code.

85. The kit of claim 84, wherein each of the microcarriers comprises: (i) a transparent polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other; (ii) an opaque polymer layer, wherein the opaque polymer layer is secured to the first surface of the transparent polymer layer and surrounds a central portion of the transparent polymer layer, and wherein the opaque polymer layer comprises a two-dimensional shape representing an analog code, wherein the analog code represents an identifier; and (iii) the probe specific for the DNA mutation, wherein the probe is coupled to at least one of the first surface and the second surface of the transparent polymer layer in at least the central portion of the transparent polymer layer.

86. The kit of claim 85, wherein each of the microcarriers further comprises: (iv) a second transparent polymer layer aligned with the first transparent polymer layer, the second transparent polymer layer having a central portion aligned with the central portion of the first transparent polymer layer, wherein the second transparent polymer layer is secured to the second surface of the first transparent polymer layer and does not extend beyond the two-dimensional shape of the first transparent polymer layer; and (v) an opaque magnetic layer surrounding a central portion of the first substantially transparent polymer layer between the opaque polymer layer and a central portion of the transparent polymer layer, wherein the opaque magnetic layer is secured between the first transparent polymer layer and the second transparent polymer layer.

87. The kit of claim 85, wherein each of the microcarriers further comprises an orientation indicator for orienting the simulated code of the opaque polymer layer.

88. A kit as described in claim 85, wherein the two-dimensional shape of the opaque polymer layer comprises a gear shape comprising a plurality of gear teeth, and wherein the simulation code is represented by one or more aspects selected from the group consisting of: the height of one or more gear teeth in the plurality of gear teeth, the width of one or more gear teeth in the plurality of gear teeth, the number of gear teeth in the plurality of gear teeth, and the arrangement of one or more gear teeth within the plurality of gear teeth.

89. The kit of claim 85, wherein each of the microcarriers further comprises: (vi) one or more pillars protruding from the first surface of the first transparent polymeric layer, wherein the one or more pillars are not within a central portion of the first transparent polymeric layer; and / or (vii) one or more pillars protruding from the second surface of the first transparent polymer layer or a surface of the second transparent polymer layer that is not fixed to the first transparent polymer layer, wherein the one or more pillars are not within a central portion of the first transparent polymer layer or the second transparent polymer layer.

90. The kit of claim 85, wherein the transparent polymer of the first transparent polymer layer or the second transparent polymer layer comprises an epoxy polymer.

91. The kit of claim 90, wherein the epoxy polymer is SU-8.

92. A kit comprising: (a) a plurality of probes, each of the plurality of probes coupled to a microcarrier having a unique identifier corresponding to the probe coupled thereto, the plurality of probes comprising a first probe comprising the sequence TTTTTTTTTTTTTTAAGGAGCTGATGG (SEQ ID NO: 47), a second probe comprising the sequence TTTTTTTTTTTTTAAGGAGCTGTTGG (SEQ ID NO: 48), a third probe comprising the sequence TTTTTTTTTTTTTAAGGAGCTAGTGG (SEQ ID NO: 86), a fourth probe comprising the sequence TTTTTTTTTAAGGAGCTGGTGACGT (SEQ ID NO: 91), a fifth probe comprising the sequence TTTTTTTTTAATTACTAGCTACAGAGAAA (SEQ ID NO: 95), a sixth probe comprising the sequence TTTTTTTATTTCTAGCTACAGAAAAAT (SEQ ID NO: 99), a seventh probe comprising the sequence TTTTTTTTTTTAAGGAGCTGTGGCAG (SEQ ID NO: 104), a seventh probe comprising the sequence TTTTTTTTTTTTTTGGAGCTGTGAT (SEQ ID NO: 106), a NO: 109), an eighth probe comprising the sequence TTTTTTTTATTACCACTCAGAAAAG (SEQ ID NO: 113), a ninth probe comprising the sequence TTTTTTTTTATTACCAATCAGAGGAGG (SEQ ID NO: 119), an eleventh probe comprising the sequence TTTTTTTTTTTTTAGAAATAAAAGATTG (SEQ ID NO: 122), a twelfth probe comprising the sequence TTTTTTGGGTGTCTAAGCACCACT (SEQ ID NO: 126), a thirteenth probe comprising the sequence TTTTTTTTTTACAAACCAAGTGAGAA (SEQ ID NO: 60), a fourteenth probe comprising the sequence TTTTTTTTACTGCTGAAAAGAGAGAGT (SEQ ID NO: 57), and a fifteenth probe comprising the sequence TTTTTTTTTTAGAGGCAGAAAAAAACT (SEQ ID NO: 61); (b) a plurality of primer pairs, each comprising a first primer pair comprising a sequence of GTACTGGTGGAGTATTTGATAGTG (SEQ ID NO: 1) and ATCGTCAAGGCACTCTTGCCTAC (SEQ ID NO: 2), a second primer pair comprising a sequence of GGACCCACTCCATCGAGATTT (SEQ ID NO: 8) and CAGATATATTTCTTCATGAAGACCTCACAGTAA (SEQ ID NO: 9), a third primer pair comprising a sequence of GGAATCCATTCTGGTGCCACT (SEQ ID NO: 13) and AGAAAATCCCTGTTCCCACTCATA (SEQ ID NO: 14), a fourth primer pair comprising a sequence of GGTGCCACTACCACAGCTCCT (SEQ ID NO: 18) and TCTCAAAACTGCATTCTGACTTTCA (SEQ ID NO: 19), a fourth primer pair comprising a sequence of TAAAAATAAAGCACCTACTGCTGAAA (SEQ ID NO: 23) and AGCTTGCTTAGGTCCACTCTCTCT (SEQ ID NO: 24), and a fifth primer pair comprising a sequence of NO:24), a fifth primer pair comprising the sequence TAGGATGTAATCAGACGACACAGGA (SEQ ID NO:27) and CAGCTGACCTAGTTCCAATCTTTTA (SEQ ID NO:28), a sixth primer pair comprising the sequence TCTCCCTCCAAAAGTGGTGCT (SEQ ID NO:31) and TGGCAATCGAACGACTCTCAA (SEQ ID NO:32), an eighth primer pair comprising the sequence GCAGAAGTAAAACACCTCCACCA (SEQ ID NO:35) and GGTGCTTTATTTTTAGGTACTTC (SEQ ID NO:36), and a ninth primer pair comprising the sequence CAGGAAAATGACAATGGGAATG (SEQ ID NO:39) and ATCTAATAGGTCCTTTTCAGAATCAATAG (SEQ ID NO:40); and (c) a plurality of blocking nucleic acids, the plurality of blocking nucleic acids comprising the following blocking nucleic acids: a first blocking nucleic acid comprising the sequence (SEQ ID NO: 3); a second blocking nucleic acid sequence comprising the sequence (SEQ ID NO: 10); a third blocking nucleic acid sequence comprising the sequence (SEQ ID NO: 15); a fourth blocking nucleic acid sequence comprising the sequence (SEQ ID NO: 20); A fifth blocking nucleic acid sequence comprising the sequence (SEQ ID NO: 25); A sixth blocking nucleic acid sequence comprising the sequence (SEQID NO: 29); A seventh blocking nucleic acid sequence comprising the sequence (SEQ ID NO:33); an eighth blocking nucleic acid sequence comprising the sequence (SEQ ID NO: 37) and a ninth blocking nucleic acid sequence comprising the sequence (SEQ ID NO:41), wherein the italicized nucleic acid represents a locked nucleic acid.

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