Devices, processes, and systems for determination of nucleic acid sequence, expression, copy number, or methylation changes using combined nuclease, ligase, polymerase, and sequencing reactions

Abstract

To provide devices, processes, and systems for determination of nucleic acid sequence, expression, copy number, or methylation changes.SOLUTION: The present invention relates to methods, devices, instruments, processes, and systems for highly specific, targeted molecular analysis of regions of human genomes and transcriptomes from blood, i.e., from cell free circulating DNA, exosomes, microRNA, IncRNA, circulating tumor cells, or total blood cells. The technology enables highly sensitive identification and enumeration of mutation, expression, copy number, translocation, alternative splicing, and methylation changes using spatial multiplexing and combined nuclease, ligation, polymerase, and sequencing reactions. Such technology may be used for non-invasive early detection of cancer, non-invasive cancer prognosis, and monitoring both treatment efficacy and disease recurrence of cancer.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] This application claims priority benefit of U.S. Provisional Patent Application No. 62 / 478,412, filed March 29, 2017, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention relates to devices, processes, and systems for determining nucleic acid sequence, expression, copy number, or methylation changes using a combination of nuclease reactions, ligation reactions, polymerase reactions, and sequencing reactions. [Background technology]

[0003] Background of the Invention Advances in DNA sequencing promise to standardize and develop noninvasive molecular diagnostics that will improve prenatal care, transplant efficacy, cancer and other disease detection, and personalized treatment. Currently, patients with predisposition to disease or early-stage disease are not identified, and those with disease are not given the best possible treatment. This is all due to failures at the diagnostic stage.

[0004] In the field of cancer, there is a need for the development of technologies that enable early detection, treatment guidance, and recurrence monitoring, all from blood samples. These include (i) highly sensitive detection of single-base mutations, small insertion mutations, and small deletion mutations in known genes (present in 1% to 0.01% of cell-free DNA); (ii) highly sensitive detection of promoter hypermethylation and hypomethylation (present in 1% to 0.01% of cell-free DNA); (iii) accurate quantification of tumor-specific mRNA, lncRNA, and miRNA isolated from tumor-derived exosomes or RISC complexes or from circulating tumor cells in the blood; (iv) accurate quantification of tumor-specific copy changes in DNA isolated from circulating tumor cells; and (v) accurate quantification of mutations, promoter hypermethylation, and hypomethylation in DNA isolated from circulating tumor cells. All of the above (except for quantification of tumor-specific copy changes in DNA isolated from circulating tumor cells) require targeted sequencing of target genes or target regions of the genome. Furthermore, determining sequence information or methylation status from both strands of the original fragments unavoidably involves the identification of rare events.

[0005] Normal plasma contains nucleic acids released from normal cells through normal physiological processes (i.e., exosomes, apoptosis). Additional nucleic acids may be released under conditions of stress, inflammation, infection, or injury. Generally, DNA released from apoptotic cells averages 160 bp in length, while DNA from fetal cells averages approximately 140 bp. Plasma from cancer patients contains nucleic acids within circulating tumor cells (CTCs) in addition to nucleic acids released from cancer cells through abnormal physiological processes. Similarly, plasma from pregnant women contains nucleic acids released from fetal cells.

[0006] Developing reliable diagnostic and screening tests poses several challenges. The first is distinguishing between markers emanating from tumors or fetuses that signal disease (i.e., early cancer) and the presence of the same markers in normal tissue. Additionally, the number of markers tested and the cost of the test must be balanced against the specificity and sensitivity of the assay. This challenge requires addressing the biological variability of diseases such as cancer. In many cases, assays must function as screening tools, allowing for secondary diagnostic follow-up (i.e., colonoscopy, amniocentesis). Complicating the biological problem is the need to reliably detect nucleic acid sequence mutations or promoter methylation differences, or to reliably quantify DNA or RNA copy numbers from very few primary cells (i.e., CTCs) or when cancer- or fetal-specific signals are present in the presence of much larger amounts of nucleic acid emanating from normal cells. Finally, there is the technical challenge of distinguishing true signals resulting from the detection of disease-specific nucleic acid differences of interest from false signals resulting from normal nucleic acid present in the sample and false signals resulting from the absence of disease-specific nucleic acid differences.

[0007] As an example, consider the challenge of detecting the presence of circulating tumor DNA in plasma that harbors mutations in the p53 gene or its methylated promoter region. Such samples contain much larger amounts of cell-free DNA originating from normal cells, and tumor DNA may comprise only 0.01% of the total cell-free DNA. Therefore, detecting the presence of such mutant DNA by whole-body sequencing would require sequencing 100,000 genomes to identify 10 genomes with mutations. This would require sequencing 300,000 GB of DNA, a task beyond the scope of current sequencing technology, not to mention the enormous data management challenges. To circumvent this problem, many groups have attempted to obtain specific target regions or PCR amplification of the region in question. Sequence acquisition suffers from the drawback of dropouts, resulting in the capture of perhaps 90–95% of the desired sequence but missing the desired fragment. Another option, PCR amplification, risks introducing rare errors that cannot be distinguished from true mutations. Furthermore, PCR loses methylation information. Bisulfite treatment has traditionally been used to determine the presence of promoter methylation, but it also destroys DNA samples and lacks the ability to identify multiple methylation changes in cell-free DNA.

[0008] There are several different approaches to reducing error rates and improving the accuracy of sequencing runs. By sequencing the same region of DNA 30–100 times, consensus accuracy can be achieved even in the presence of high error rates. However, high error rates make it extremely difficult to identify low-abundance sequence variants, for example, when attempting to identify cancer mutations in the presence of normal DNA. Therefore, low error rates are required to detect relatively low-abundance mutations. The first approach, called tagged amplicon deep sequencing (TAm-Seq) (Forshew et al., “Noninvasive Identification and Monitoring of Cancer Mutations by Targeted Deep Sequencing of Plasma DNA,” Sci Transl Med. 4(136):136 (2012)), is based on primer design that amplifies 5995 bases targeting selected regions of cancer-related genes, including TP53, EGFR, BRAF, and KRAS. This approach can identify mutations in the p53 gene at frequencies ranging from 2% to 65%. In this method, primers are designed to preamplify DNA (15 cycles) in a multiplex reaction with multiple PCR primers. Preamplification generates both desired and undesired products, which are then further amplified by singleplex PCR. These fragments undergo a final barcoding PCR step followed by standard next-generation sequencing. The advantage of this method is that it utilizes time-tested multiplex PCR-PCR, which excels at amplifying a small number of starting nucleic acids. A disadvantage is that this method cannot distinguish between true mutations and PCR errors in the initial rounds of amplification. Therefore, a sensitivity of 2% (i.e., detection of one mutant allele in 50 wild-type alleles) is sufficient for late-stage cancer assessment before treatment decisions are made, but is insufficient for early detection.

[0009] A variation of the first approach, called the Safe-Sequencing System ("Safe-SeqS") (Kinde et al., "Detection and Quantification of Rare Mutations with Massively Parallel Sequencing," Proc Natl Acad Sci USA 108(23):9530-5 (2011)), involves adding randomly fragmented genomic DNA to the ends of linkers, which are then ligated to the genomic DNA. This approach demonstrated that most mutations revealed by genomic sequencing are actually errors, reducing estimated sequencing errors by at least 70-fold. Similarly, a technique called ultrasensitive deep sequencing (Narayan et al., "Ultrasensitive Measurement of Hotspot Mutations in Tumor DNA in Blood Using Error-Suppressed Multiplexed Deep Sequencing," Cancer Res. 72(14):3492-8 (2012)) involves adding barcodes to primers for nested PCR amplification. As expected, similar systems that rely on bioinformatics tools to add barcodes to detect rare mutations and copy number variations have been developed (Talasaz, A.; Systems and Methods to Detect Rare Mutations and Copy Number Variations, U.S. Patent Application Publication No. US 2014 / 0066317 A1). Using paired-end reads, regions containing putative mutations are covered. This method has been used to track known mutations in the plasma of patients with late-stage cancer. These techniques require a large number of reads to establish a consensus sequence. Because these methods require extension of the entire target DNA, they cannot distinguish between true mutations and polymerase errors, especially when damaged bases such as deaminated cytosines are copied. Finally, these methods do not provide information on the methylation status of CpG sites within the fragment.

[0010] The second approach, called duplex sequencing (Schmitt et al., "Detection of Ultra-Rare Mutations by Next-Generation Sequencing," Proc Natl Acad Sci USA 109(36):14508-13 (2012)), is based on the use of double-stranded linkers containing 12-base random tags. By amplifying both the top and bottom strands of the input target DNA, a given fragment acquires a unique identifier (consisting of 12 bases at each end) that can be tracked by sequencing. Sequence reads that share a unique tag set are grouped into paired families, including members with the identifier in either the top or bottom strand orientation. Each family pair represents the amplification of a single double-stranded DNA fragment. Mutations present in only one or a few family members represent sequencing mistakes that occur later in the amplification or errors introduced during PCR. Mutations occurring in many or all members of a family pair result from PCR errors during the first round of amplification, such as can occur when mutagenic DNA damage sites are copied. In contrast, true mutations present on both strands of a DNA fragment appear in all members of a family pair. Although artifactual mutations can co-occur with true mutations in a family pair, all but those occurring in the first round of PCR amplification can be individually identified and excluded when creating an error-corrected single-stranded consensus sequence. Sequences from each of the two strands of each individual DNA duplex can then be compared to obtain a double-stranded consensus sequence that eliminates any remaining errors that occurred during the first round of PCR. The advantage of this approach is that it clearly distinguishes true mutations from PCR errors or mutagenic DNA damage, resulting in a 3.8 × 10 -10The goal of this approach is to achieve an extremely low error rate. A drawback of this method is that it requires sequencing a large number of fragments to obtain each strand of at least five members of a family pair (i.e., a minimum of 10 sequence reads per original fragment, but due to variability, many more may be required). Furthermore, this method has not been tested with cfDNA, which tends to have smaller fragments than those generated from intact genomic DNA, so sequencing a larger number of fragments would be required to cover all potential mutations. Finally, this method does not provide information about the methylation status of CpG sites within the fragments.

[0011] A third approach, called single-molecule molecular inversion probes (smMIPs) (Hiatt et al., "Single Molecule Molecular Inversion Probes for Targeted, High-Accuracy Detection of Low-Frequency Variation," Genome Res. 23(5):843-54 (2013)), combines single-molecule tagging and multiplex capture to enable highly sensitive detection of low-frequency subclonal variations. This method has an error rate of 2.6 × 10 in clinical specimens. -5 The authors claim that this approach requires sequencing a large number of fragments to obtain each strand of at least five members of a family pair (i.e., a minimum of 10 sequence reads per original fragment, but due to variability, many more may be required). Furthermore, because this method requires extension of the entire target DNA, it cannot distinguish between true mutations and polymerase-induced errors, especially when damaged bases such as deaminated cytosines are copied. Furthermore, this method has not been tested with cfDNA, which tends to be smaller than fragments generated from intact genomic DNA, so sequencing a larger number of fragments would be required to cover all potential mutations. Finally, this method does not provide information about the methylation status of CpG sites within the fragments.

[0012] A fourth method, called circle sequencing (Lou et al., "High-throughput DNA Sequencing Errors are Reduced by Orders of Magnitude Using Circle Sequencing," Proc Natl Acad Sci USA 110(49):19872-7(2013) (Non-Patent Document 6); Acevedo et al., "Mutational and Fitness Landscapes of an RNA Virus Revealed Through Population Sequencing," Nature 2014 505(7485):686-90(2014) (Non-Patent Document 7); and Acevedo et al., "Library Preparation for Highly Accurate Population Sequencing of RNA Viruses," Nat Protoc. 9(7):1760-9(2014) (Non-Patent Document 8)), involves fragmenting DNA or RNA to approximately 150 bases, denaturing it to form single strands, circularizing the single strands, and then annealing them with random hexamer primers and phi29. This method is based on rolling circle amplification using DNA polymerase (in the presence of uracil-DNA glycosylase and formamidopyrimidine-DNA glycosylase), which is then refragmented to approximately 500 bases before standard next-generation sequencing. The advantage of this technique is that rolling circle amplification creates multiple tandem copies while stripping away the original target DNA, so polymerase errors appear in only one copy, while true mutations can appear in all copies. Because copies are physically interconnected, the read family size averages three copies. This method also uses uracil-DNA glycosylase and formamidopyrimidine-DNA glycosylase to remove targets containing damaged bases, eliminating such errors. The advantage of this technique is that it achieves high resolution, compared to current levels of approximately 0.1–1×10. -2 From 7.6 × 10 -6The key to success is achieving a low sequencing error rate. Currently, this latter error rate is sufficient to identify cancer mutations in plasma when wild-type DNA is present in 100- to 10,000-fold excess. An additional advantage is that because two to three copies of the same sequence are physically linked, sequence data generated from a single fragment can verify true mutations. This contrasts with the requirement for at least 10 fragments when using duplex sequencing approaches. However, this method does not provide the ability to distinguish between copy number changes, nor does it provide information about the methylation status of CpG sites within the fragment.

[0013] A fifth approach, developed by Complete Genomics (Drmanac et al., "Human Genome Sequencing Using Unchained Base Reads on Self-Assembling DNA Nanoarrays," Science 327(5961):78-81(2010)), is based on the use of ligation reads on nanoball arrays. Approximately 400 nucleotides of genomic DNA are circularized with linkers, cleaved, recircularized with linkers, and finally recircularized until approximately four linkers are included. Phi29 DNA polymerase is used to perform rolling circle amplification of the DNA to generate nanoballs, which are then arrayed and sequenced using a ligation-based approach. What makes this approach particularly relevant here is its ability to generate multiple tandem copies of the same sequence, followed by sequencing from a single rolling circle amplification product. Because the same sequence is interrogated multiple times by either ligase or polymerase (by combining rolling circle with other sequencing-by-synthesis approaches), the error rate per base can be significantly reduced. Therefore, sequencing directly from rolling circle products offers many of the same advantages as the circle sequencing approach described above.

[0014] A sixth approach, called SMRT-1 molecular real-time sequencing (Flusberg et al., "Direct Detection of DNA Methylation During Single-Molecule, Real-Time Sequencing," Nat Methods 7(6):461-5 (2010)), relies on adding a hairpin loop to the end of a DNA fragment and then extending a DNA polymerase with strand displacement activity around the covalently closed loop to obtain sequence information about the two complementary strands. Specifically, a single polymerase molecule catalyzes the incorporation of a fluorescently labeled nucleotide into the complementary nucleic acid strand. Upon incorporation of the nucleotide opposite the methylated base, the polymerase slows down or "stutters," and the resulting fluorescence pulse allows the detection of N 6 Modified nucleotides within a DNA template, including 5-methyladenine, 5-methylcytosine, and 5-hydroxymethylcytosine, can be directly detected. The accuracy of this method is improving, particularly as the polymerase can traverse the closed loop several times to determine a consensus sequence. While the technique is designed to obtain sequence information for "dumbbell"-shaped substrates (mostly consisting of two complementary sequences of a linear piece of DNA), it can also be applied to single-stranded circular substrates.

[0015] Several research groups and companies are developing kits that amplify specific target sequences while attaching a unique molecular identifier (UMI) or barcode to each fragment.

[0016] A simple method called SiMSen-Seq (SiMSen-Seq: DNA barcoding by simple multiplex PCR for sensitive mutation detection by sequencing) uses two rounds of PCR with a high-fidelity polymerase to add hairpin-protected barcodes to each fragment as well as to an external universal primer (Stahlberg et al., "Simple, Multiplexed, PCR-based Barcoding of DNA Enables Sensitive Mutation Detection in Liquid Biopsies Using Sequencing," Nucleic Acids Res. 44(11):e105) (2016)). In this method, one primer contains an adapter stem that "hides" the barcode from the target DNA, preventing random bases in the barcode sequence from misdirecting hybridization of the primer to the target. The other primer is a regular primer with an Illumina adapter sequence at its end. After two rounds of amplification with a high-fidelity polymerase, the adapter and barcode are added to the target fragment. After protease treatment and dilution, a second PCR is performed using Illumina adapters containing patient identifier barcodes. This method previously identified hotspot locations for undetected sequencing errors but is now designed to barcode only one strand.

[0017] In the ThruPLEX Tag-seq kit (Rubicon Genomics), stem-loop adapters are ligated to the ends of double-stranded DNA. As with standard Y adapters, the genomic DNA is repaired to create blunt ends. In the next step, stem-loop adapters containing 5'-blocked unique molecular tags (UMIs) are ligated to the 5' end of the DNA, leaving a nick at the 3' end of the target fragment. Stem-loop adapters lack single-stranded overhangs, making them less susceptible to ligation with each other. Both of these factors contribute to the nonspecific background seen in many other NGS preparations. Instead, stem-loop adapters contain cleavable replication-terminating bases. In the final step, library synthesis is completed by extending the 3' end of the DNA, and high-fidelity amplification adds Illumina-compatible indexes. Any remaining empty adapters are degraded. Ligation reactions can be inefficient, potentially reducing yields when the input of mutant samples is limiting. Furthermore, this method lacks selectivity for specific targets.

[0018] In the NEBNext Direct target enrichment method (New England Biolabs), DNA is fragmented to lengths of approximately 150–200 bp. The fragmented DNA is rapidly hybridized to biotinylated oligonucleotide "baits" that define the 3' end of each target of interest. These oligonucleotide baits are designed for both the top and bottom strands of each target. The bait-target hybrids are then bound to streptavidin beads, and any 3' off-target sequences are enzymatically trimmed to generate blunt ends. This combination of short hybridization times and enzymatic removal of 3' off-target sequences allows for improved sequencing efficiency compared to traditional hybridization-based enrichment methods. The trimmed targets are then converted into Illumina-compatible libraries containing unique molecular identifiers (UMIs) and sample barcodes. This conversion is performed as follows: The blunt ends are dA-tailed with terminal transferase, allowing for ligation of hairpin loop sequences to single-stranded dA overhangs. The probe is then extended with DNA polymerase to generate copies of the original fragment, generating double-stranded DNA with random 5' ends. These ends are then blunted (using T4 polymerase or DNA polymerase 1) to either include a phosphate from the initial fragmentation or add a phosphate using T4 kinase. This new end is now suitable for adapter ligation to the original target strand, which contains a UMI sequence. The adapter hairpin loop is then cleaved to generate a top strand consisting of the 5' adapter sequence, the UMI sequence, a stretch of the 5' target sequence, the target region of interest up to the 3' end complementary to the bait, a polydA sequence, and then a further 3' adapter sequence. This top strand can then be lysed to remove the streptavidin beads, and purified, making it suitable for amplification with Illumina or Ion Torrent adapters containing patient identifier barcodes. Sequence-matched libraries are generated within a day. This procedure is compatible with most automated liquid handling systems.Although this technique is designed to be highly efficient at capturing only the fragment of interest, it is also a lengthy, multi-step procedure that can result in low yields when the input amount of mutant sample is limiting.

[0019] The QIAseq targeted RNA sequencing method (Qiagen Inc.) adds a unique identifier to RNA sequences, enabling their accurate counting. After RNA sample purification, reverse transcriptase is used to synthesize cDNA. A composite primer consisting of an initial 5' consensus sequence, an internal 12-base molecular tag (i.e., UMI), and a gene-specific 3' portion is used to generate an extension product from the cDNA. After extension, the reaction is purified to remove unreacted primers. This is followed by a first-stage PCR using a universal primer and a second gene-specific primer containing a second 5' consensus sequence. According to the manufacturer, the first and second gene-specific primers "do not recognize each other, thereby minimizing primer dimerization." An additional reaction purification step follows the first PCR. This is followed by a second-stage PCR using a universal adapter sequence to add Illumina or Ion Torrent adapters containing patient identifier barcodes. Sequence-compatible libraries are generated within six hours. Because each initial cDNA molecule is expected to be extended by a primer containing a UMI, transcripts can be matched to unique UMIs to count the number of original transcripts present for each RNA molecule, distinguishing five copies of a single transcript from five unique transcripts of the same gene. This technique is similar to RNA-seq but is designed to count RNA fragments for highly specific fragments of interest. This technique is also adaptable to identifying low-abundance mutations, although multiple wash steps can reduce yields when the input mutant sample is limited.

[0020] The Oncomine Cell-Free DNA Assay (ThermoFisher Scientific) for clinical studies of liquid biopsies uses a two-step PCR reaction to directly amplify target sequences from cfDNA. Both the forward and reverse composite primers contain a first and second 5' consensus sequence, an internal unique molecular tag (i.e., UMI), and a gene-specific 3' portion. After exactly two cycles of PCR, two composite double-stranded products are formed. The first product contains the top-strand primer extension product, the top-strand target sequence, and the complement of the bottom-strand primer containing the second consensus sequence. This hybridizes to the initial extension of the bottom-strand primer containing the bottom-strand target sequence. The second product contains the bottom-strand primer extension product, the bottom-strand target sequence, and the complement of the top-strand primer containing the first consensus sequence. This hybridizes to the initial extension of the top-strand primer containing the top-strand target sequence. Thus, both the top and bottom strands contain a universal adapter sequence and a unique UMI sequence resulting from each initial target strand. Next, target amplicons purified from gene-specific primers are captured onto a solid support. After release of the products from the solid support, appropriate second-stage PCR is performed using universal adapter sequences to add Ion Torrent adapters containing patient identifier barcodes. Sequence-aware libraries are generated within hours and can then be combined with additional template preparations using emulsion PCR on beads. While this approach is very rapid and robust, it requires physical removal of the initial gene-specific primers and an extra step of purification / size selection after the second PCR step (presumably to eliminate primer dimers). It is unclear whether this procedure could potentially reduce yields when the input amount of mutant sample is limiting.

[0021] The present invention is directed to overcoming these and other deficiencies in the art. [Prior art documents] [Patent documents]

[0022] [Patent Document 1] U.S. Patent Application Publication No. US 2014 / 0066317 A1 [Non-patent literature]

[0023] [Non-Patent Document 1] Forshew et al., “Noninvasive Identification and Monitoring of Cancer Mutations by Targeted Deep Sequencing of Plasma DNA,” Sci Transl Med.4(136):136(2012) [Non-patent document 2] Kinde et al., “Detection and Quantification of Rare Mutations with Massively Parallel Sequencing,” Proc Natl Acad Sci USA 108(23):9530-5(2011) [Non-patent document 3] Narayan et al., “Ultrasensitive Measurement of Hotspot Mutations in Tumor DNA in Blood Using Error-suppressed Multiplexed Deep Sequencing,” Cancer Res.72(14):3492-8(2012) [Non-patent document 4] Schmitt et al., “Detection of Ultra-Rare Mutations by Next-Generation Sequencing,” Proc Natl Acad Sci USA 109(36):14508-13(2012) [Non-patent document 5] Hiatt et al., “Single Molecule Molecular Inversion Probes for Targeted,High-Accuracy Detection of Low-Frequency Variation,” Genome Res.23(5):843-54(2013) [Non-patent document 6] Lou et al., “High-throughput DNA Sequencing Errors are Reduced by Orders of Magnitude Using Circle Sequencing,” Proc Natl Acad Sci USA 110(49):19872-7(2013) [Non-Patent Document 7] Acevedo et al., “Mutational and Fitness Landscapes of an RNA Virus Revealed Through Population Sequencing,” Nature 2014 505(7485):686-90(2014) [Non-patent document 8] Acevedo et al., “Library Preparation for Highly Accurate Population Sequencing of RNA Viruses,” Nat Protoc.9(7):1760-9(2014) [Non-Patent Document 9] Drmanac et al., “Human Genome Sequencing Using Unchained Base Reads on Self-Assembling DNA Nanoarrays,” Science 327(5961):78-81(2010) [Non-Patent Document 10] Flusberg et al., “Direct Detection of DNA Methylation During Single-Molecule,Real-Time Sequencing,” Nat Methods 7(6):461-5(2010) [Non-Patent Document 11] Stahlberg et al., “Simple, Multiplexed, PCR-based Barcoding of DNA Enables Sensitive Mutation Detection in Liquid Biopsies Using Sequencing,” Nucleic Acids Res.44(11):e105) (2016) Summary of the Invention

[0024] One aspect of the present invention relates to a system for identifying multiple nucleic acid molecules in a sample. The system includes an inlet and a cartridge. The cartridge defines a space containing multiple primary reaction chambers fluidly connected to the inlet, which receive material from the inlet and generate primary reaction chamber products from the material. The space also includes a product capture housing containing a solid support with multiple individual rows of multiple product capture subunits, each individual product capture subunit containing an array of multiple individual hydrophilic micropores or microwells separated by a hydrophobic surface, where the primary reaction products are further reacted to generate array products. The array products are detected in the micropores or microwells, where one or more rows of individual product capture subunits receive material that has passed through one of the multiple primary reaction chambers.

[0025] Another aspect of the present invention relates to a system for identifying multiple nucleic acid molecules in a sample. The system includes a cartridge including an inlet, an outlet, and an array of micropores or microwells, the cartridge being fluidically connected to the inlet and the outlet. The cartridge defines a volume including multiple primary reaction chambers in fluid communication with the inlet, which receive material from the inlet and produce a primary reaction-chamber product from the material. The volume also includes multiple secondary reaction chambers, one or more of which are fluidly connected to one of the multiple primary reaction chambers and receive material from one of the multiple primary reaction chambers and produce a secondary reaction-chamber product. At least some of the multiple primary and secondary reaction chambers are configured to hold troughs of fluid within the multiple primary and secondary reaction chambers, facilitating mixing of the sample, reagents, and / or product reactants for subsequent reactions to produce chamber or array products. The space also includes a plurality of mixing chambers, each fluidly coupled to one of the plurality of secondary reaction chambers, for receiving material from one of the plurality of secondary reaction chambers and discharging material into the product capture housing, such that each row of individual product capture subunits is fluidly coupled to one of the one or more mixing chambers and receives material from one of the one or more mixing chambers. The space also includes a product capture housing containing a solid support with a plurality of individual rows of multiple product capture subunits, each individual product capture subunit comprising an array of a plurality of individual hydrophilic micropores or microwells separated by a hydrophobic surface, where the secondary reaction products are further reacted to produce array products. The array products are detected in the micropores or microwells, where one or more rows of individual product capture subunits receive material that has passed through one of the plurality of primary reaction chambers.

[0026] Another aspect of the present invention relates to a system for identifying multiple nucleic acid molecules in a sample. The system includes an inlet, an outlet, and a cartridge fluidly coupled to the inlet and the outlet. The cartridge defines a space containing a product capture housing containing a solid support with multiple individual rows of product capture subunits. Each individual product capture subunit includes an array of multiple individual hydrophilic micropores separated by a hydrophobic surface, each micropore having opposing first and second open ends, the first end having a larger diameter and the second end having a smaller diameter than the first end. The product capture housing includes multiple fluid flow paths located above and below the solid support so that substances can pass from the inlet through the row of product capture subunits, contact the array of micropores within the subunits, and then to the outlet.

[0027] A further aspect of the invention relates to a method for preparing a system for identifying a plurality of nucleic acid molecules in a sample, the method comprising providing a system of the invention and adding universal tags or capture oligonucleotide primers or probes to micropores or microwells of product capture subunits on a solid support within a product capture housing, such that the universal tags or capture oligonucleotide primers or probes are retained within the micropores or microwells.

[0028] Another embodiment of the present invention relates to a process for identifying multiple nucleic acid molecules in a sample using the system of the present invention, wherein the process, following filling of one or more primary reaction chambers and / or one or more secondary reaction chambers (if present), includes running the primary and / or secondary reactions in the system and detecting the presence of target nucleic acid molecules in the sample in the microwells or micropores based on the running of the primary and / or secondary reactions.

[0029] Another embodiment of the present invention relates to a process for identifying multiple nucleic acid molecules in a sample using the system of the present invention, wherein after performing primary and / or secondary reactions, the products of such reactions are amplified in microwells or micropores under conditions where a polymerase, exonuclease, endonuclease, or ribonuclease cleaves one or more probes containing a quencher group and a fluorescent group in a target-specific manner, liberating the fluorescent group and generating a signal if the target nucleic acid molecule is present in the sample.

[0030] Another embodiment of the present invention relates to a process for identifying multiple nucleic acid molecules in a sample using the system of the present invention. This process involves providing a sample containing multiple target nucleic acid molecules and then contacting the sample with a set of primary oligonucleotide primers having a first portion complementary to a portion of the target nucleic acid molecule and a second portion, and a polymerase to form a polymerase chain reaction mixture. This mixture is subjected to a polymerase chain reaction in a primary reaction chamber to produce a set of amplification products. The amplification products are delivered to a product capture housing containing a solid support with multiple individual rows of multiple capture subunits. Each individual product capture subunit contains an array of multiple individual micropores containing immobilized capture probes complementary to the second portion. The target nucleic acid molecules are captured by the immobilized capture probes and copied. The nucleotide sequences of the immobilized target nucleic acid molecules are obtained by performing a sequencing reaction in the micropores.

[0031] The present invention also relates to a process for preparing a microtiter plate for identifying multiple nucleic acid molecules in a sample. This process involves providing a microtiter plate with a plurality of individual rows and columns of product capture subunits, each of which contains an array of multiple individual hydrophilic microwells separated by a hydrophobic surface. The microwells of the microtiter plate are filled with an aqueous liquid containing oligonucleotide primers and / or probes. The microtiter plate is centrifuged to distribute the aqueous liquid into the unfilled microwells of each individual product capture subunit in the microtiter plate. Thereafter, when the centrifugation is terminated, the aqueous liquid is no longer in contact with the hydrophobic surface. The aqueous liquid is evaporated, and the microwells are dried, leaving the oligonucleotide primers in the microwells.

[0032] Another aspect of the present invention relates to a system for identifying multiple nucleic acid molecules in a sample. The system includes an inlet, an outlet, and a cartridge fluidly coupled to the inlet and the outlet. The cartridge defines a space containing a product capture housing containing a solid support with multiple individual rows of product capture subunits. Each individual product capture subunit includes an array of multiple individual hydrophilic micropores separated by a hydrophobic surface, each micropore having opposing first and second open ends, the first end having a larger diameter and the second end having a smaller diameter than the first end. The product capture housing includes multiple fluid flow paths located above and below the solid support so that substances can pass from the inlet through the row of product capture subunits, contact the array of micropores within the subunits, and then to the outlet.

[0033] The present invention provides a series of devices, chambers, and assays for determining the cause of disease directly from a blood sample. Nucleic acids are purified from clinical samples, target regions are subjected to a series of amplification reactions, and targets are identified or enumerated using either real-time PCR or sequencing as readouts.

[0034] The present invention aims to help address a major diagnostic clinical challenge facing the United States and the world. The greatest unmet need is detecting cancer at its earliest stages. Accessible, accurate early detection testing could save over 300,000 lives annually in the United States and over 4,000,000 lives worldwide, saving the United States $300 billion annually in healthcare costs. One promising solution to this challenge is providing a process and system, as described in this application, for simultaneously assaying multiple DNA mutations and methylation changes with single-molecule sensitivity. The same assay can also be used to monitor the "cancer marker burden" in the blood and how effectively a given treatment is killing residual cancer cells after surgery. A related challenge is monitoring patients for early recurrence of cancer, at a time when alternative treatments may still be effective. The present invention provides the flexibility to use Taqman™ assays, sequencing, or both to track cancer markers.

[0035] Infectious disease testing is shifting from single pathogen detection to symptom-based or bloodborne pathogen detection. This invention potentially provides accurate viral or bacterial load values ​​for hundreds of targets simultaneously, guiding physicians to make clinically actionable decisions. For example, patients suffering from respiratory illnesses can be simultaneously tested for all strains of influenza and parainfluenza viruses, adenoviruses, coronaviruses, rhinoviruses, enteroviruses, respiratory syncytial viruses, Mycobacterium tuberculosis, Streptococcus pneumoniae, Group A Streptococcus, Mycoplasma pneumoniae, and Haemophilus influenzae. In the case of blood borne pathogens, the present invention can be used to distinguish between Staphylococcus, MRSA, Streptococcus, Enterococcus (VRE), Listeria, Acinetobacter, Enterobacter, E. coli (including toxin producers), Klebsiella (including those in KPC), Pseudomonas, Proteus, Candida, Cryptococcus, Neisseria, Haemophilus, and the like. Testing international travelers with fever can distinguish Zika virus from viral hemorrhagic fevers (dengue, yellow fever, West Nile, arenaviruses, filoviruses, bunyaviruses, and other flaviviruses) or other viral (influenza, RSV, SARS, chikungunya, rubella, measles, parvovirus, enterovirus, adenovirus, and alphavirus infections) or parasitic (malaria) or bacterial (group A streptococcus, rickettsia, borrelia, leptospirosis) causes.

[0036] Non-invasive prenatal testing is currently used to identify chromosome copy abnormalities using either direct sequencing to count chromosome fragments or ligation-based detection with array-based quantification. The ability of the present invention to accurately identify and count targets at the single molecule level is believed to provide the opportunity to provide highly accurate results at a lower cost. For example, more complete blood tests are possible for life-threatening autosomal and X-linked recessive Mendelian genetic diseases; trisomy 21, trisomy 18, trisomy 13, Turner syndrome, Klinefelter syndrome (chromosome copy abnormalities); Duchenne and Becker muscular dystrophy, cystic fibrosis, and other genetic diseases. [The present invention 1001] Inlet and A cartridge defining a space; 1. A system for identifying a plurality of nucleic acid molecules in a sample, comprising: The space is a plurality of primary reaction chambers in fluid communication with the inlets for receiving materials from the inlets and producing primary reaction chamber products from the materials; a product capture housing containing a solid support comprising a plurality of individual rows of a plurality of product capture subunits, each individual product capture subunit comprising an array of a plurality of individual hydrophilic micropores or microwells separated by a hydrophobic surface, in which primary reaction products are further reacted to produce array products which are detected in said micropores or microwells, wherein one or more of said rows of individual product capture subunits receive material that has passed through one of said plurality of primary reaction chambers; Including, The system. [The present invention 1002] 1001 , the system of claim 10 further comprising an outlet for expelling material from said product capture housing. [The present invention 1003] The space defined by the cartridge is one or more initial reaction chambers into which the inlet discharges material and from which the material discharges into the plurality of primary reaction chambers; The system of the present invention 1001 further includes: [The present invention 1004] The space defined by the cartridge is a plurality of secondary reaction chambers, one or more of which are in fluid communication with one of the plurality of primary reaction chambers for receiving material from one of the plurality of primary reaction chambers; a plurality of mixing chambers each fluidly coupled to one of the plurality of secondary reaction chambers, each mixing chamber receiving material from one of the plurality of secondary reaction chambers and discharging material into the product capture housing, whereby each row of individual product capture subunits is fluidly coupled to one of the one or more mixing chambers for receiving material from the one or more mixing chambers; The system of the present invention 1001 further includes: [The present invention 1005] 10. The system of claim 10, wherein at least some of said plurality of primary and secondary reaction chambers are configured to hold a trough of liquid within said plurality of primary and secondary reaction chambers. [The present invention 1006] 10. The system of claim 10, wherein each of said plurality of primary and secondary reaction chambers has an internal baffle that retains a trough of liquid within said plurality of primary and secondary reaction chambers. [The present invention 1007] 10. The system of claim 10, wherein each of said plurality of primary and / or secondary reaction chambers has one or more internal baffles that hold a plurality of troughs of liquid within said plurality of primary and secondary reaction chambers. [The present invention 1008] 10. The system of claim 10, wherein each of said mixing chambers includes a partition extending from near where the material enters said mixing chamber to near where the material exits said mixing chamber. [The present invention 1009] each of the mixing chambers includes a first surface that is more hydrophobic and a second surface that is spaced apart from the first surface and is less hydrophobic than the first surface; the first and second surfaces extend from near where the material enters the mixing chamber to near where the material exits the mixing chamber; The system of the present invention 1004. [The present invention 1010] The system of the present invention 1004, wherein said primary reaction chamber and / or said secondary reaction chamber comprises an interior surface onto which oligonucleotide primers or probes can be spotted. [The present invention 1011] The system of the present invention 1001, wherein the product capture subunit comprises an array of a plurality of individual micropores, each of the micropores having opposing first and second open ends, the first end having a larger diameter and the second end having a smaller diameter than the diameter of the first end. [The present invention 1012] a mesh screen covering the second end of the micropore within the product capture housing; The system of the present invention 1011 further includes: [The present invention 1013] beads disposed within the individual micropores The system of the present invention 1011 further includes: [The present invention 1014] 1001. The system of claim 1001, wherein said product capture subunit comprises an array of a plurality of individual microwells, each having an open end and a closed end. [The present invention 1015] The product capture housing comprises: a plurality of fluid flow paths that allow materials to pass from the plurality of mixing chambers through the array of product capture subunits and into contact with the array of micropores or microwells within those subunits; The system of the present invention 1004 includes: [The present invention 1016] The system of the present invention 1015, wherein the plurality of fluid channels are located above and below the solid support. [The present invention 1017] The system of the present invention 1015, wherein the plurality of fluid channels are located above the solid support. [The present invention 1018] one or more valves for selecting whether to direct reagents or reactants into or out of the cartridge through the inlet; The system of the present invention 1001 further includes: [The present invention 1019] one or more valves for selecting whether to direct reagents or reactants into or out of the product capture housing through the outlet and / or a location within the product capture housing remote from the outlet; The system of the present invention 1001 further includes: [The present invention 1020] 1001. The system of claim 10, further comprising one or more heating elements within said cartridge proximate said primary reaction chamber and / or said product capture housing. [The present invention 1021] 1003. The system of claim 1003, further comprising one or more heating elements within said cartridge proximate said initial reaction chamber. [The present invention 1022] The system of claim 1004, further comprising one or more heating elements within said cartridge proximate one of said secondary reaction chambers and / or one or more of said mixing chambers. [The present invention 1023] 1. A method for preparing a system for identifying a plurality of nucleic acid molecules in a sample, comprising: Providing a system of the present invention 1001; adding a universal tag or capture oligonucleotide primer or probe to the micropore or microwell of the product capture subunit on the solid support within the product capture housing, thereby retaining the universal tag or capture oligonucleotide primer or probe within the micropore or microwell; The method comprising: [The present invention 1024] 1023. The method of claim 1023, further comprising filling said one or more primary reaction chambers with primary reaction oligonucleotide probes or primers, each having a first portion comprising a nucleotide sequence complementary to a portion of a target nucleic acid in said sample. [The present invention 1025] the primary reaction oligonucleotide probe or primer is a second portion comprising a nucleotide sequence identical to or complementary to a portion of a universal tag or capture oligonucleotide primer retained within said micropore or microwell. The method of the present invention 1024 further comprises: [The present invention 1026] 1. A method for preparing a system for identifying a plurality of nucleic acid molecules in a sample, comprising: Providing a system of the present invention 1004; adding a universal tag or capture oligonucleotide primer or probe to the micropore or microwell of the product capture subunit on the solid support within the product capture housing, thereby retaining the universal tag or capture oligonucleotide primer or probe within the micropore or microwell; The method comprising: [The present invention 1027] The method of claim 1026, further comprising filling said one or more primary and / or secondary reaction chambers with primary or secondary reaction oligonucleotide probes or primers, each having a first portion comprising a nucleotide sequence complementary to a portion of a target nucleic acid in said sample. [The present invention 1028] the primary or secondary reaction oligonucleotide probe or primer is a second portion comprising a nucleotide sequence identical to or complementary to a portion of a universal tag or capture oligonucleotide primer retained within said micropore or microwell. The method of the present invention 1027 further comprising: [The present invention 1029] the product capture subunit comprises an array of individual micropores; each of the micropores having opposing first and second open ends; the first end has a larger diameter and the second end has a smaller diameter than the diameter of the first end; a first passageway in fluid communication with the first end of the micropore and a second passageway in fluid communication with the second end of the micropore; wherein said universal tag or capture oligonucleotide primer or probe is subjected to the following steps in the order shown: passing the universal tag or capture oligonucleotide primer or probe through the first passageway and from the first open end to the micropore while passing a hydrophobic liquid through the second passageway; passing a hydrophobic liquid through the first passage while passing a hydrophobic liquid through the second passage; passing a volatile solvent through the first passageway while passing a hydrophobic liquid through the second passageway; and passing air through the first passageway while passing heat, a hydrophobic liquid, a volatile solvent, and air through the second passageway. is added to the micropores by a method comprising: The method of the present invention 1023 or the method of the present invention 1026. [The present invention 1030] the product capture subunit comprises an array of individual micropores; each of the micropores having opposing first and second open ends; the first end has a larger diameter and the second end has a smaller diameter than the diameter of the first end; a first passageway in fluid communication with the first ends of the micropores; and a second passageway separated from the second ends of the micropores by a mesh screen covering the second ends and in fluid communication with the second passageway; wherein said detection or capture oligonucleotide primers or probes are subjected to the following steps in the order shown: passing the universal tag or capture oligonucleotide primer or probe through the first passageway and from the first open end into the micropore; passing a hydrophobic liquid through the first passage while expelling the universal tag or capture oligonucleotide primer or probe from the first passage; passing a hydrophobic liquid through the first passage while passing a hydrophobic liquid through the second passage; passing a volatile solvent through the first passageway while passing a hydrophobic liquid through the second passageway; and passing air through the first passageway while passing heat, a hydrophobic liquid, a volatile solvent, and air through the second passageway. is added to the micropores by a method comprising: The method of the present invention 1023 or the method of the present invention 1026. [The present invention 1031] A process for identifying a plurality of nucleic acid molecules in a sample using a system prepared by the method of the present invention 1025 or the method of the present invention 1028, comprising: charging said one or more primary reaction chambers and, if present, optionally charging said one or more secondary reaction chambers; conducting the primary and / or secondary reactions in the system; detecting the presence of a target nucleic acid molecule in the sample in the microwell or micropore based on the performance of the primary reaction and / or secondary reaction; The process further comprises: [The present invention 1032] The detecting amplifying the products of the primary and / or secondary reactions in the microwells or micropores under conditions where a polymerase, exonuclease, endonuclease, or ribonuclease cleaves one or more probes comprising a quencher group and a fluorescent group in a target-specific manner, such that if a target nucleic acid molecule is present in the sample, the fluorescent group is released and a signal is generated. The process of the present invention 1031, comprising: [The present invention 1033] Carrying out the primary reaction and / or secondary reaction providing a sample containing a plurality of target nucleic acid molecules; contacting the sample with a set of primary oligonucleotide primers having a first portion complementary to, or the complement of, a portion of the target nucleic acid molecule, and a polymerase to form a first polymerase extension or chain reaction mixture; subjecting the first polymerase extension or chain reaction mixture to a first polymerase extension or chain reaction in one or more initial or primary reaction chambers to produce a first set of extension products or amplification products; contacting the first set of extension products or amplification products with a set of secondary oligonucleotide primers having first portions complementary to portions of the primary extension products or primary amplification products, and a polymerase to form a second polymerase chain reaction mixture; subjecting the second polymerase chain reaction mixture to a second polymerase chain reaction in the primary or secondary reaction chamber to produce a second set of amplification products, each secondary amplification product comprising a 5' second partial sequence, a target nucleotide sequence specific portion or its complement, and a 3' second partial complementary sequence; The process of the present invention 1031, comprising: [The present invention 1034] Carrying out the primary reaction and / or secondary reaction providing a sample containing a plurality of target nucleic acid molecules; contacting the sample with a set of primary oligonucleotide primers having a portion complementary to a portion of the target nucleic acid molecule or its extension product, and with a polymerase to form a first polymerase chain reaction mixture; subjecting the first polymerase chain reaction mixture to a first polymerase chain reaction in one or more initial or primary reaction chambers to produce a first set of amplification products; contacting the first set of amplification products with a set of oligonucleotide probes having a first portion complementary to a portion of the first set of amplification products and a second portion, and with a ligase to form a ligase detection reaction mixture; subjecting the ligase detection reaction mixture to a ligase detection reaction in the primary or secondary reaction chamber to produce a set of ligation products, each ligation product comprising a 5' second subsequence, a target nucleotide sequence specific portion or its complement, and a 3' second subsequence; The process of the present invention 1031, comprising: [This invention 1035] The charging of the one or more primary reaction chambers, if present, the one or more secondary reaction chambers, and carrying out the primary and / or secondary reactions in the system comprises the following steps in the order shown: passing a hydrophobic liquid through the system through the inlet; delivering primary reaction oligonucleotide probes or primers, and reverse transcription and / or polymerase chain reaction reagents, as well as a hydrophobic liquid, through the inlet into the system; performing a polymerase extension or chain reaction in said system; discharging material from the system through the inlet; delivering a hydrophobic liquid, polymerase chain reaction reagents or ligase detection reaction reagents, and a hydrophobic liquid through the inlet to the system; and performing a polymerase chain reaction or a ligase detection reaction on said system; The process of the present invention 1031 is performed by a process including: [The present invention 1036] the product capture subunit comprises an array of individual micropores; each of the micropores having opposing first and second open ends; the first end has a larger diameter and the second end has a smaller diameter than the diameter of the first end; a first passageway in fluid communication with a first end of the micropore and a second passageway in fluid communication with the second end of the micropore; wherein conducting the secondary reaction in the system comprises the following steps in the order shown: passing the products of a polymerase chain reaction or ligase detection reaction through the first passageway to the product capture housing while passing a hydrophobic liquid through the second passageway; passing a hydrophobic liquid through the first and second passages; and subjecting the products of the polymerase chain reaction or ligase detection reaction to a polymerase chain reaction with a universal tag primer and probe within the micropore of the product capture subunit. executed by a process containing The process of the present invention 1031. [This invention 1037] the product capture subunit comprises an array of individual micropores; each of the micropores having opposing first and second open ends; the first end has a larger diameter and the second end has a smaller diameter than the diameter of the first end; a first passageway in fluid communication with a first end of the micropore; and a second passageway in fluid communication with and separated from the second end of the micropore by a mesh screen covering the second end of the micropore; Carrying out the secondary reaction in the system comprises the following steps in the order shown: directing the product of a polymerase chain reaction or a ligase detection reaction through the first passageway to the product capture housing; passing a hydrophobic liquid through the first passageway; passing a hydrophobic liquid through said first and second passageways; subjecting the products of the polymerase chain reaction or ligase detection reaction to a polymerase chain reaction with a universal tag primer and probe within the micropore of the product capture subunit. executed by a process containing The process of the present invention 1031. [The present invention 1038] The process of the present invention 1031, wherein a plurality of nucleic acid molecules are identified in a sample, the plurality of nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other nucleic acid molecules in said sample or in other samples by one or more nucleotides, one or more nucleotide insertions or deletions, one or more copy numbers, one or more transcript sequences, one or more translocations, and / or one or more methylated residues. [This invention 1039] 10. A process for identifying a plurality of nucleic acid molecules in a sample using a system prepared by the method of the present invention 1024, wherein following filling the one or more primary reaction chambers, the process comprises: conducting the primary reaction in the system; obtaining the nucleotide sequence of the target nucleic acid molecule in the sample following the primary reaction; The process further comprises: [The present invention 1040] conducting the primary reaction, providing a sample containing a plurality of target nucleic acid molecules; contacting the sample with a set of primary oligonucleotide primers having a first portion complementary to a portion of the target nucleic acid molecule and a second portion, and with a polymerase to form a polymerase chain reaction mixture; subjecting the polymerase chain reaction mixture to polymerase chain reaction in the primary reaction chamber to produce a set of amplification products; delivering the amplification products to the product capture housing containing a solid support comprising a plurality of individual rows of a plurality of capture subunits, the solid support comprising an array of a plurality of individual micropores, each individual product capture subunit comprising an immobilized capture probe complementary to the second portion, wherein obtaining the nucleotide sequence of the target nucleic acid molecule in the sample is performed within the micropores; The process of the present invention 1039, comprising: [This invention 1041] 1040. The process of claim 1040, wherein the product capture subunit comprises an array of a plurality of individual micropores, each of the micropores having opposite first and second open ends, the first end having a larger diameter and the second end having a smaller diameter than the diameter of the first end. [The present invention 1042] a mesh screen covering the second end of the micropore within the product capture housing; The process of the present invention 1041 further comprises: [This invention 1043] beads containing the immobilized capture probes disposed within the individual micropores; The process of the present invention 1040 further comprising: [This invention 1044] The process of claim 1040, further comprising removing at least one second portion from said amplification product before obtaining said nucleotide sequence and after subjecting said polymerase chain reaction mixture to polymerase chain reaction. [This invention 1045] 1044. The process of claim 1044, wherein said removing is carried out using uracil DNA glycosylase, apurinic / apyrimidinic endonuclease, endonuclease III, endonuclease IV, endonuclease V, alkyladenine DNA glycosylase, formamidopyrimidine DNA glycosylase, or 8-oxyguanine DNA glycosylase, or a combination thereof. [The present invention 1046] the product capture subunit comprises an array of individual micropores; each of the micropores having opposing first and second open ends; the first end has a larger diameter and the second end has a smaller diameter than the diameter of the first end; a first passageway in fluid communication with a first end of the micropore; and a second passageway in fluid communication with and separated from the second end of the micropore by a mesh screen covering the second end of the micropore; wherein obtaining the nucleotide sequence comprises the following steps in the order shown: directing the product of a polymerase chain reaction through the first passageway to the product capture housing; passing a hydrophobic liquid through the first passageway so as to distribute the product into individual microwells; passing a hydrophobic liquid through said first and second passageways; amplifying said products in a polymerase chain reaction and / or isothermal reaction using said capture oligonucleotide primers immobilized on the interior surface of said microwells under conditions to produce amplification products; passing a volatile solvent through the first passageway while passing a hydrophobic liquid through the second passageway; denaturing the products of the polymerase chain reaction and / or isothermal reaction while passing a hydrophobic liquid through the second passageway so as to isolate the products into individual microwells, and washing unimmobilized nucleic acid molecules away from the first passageway; passing a hydrophobic liquid having a density greater than water through the first passageway while passing a volatile solvent, air, and sequencing reagents through the second passageway; and performing a sequencing reaction on said product capture subunit. executed by a process containing The process of the present invention 1039. [This invention 1047] 1. A process for preparing a microtiter plate for identifying a plurality of nucleic acid molecules in a sample, comprising: providing a microtiter plate comprising a plurality of individual rows and columns of product capture subunits, each individual product capture subunit comprising an array of a plurality of individual hydrophilic microwells separated by a hydrophobic surface; filling a set of aqueous liquids containing oligonucleotide primers and / or probes into the set of microwells of the microtiter plate; centrifuging the microtiter plate to distribute the aqueous liquid into the unfilled microwells of each individual product capture subunit within the microtiter plate; terminating the centrifugation so that the aqueous liquid is not in contact with the hydrophobic surface; evaporating the aqueous liquid; drying the microwells so as to leave the oligonucleotide primers in the microwells; The process comprising: [This invention 1048] 1. A process for identifying a plurality of nucleic acid molecules in a sample using a microtiter plate prepared by the process of the present invention 1047, comprising: depositing an aqueous sample into said microtiter plate; applying a hydrophobic liquid to the microtiter plate such that the hydrophobic liquid is positioned above the aqueous sample; centrifuging the microtiter plate to distribute the aqueous liquid into unfilled microwells within the microtiter plate; terminating the centrifugation so that the sample is not in contact with the hydrophobic surface; conducting an amplification reaction of the nucleic acid molecule under conditions in which a polymerase, exonuclease, endonuclease, or ribonuclease cleaves one or more probes comprising a quencher group and a fluorescent group in a target-specific manner, such that when the target nucleic acid molecule is present in the sample, the fluorescent group is released and a signal is generated; The process comprising: [This invention 1049] The process of the present invention 1048, wherein a plurality of nucleic acid molecules are identified in a sample, the plurality of nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other nucleic acid molecules in the sample or in other samples by one or more nucleotides, one or more nucleotide insertions or deletions, one or more copy numbers, one or more transcript sequences, one or more translocations, and / or one or more methylated residues. [The present invention 1050] with filler port; Exit and; a cartridge in fluid communication with the inlet and the outlet and defining a space; 1. A system for identifying a plurality of nucleic acid molecules in a sample, comprising: The space is a product capture housing containing a solid support comprising a plurality of individual rows of product capture subunits, each individual product capture subunit comprising an array of a plurality of individual hydrophilic micropores separated by a hydrophobic surface, each of the micropores having opposite first and second open ends, the first end having a larger diameter and the second end having a smaller diameter than the diameter of the first end; the product capture housing comprising a plurality of fluid flow paths located above and below the solid support such that a substance can pass from the inlet through the row of product capture subunits to contact the array of micropores within the subunits and to the outlet; Including, The system. [This invention 1051] one or more valves for selecting whether to direct reagents and / or reactants into or out of the product capture housing through the inlet or the outlet; The system of the present invention 1050 further includes: [This invention 1052] The system of claim 1050, further comprising one or more heating elements within said cartridge proximate said product capture housing. [This invention 1053] 1. A method for preparing a system for identifying a plurality of nucleic acid molecules in a sample, comprising: Providing a system of the present invention 1050; adding a capture oligonucleotide primer or probe to the micropore of the product capture subunit on the solid support within the product capture housing, thereby retaining the capture oligonucleotide primer or probe within the micropore or microwell; The method comprising: [This invention 1054] 10. A process for identifying a plurality of nucleic acid molecules in a sample using a system prepared by the method of the present invention 1053, wherein following said adding capture oligonucleotide primers or probes to said micropores, said process comprises: carrying out the reaction in the system; and detecting the presence of a target nucleic acid molecule in the sample within the micropore based on the performance of the reaction; The process comprising: [Brief explanation of the drawings]

[0037] [Figure 1]Figures 1A-1D show schematic diagrams of a solid support composed of subcompartments suitable for fluidic coupling to a cartridge. Each subcompartment contains a micropore or microwell for subsequent qPCR, UniTaq, FRET, qLDR, or sequencing reactions and target identification. In Figure 1A, each subcompartment is 400 microns wide by 600 microns long (shown as a rectangular cross-section) and contains 24 micropores or microwells with a diameter of 50 microns. Additional 100-micron-wide ridges are used between subcompartments to provide spacing and additional structural support. These are represented by the "white" areas between the rows and columns of the rectangular subcompartments. In Figure 1B, each subcompartment is 600 microns wide by 400 microns long (shown as a rectangular cross-section) and contains 2,760 micropores or microwells with a diameter of 5 microns. Additional 100-micron-wide ridges are used between subcompartments to provide spacing and additional structural support. In Figure 1C, each subcompartment is 800 microns wide by 1,200 microns long (shown as a rectangular cross-section) and contains 96 micropores or microwells with a diameter of 50 microns. An additional 200 micron wide ridge is used between the subcompartments to provide spacing and additional structural support. In Figure 1D, each subcompartment is 400 microns wide by 600 microns long (shown as a rectangular cross-section) and contains 2,760 micropores or microwells with a diameter of 5 microns. An additional 100 micron wide ridge is used between the subcompartments to provide spacing and additional structural support. [Figure 2]A schematic front view of the fluidic connection of a microchannel to an array of 50-micron diameter microwells or micropores is shown. Figure 2 shows a schematic front view of an exemplary design of a pre-chamber that allows for fluidic transfer of liquids to chambers containing thousands of microwells or micropores. In this illustration, the input sample is fluidically connected to a large hexagonal chamber (bottom), which is fluidically connected to a first set of 12 diamond-shaped chambers (every fourth chamber contains a large trough, a medium trough, and a small trough), which is fluidically connected to a second set of 24 diamond-shaped chambers (every third chamber contains a large trough and a small trough), which is fluidically connected to 24 elongated mixing chambers, which are fluidically connected to chambers containing microwells or micropores (only two rows are shown at the top of the panel in the enlarged front view). [Figure 3] A schematic front view of the microchannel fluidic connection to an array of 5-micron diameter micropores is shown. Figure 3 shows a schematic front view of another exemplary design of a pre-chamber that allows fluid transfer of liquid to a chamber containing millions of micropores, suitable for Taqman™ or sequencing reactions. In this illustration, the input sample is fluidically connected to a large hexagonal chamber (bottom), which is fluidically connected to a first set of eight hexagonal chambers (every fourth chamber contains a large trough and a small trough), which is fluidically connected to a second set of 16 hexagonal chambers (every third chamber contains a large trough and a small trough), which is fluidically connected to 16 elongated mixing chambers, which are fluidically connected to chambers containing microwells or micropores (only two rows are shown at the top of the panel in the enlarged front view). [Figure 4]Figures 4A-4C show a schematic front view (Figure 4A), a cross-section along line BB in Figure 4A (Figure 4B), and a cross-section along line CC in Figure 4A (Figure 4C) of a 50 micron microwell in a solid support, illustrating how ridges between chambers connect the plates to help direct fluid flow and provide structural stability. This illustration also applies to 5 or 2.5 micron micropores, although more micropores are depicted within each chamber. In one embodiment, the vertical ridges are flush with the top and bottom plates, and the horizontal ridges have indentations or channels that allow fluid to flow up the columns but not from one column to the next. [Figure 5] Figures 5A-5C show a schematic front view (Figure 5A), a cross-section along line BB in Figure 5A (Figure 5B), and a cross-section along line CC in Figure 5A (Figure 5C) of a 50-micron microwell in a solid support, illustrating how the ridges between the chambers connect the two plates to facilitate fluid flow direction and provide structural stability. This illustration also applies to 5 or 2.5 micron micropores, although more micropores are depicted within each chamber. In this illustration, the front of the chamber is the area between the light-colored plate and the wide-diameter side of the micropore, and the back of the chamber is the area between the dark-colored plate and the narrow-diameter side of the micropore. The back plate can be pressed against a heating element to enable temperature control, heating, and / or thermal cycling. In one embodiment, the vertical ridges are flush with the top and bottom plates, and the horizontal ridges have indentations or channels that allow fluid to flow up the column but not from one column to the next. [Figure 6]Figures 6A-6C show a schematic front view (Figure 6A), a cross-section along line BB in Figure 6A, and a cross-section along line CC in Figure 6A, similar to Figure 13. However, in this figure, it is shown how the bottom of the 50, 5, or 2.5 micron micropores comprises another layer of 0.5 micron holes on a 200-400 nanometer thick silicon nitride substrate. This layer allows the 5 or 2.5 micron micropores to be filled with liquid from the front, while the 0.5 micron pores on the back allow air to pass through but prevent liquid from leaking. In this figure, the front of the chamber is the area between the light plate and the wide-diameter side of the micropore, and the back of the chamber is the area between the dark plate and the narrow-diameter side of the micropore. The back plate can be pressed against a heating element to allow temperature control, heating, and / or thermal cycling. [Figure 7]7A-7I show schematic front views of various pre-chamber designs that can perform various operations, including mixing different reagents, performing various amplification reactions, or storing portions of the amplification reactions for later use in subsequent reactions or for fluid transfer to a chamber containing microwells or micropores. Figure 7A shows a chamber with a trough for retaining small amounts of reactants after draining. Figure 7B shows a chamber with a trough for retaining medium amounts of reactants after draining. Figure 7C shows a chamber with a trough for retaining large amounts of reactants after draining. Figure 7D shows a chamber with two troughs for retaining one or two small amounts of reactants after draining. Figure 7E shows a chamber with two troughs for retaining one medium amount and / or one small amount of reactant after draining. Figure 7F shows a chamber with a trough for retaining large amounts of reactants after draining and an additional barrier to ensure the second reaction fluid moves downward and thoroughly mixes with the remaining products from the first reaction. Figure 7G is similar to Figure 7A, but reagents are loaded from the side of the chamber rather than the bottom. Figure 7H is similar to Figure 7G, but more of the product is retained at the bottom of the chamber. Figure 7I is similar to Figure 7H, but with some additional sections that allow for the aqueous and oil layers to move independently. In Figure 7I, the chamber is similar to Figure 7H, but with some additional sections. [Figure 8]Figures 8A-8C show schematic front views of various pre-chamber designs that allow fluid transfer of liquids into chambers containing microwells or micropores. Figure 8A shows an example in which primers and / or probes (gray circles) fluidically coupled within eight chambers exit the chambers, enter a more elongated chamber, and then into a row of microwells or micropores, ultimately drying within the microwells or micropores and covalently bonding to the interior surface. Figure 8B shows an example in which reagents fluidically coupled to a 4+4 chamber exit the chambers and enter a more elongated chamber. The left side is made of a coated or extremely hydrophobic plastic, while the right side is slightly hydrophobic or somewhat hydrophilic. Figure 8C is similar to Figure 8A, but has only four chambers and includes additional plastic ridges or dividers. [Figure 9] Figures 9A-9B show schematic side views of an embodiment for filling a micropore, as illustrated in Figures 5A and 6B. Figure 9A shows a micropore that is open on both the top and bottom. Primers (and probes) are fluidically introduced into the micropore from the top while oil is introduced from the bottom. The oil then sweeps the aqueous solution away from the top region, fluidically separating the primers / probes. The primers are then fixed and allowed to dry. Figure 9B shows a micropore that is open on the top side and has a separate layer of 0.5 micron holes on top of a 200-400 nanometer thick silicon nitride. This layer allows liquid to be filled from the front of the 50, 5, or 2.5 micron micropores, while the 0.5 micron holes on the bottom allow air to pass through but prevent liquid from leaking. [Figure 10]A schematic front view of an embodiment in which reaction chambers are filled before filling microwells or micropores is shown. This configuration consists of two sets of reaction chambers, each with a trough, with the second set pre-spotted with the appropriate ligation probe oligonucleotides (gray circles). After a light oil cap is introduced at the bottom, an aqueous liquid containing the target, PCR primers, and PCR reagents is introduced and then fluidically transferred to the first set of reaction chambers using heavy oil. After the PCR step, the oil and most of the aqueous reactants are drained, leaving a portion of the product in the troughs of the first two chambers. After filling the chambers again with light oil, LDR reagents and enzymes are loaded, and then this aqueous reaction mixture is fluidically transferred to the second set of reaction chambers (where it mixes with the pre-spotted LDR primers) using heavy oil. [Figure 11]Figures 11A-11B show schematic front-on views of an embodiment for filling micropores, such as those illustrated in Figures 5A and 6B, to perform real-time PCR reactions, such as Taqman™ or UniTaq reactions. The figures begin with a micropore that has been pre-filled and dried with one to four UniTaq primer sets (or alternatively, one to four universal tag primer sets with target-specific Taqman™ probes). The figures are not to scale and are for illustrative purposes. In Figure 11A, tailed target or ligated probes are fluidly introduced into the micropore from the bottom front while oil is introduced from the bottom back. The oil inflow from the front subsequently forces aqueous liquid out of the non-productive volume and into the micropore, simultaneously coating each individual micropore at the front. In Figure 11B, all surfaces are hydrophobic except for the inner surface of the micropore and the silicon nitride with 0.5 micron pores. When aqueous fluid is injected from the bottom front, it enters the micropores from the front and pushes out the air from the back, but does not pass through the 0.5 micron silicon nitride pores. When aqueous fluid fills the micropores from the front, oil flows in from the front, pushing the aqueous fluid out of the non-productive volume and into the micropores, simultaneously covering each individual micropore on the front with oil. Oil may also be filled into the back of the chamber. Each micropore is fluidically isolated, suitable for subsequent independent amplification and thermal cycling reactions. [Figure 12]A schematic side view of an embodiment for filling micropores, such as that illustrated in Figure 6, to perform sequencing reactions is shown. In this example, all surfaces are hydrophobic except for the inner surface of the micropore and the silicon nitride with 0.5 micron holes. Aqueous fluid is injected from the lower front face, entering the micropore from the front and pushing air out the back face, but not through the 0.5 micron silicon nitride holes. When aqueous fluid is filled into the micropore from the front face, oil flows in from the front face, pushing the aqueous fluid out of the non-productive volume and into the micropore, simultaneously covering each individual micropore at the front face with oil. Oil is also filled at the back face. Each micropore is fluidically isolated and suitable for subsequent independent thermocycling reactions that amplify template strands and anchor them to solid supports on the inner surface of the micropore. The oil is then pumped out of the front chamber, denaturing the opposite strand products, while the other products and primers are washed away. A heavy oil plug is used to seal the bottom of the front chamber while rinsing the back, delivering target strands clonally propagated and immobilized in micropores suitable for sequencing to the array. [Figure 13]Figures 13A-13B show schematic front views of chamber formats using the microwells or micropores described in Figures 1 and 6. Figure 13A shows a microwell format in which the subcompartments are 800 microns wide by 1200 microns long (shown as rectangular cross-sections) and contain 96 microwells with a diameter of 50 microns. Additional 200-micron-wide ridges are used between the subcompartments to provide spacing and additional structural support. These are represented as "white" areas between the rows and columns of the rectangular subcompartments. Figure 13B is a schematic of a microfluidic chamber for sequencing on an array of micropores in a microtiter plate format. The close-up shows only two double columns and one double row of subcompartments, each containing 2,072 micropores. In one embodiment, the micropore-containing chambers are fed using acoustic droplet ejection through a series of individual openings that can be fluidically closed or fluidically opened, allowing reagents, enzymes, targets, or pre-amplified targets to flow into all chambers in a given row. When using acoustic droplet ejection, droplets can be fed into a series of hydrophilic input chambers, which then feed into an array of micropores, facilitating fluid flow into individual openings. In this schematic, each individual opening is connected to a hydrophilic input chamber, which feeds two rows of micropores. The chambers are also fluidically connected to allow reagents to flow into all of the chambers through a single inlet and out a single waste or outlet port on the other side. Once the hydrophilic input chambers are properly filled with reagents, enzymes, targets, or pre-amplified targets, these openings are closed, and oil or other reagents are then added through a single inlet, allowing the input solution to fluidically move into the micropores for further reaction. [Figure 14]A schematic side view of a microtiter plate format using microwells within chambers as described in Figure 13A is shown, suitable for pre-filling with appropriate primers and probes. Step A shows a side view of one chamber within a hydrophobic plate. This contains a 50-micron hydrophilic well with ridges on both sides. In step B, the plate is inverted and filled with one to four UniTaq primer sets (or alternatively, one to four universal tag primer sets equipped with mutation- or methylation-specific Taqman™ probes) using acoustic droplet ejection. In step C, the plate is centrifuged, dispersing the aqueous liquid into the empty microwells. In contrast, step D shows droplets formed on the microwells after centrifugation, ensuring that the aqueous solution avoids the hydrophobic surface. In step E, the aqueous solution is evaporated, leaving the wells with dried primer / probe sets (shown in step F). [Figure 15] Figure 13A shows a schematic side view of a microtiter plate format using microwells within the chambers described above, pre-filled with the appropriate Taqman™ or UniTaq primers and probes. Step A shows a side view of one chamber within a hydrophobic plate. This contains 50-micron hydrophilic wells with ridges on both sides. In step B, the plate is inverted and filled with reagents appropriate for real-time amplification (i.e., Taqman™ reaction) and target DNA using acoustic droplet ejection. In step C, the aqueous layer is overlaid with hydrophobic mineral oil. In step D, the plate is transferred to a swinging bucket rotor and centrifuged. The concentrated aqueous liquid disperses into the empty microwells. In step E, the plate is transferred to a thermocycler. The droplets are separated into individual microwells covered with mineral oil and suitable for amplification. [Figure 16] 1 shows an exemplary PCR-PCR-qPCR procedure with Taqman™ readout for identification or relative quantification of unknown pathogens. [Figure 17] An exemplary PCR-PCR-qPCR procedure using UniTaq readout for identification or relative quantification of unknown pathogens is shown. [Figure 18]An exemplary PCR-PCR-qPCR procedure using split-probe UniTaq (UniRq) readout for identification or relative quantification of unknown pathogens is shown. [Figure 19] 1 shows an exemplary PCR-LDR-qPCR procedure with Taqman™ readout for identification or relative quantification of unknown pathogens. [Figure 20] An exemplary PCR-LDR-qPCR procedure with UniTaq readout for identification or relative quantification of unknown pathogens is shown. [Figure 21] An exemplary PCR-LDR-qPCR procedure using split-probe UniTaq (UniSpTq) readout for identification or relative quantification of unknown pathogens is shown. [Figure 22] An exemplary PCR-qLDR (UniLDq) procedure using universal split-probe readout for identification or relative quantification of unknown pathogens is shown. [Figure 23] An exemplary PCR-qLDR (TsLDq) procedure using target-specific split-probe readout for identification or relative quantification of unknown pathogens is shown. [Figure 24] A schematic front view of a portion of an exemplary design for pre-chamber filling, which allows for fluid transfer of liquid into a chamber containing microwells or micropores, is shown. The design shows a chamber structure and microwells or micropores suitable for performing multiplex PCR-nested PCR-UniTaq detection (or multiplex PCR-nested PCR-real-time PCR using target-specific Taqman™ probes) for the identification and quantification of unknown pathogens. Gray circles symbolize row or column regions that are pre-filled with different primer or probe sets. [Figure 25]Figures 25A-25B show schematic side views of a cartridge and valve configuration for performing multiplex PCR-nested PCR-real-time PCR assays using UniTaq or target-specific Taqman™ probes using a micropore plate containing thousands of micropores. Figure 25A is a schematic front view showing the fluidic connection of the microchannels to an array of 50-micron diameter microwells or micropores. In Figure 25B, the micropore plate is fluidically accessible from both sides of the pores. The first side (top of the plate, shown on the left side of the plate) is in communication with valves 1, 2, and 3, while the second side (bottom of the plate, shown on the right side of the plate) is in communication with valves 4 and 5. [Figure 26] An exemplary PCR-PCR-qPCR procedure with Taqman™ readout is shown for identification or relative quantification of unknown pathogens directly from blood. [Figure 27] An exemplary PCR-PCR-qPCR procedure with UniTaq readout for identification or relative quantification of unknown pathogens directly from blood is shown. [Figure 28] 1 shows an exemplary PCR-LDR-qPCR carryover prevention reaction with Taqman™ readout for identifying or relative quantitation of low level mutations. [Figure 29] 1 shows an exemplary PCR-LDR-qPCR carryover prevention reaction with UniTaq readout for identifying or relative quantitation of low-level mutations. [Figure 30]Figure 1 shows a front view of a portion of an exemplary design for pre-chamber filling, which allows for fluid transfer of liquid into a chamber containing microwells or micropores. This design shows a chamber structure and microwells or micropores suitable for performing multiplex PCR-LDR-UniTaq detection to identify and quantitate low levels of unknown mutations in plasma. (Alternatively, multiplex PCR-LDR-real-time PCR using mutation-specific Taqman™ probes is used.) Gray circles symbolize row or column regions pre-filled with different primer or probe sets. [Figure 31] 1 shows an exemplary PCR-LDR-qPCR (optionally with carryover prevention) reaction using Taqman™ readout to identify or relative quantitate low-level methylation. [Figure 32] 1 shows an exemplary PCR-LDR-qPCR (optionally with carryover prevention) reaction using UniTaq readout to identify or relative quantitate low-level methylation. [Figure 33] 1 shows an exemplary RT-PCR-LDR-qPCR reaction with UniTaq readout for identifying or relative quantitation of wild-type and alternatively spliced ​​mRNA transcripts. [Figure 34] Figure 1 shows a front view of a portion of an exemplary design for pre-filling chambers, which allows for fluid transfer of liquid into chambers containing microwells or micropores. This design shows a chamber structure and microwells or micropores suitable for performing multiplex RT-PCR-LDR-UniTaq detection to identify and quantify both rare and overexpressed lncRNAs, mRNAs, or splice variants. (Alternatively, multiplex PCR-LDR-real-time PCR using target-specific Taqman™ probes is used.) Gray circles symbolize row or column regions pre-filled with different primer or probe sets. [Figure 35]An exemplary fragment identifier PCR method using sequencing-by-synthesis readout to identify mutations in one strand of an unknown pathogen is shown. In this example, the product is dispensed into a micropore containing an immobilized second tag sequence primer, or onto a bead in the micropore. [Figure 36] An embodiment of a fragment identifier PCR method is shown in which the first tag primer is present in greater abundance than both the solution and the immobilized second tag primer (which is longer than the first tag primer) to maximize product yield per micropore. [Figure 37] Another embodiment of the fragment identifier PCR method is shown in which the first tag primer in solution contains a primer with two different 5' portions and a 5' extension portion and is present in greater amount than both the solution and the immobilized second tag primer (which is longer than the first tag primer) to maximize product yield per micropore. [Figure 38] Another embodiment of the fragment identifier PCR method is shown in which the first tag primer in solution contains dA35 and a primer with dA35 added to a GC-rich scaffold, and is present in greater amount than both the solution and the immobilized second tag primer (which is longer than the first tag primer) to maximize product yield per micropore. [Figure 39]

[0023] Figure 1 shows a front view of a portion of an exemplary design of a pre-chamber filling system that allows fluid transfer of liquid into a chamber containing microwells or micropores. This design shows a chamber structure and microwells or micropores suitable for performing multiplex PCR-nested PCR-sequencing for unknown pathogen identification. Gray circles symbolize row or column regions that are pre-filled with different primer or probe sets. The figure is not to scale and is for illustrative purposes. [Figure 40]An exemplary fragment identifier PCR method using sequencing-by-synthesis readout to identify low-abundance mutations in one target strand of cfDNA is shown. In this example, the product is dispensed into a micropore containing an immobilized second tag sequence primer, or onto beads. [Figure 41] An exemplary fragment identifier PCR method using sequencing-by-synthesis readout to identify low-abundance mutations in one target strand across overlapping fragments of cfDNA is shown. In this example, a second tag sequence primer is biotinylated and captured onto streptavidin-coated beads and dispensed into a micropore, or dispensed directly into a streptavidin-coated micropore. [Figure 42] An exemplary fragment identifier PCR method using sequencing-by-synthesis readouts to identify low-abundance mutations in one target strand across overlapping fragments of cfDNA is shown. In this example, the product is dispensed into a micropore containing an immobilized second tag sequence primer, or onto beads. [Figure 43] Further details of PCR amplification using biotinylated or immobilized second tag sequence primers are presented, demonstrating that shorter amplicons form panhandle structures that do not amplify, while the desired longer product amplifies on the solid support. [Figure 44] Figure 1 shows another embodiment of an exemplary fragment identifier PCR method using sequencing-by-synthesis readout to identify low-abundance mutations in one target strand across overlapping fragments of cfDNA. In this figure, two target-specific primers containing a second tag sequence are shown. In this example, the product is dispensed into a micropore containing an immobilized second tag sequence primer, or a bead is dispensed into the micropore. [Figure 45]Figure 1 shows another embodiment of an exemplary fragment identifier PCR method using sequencing-by-synthesis readout to identify low-abundance mutations in one target strand across overlapping fragments of cfDNA. In this figure, two target-specific primers containing a first tag sequence are shown. In this example, the product is dispensed into a micropore containing an immobilized second tag sequence primer, or a bead is dispensed into the micropore. [Figure 46] An exemplary fragment identifier PCR method using sequencing-by-synthesis reads to identify low-abundance mutations in both target strands across overlapping fragments of cfDNA is shown. In this example, the product is dispensed into a micropore containing a fixed first tag sequence primer, or beads are dispensed into a micropore. By using a different nested primer containing a second tag sequence, the region amplified from the top strand differs from the region amplified from the bottom strand, allowing the reads obtained from the top and bottom strand sequences to be distinguished. [Figure 47] An exemplary fragment identifier PCR method using sequencing-by-synthesis reads is shown for identifying SNPs and counting the copy number of both locus-specific strands of cfDNA. In this example, the product is dispensed into a micropore containing an immobilized first tag sequence primer, or a bead into the micropore. By using a different nested primer containing a second tag sequence, the region amplified from the top strand differs from the region amplified from the bottom strand, allowing the reads obtained from the top and bottom strand sequences to be distinguished. [Figure 48]Figure 1 shows a front view of a portion of an exemplary design for pre-filling chambers containing microwells or micropores, allowing fluid transfer of liquids into chambers containing microwells or micropores. This design shows a chamber structure and microwells or micropores suitable for performing multiplex PCR-nested PCR-sequencing to identify low-abundance unknown mutations in plasma or for noninvasive prenatal testing of trisomies in plasma. Gray circles represent row or column regions pre-filled with different primer or probe sets. [Figure 49] Figures 49A-49B show a schematic side view of a cartridge and valve configuration for identifying low-abundance, unknown mutations in plasma using fragment identifier PCR-sequencing. Figure 49A is a schematic front view showing the fluidic connection of a microchannel to an array of 5-micron diameter micropores. Figure 49B shows a fragment identifier PCR-sequencing fluidic system using a micropore plate containing millions of micropores. The micropore plate is fluidically accessible from both sides of the pores. The first side (top of the plate, shown on the left side of the plate) is in communication with valves 1, 2, and 3, while the second side (bottom of the plate, shown on the right side of the plate) is in communication with valves 4, 5, and 6. [Figure 50] Figure 1 shows a schematic side view of a cartridge and valve configuration for identifying low-abundance, unknown mutations in plasma using fragment identifier PCR-sequencing. Step A involves providing a microplate with microfluidic channels fluidically connected to an array of 5-micron diameter micropores. Step B shows performing initial reactions in individual wells, followed by the use of acoustic droplet ejection to flow appropriate reagents, enzymes, buffers, target, and / or pre-amplified target into an input chamber and openings leading to an array containing millions of micropores. Step C shows the plate fluidically connected to four valves. The micropore plate is fluidically accessible from both sides of the pores. The first side (shown on the top of the plate) is in communication with valves 1 and 3, while the second side (shown on the bottom of the plate) is in communication with valves 2 and 4. [Figure 51]An exemplary Bsh1236I-bisulfite-fragment discriminator PCR method using sequencing-by-synthesis readout to identify low-abundance methylations in one target strand of cfDNA is shown. In this example, the product is dispensed into a micropore containing an immobilized second tag sequence primer, or onto beads. [Figure 52] FIG. 1 shows a front view of a portion of an exemplary design of a pre-chamber loading device that allows fluid transfer of liquid into a chamber containing microwells or micropores. The design shows a chamber structure and microwells or micropores suitable for performing multiplex PCR-nested PCR-sequencing and Bsh1236I-bisulfite-multiplex PCR-nested PCR-sequencing to identify low-abundance unknown mutations and methylations in plasma. [Figure 53] An exemplary fragment discriminator RT-PCR method using sequencing-by-synthesis readouts to identify low- and medium-abundance lncRNAs, mRNAs, and splice site variants isolated from CTCs or exosomes is shown. In this example, the product is dispensed into a micropore containing an immobilized second tag sequence primer, or onto beads. [Figure 54] Figure 1 shows a front view of a portion of an exemplary design for pre-filling chambers, allowing fluid transfer into chambers containing microwells or micropores. This design shows a chamber structure and microwells or micropores suitable for performing multiplex RT-PCR-nested PCR-UniTaq detection to identify low- and medium-abundance lncRNAs, mRNAs, and splice site variants isolated from CTCs or exosomes. (Alternatively, multiplex PCR-nested PCR-real-time PCR using transcript-specific Taqman™ probes.) Gray circles represent row or column regions pre-filled with different primer or probe sets. DETAILED DESCRIPTION OF THE INVENTION

[0038] Detailed Description of the Invention One aspect of the present invention relates to a system for identifying multiple nucleic acid molecules in a sample. The system includes an inlet and a cartridge. The cartridge defines a space containing multiple primary reaction chambers fluidly connected to the inlet, which receive material from the inlet and generate primary reaction chamber products from the material. The space also includes a product capture housing containing a solid support with multiple individual rows of multiple product capture subunits, each individual product capture subunit containing an array of multiple individual hydrophilic micropores or microwells separated by a hydrophobic surface, where the primary reaction products are further reacted to generate array products. The array products are detected in the micropores or microwells, where one or more rows of individual product capture subunits receive material that has passed through one of the multiple primary reaction chambers.

[0039] In one embodiment, the system for identifying a plurality of nucleic acid molecules in a sample of the present invention further comprises an outlet for expelling material from the product capture housing.

[0040] In another embodiment of the system for identifying multiple nucleic acid molecules in a sample of the present invention, the space defined by the cartridge further includes one or more initial reaction chambers into which materials are discharged from an inlet, from which materials are discharged into multiple primary reaction chambers.

[0041] In yet another embodiment of the system for identifying multiple nucleic acid molecules in a sample of the present invention, the space defined by the cartridge further includes a plurality of secondary reaction chambers, one or more of which are fluidly coupled to one of the plurality of primary reaction chambers and receive material from one of the plurality of primary reaction chambers. The space also includes a plurality of mixing chambers, each fluidly coupled to one of the plurality of secondary reaction chambers, into which material from one of the plurality of secondary reaction chambers is received and which discharges material into the product capture housing, such that each row of individual product capture subunits is fluidly coupled to one of the one or more mixing chambers and receives material from one of the one or more mixing chambers.

[0042] According to this embodiment of the system for identifying multiple nucleic acid molecules in a sample of the present invention, at least some of the multiple primary and secondary reaction chambers are configured to hold troughs of liquid within the multiple primary and secondary reaction chambers.

[0043] In another embodiment of the system for identifying multiple nucleic acid molecules in a sample of the present invention, wherein the space defined by the cartridge further includes multiple secondary reaction chambers and multiple mixing chambers, each of the multiple primary and / or secondary reaction chambers has an internal baffle that holds a trough of liquid within the multiple primary and secondary reaction chambers.

[0044] In yet another embodiment of the system for identifying multiple nucleic acid molecules in a sample of the present invention, wherein the space defined by the cartridge further includes multiple secondary reaction chambers and multiple mixing chambers, each of the multiple primary and / or secondary reaction chambers has one or more internal baffles that hold multiple troughs of liquid within the multiple primary and secondary reaction chambers.

[0045] In a further embodiment of the system for identifying multiple nucleic acid molecules in a sample of the present invention, wherein the space defined by the cartridge further includes multiple secondary reaction chambers and multiple mixing chambers, each of the mixing chambers includes a partition extending from near where the material enters the mixing chamber to near where the material exits the mixing chamber.

[0046] In another embodiment of the system for identifying multiple nucleic acid molecules in a sample of the present invention, in which the space defined by the cartridge further includes multiple secondary reaction chambers and multiple mixing chambers, each of the mixing chambers includes a first surface that is highly hydrophobic and a second surface that is spaced apart from the first surface and is less hydrophobic than the first surface, and the first and second surfaces extend from near where the substance enters the mixing chamber to near where the substance exits the mixing chamber.

[0047] In yet another embodiment of the system for identifying multiple nucleic acid molecules in a sample of the present invention, wherein the space defined by the cartridge further comprises multiple secondary reaction chambers and multiple mixing chambers, the primary reaction chamber and / or the secondary reaction chamber comprises an interior surface onto which oligonucleotide primers or probes can be spotted.

[0048] In another embodiment of a system for identifying multiple nucleic acid molecules in a sample according to the invention, the product capture subunit comprises an array of multiple individual micropores, each having opposite first and second open ends, the first end having a larger diameter and the second end having a smaller diameter than the diameter of the first end.

[0049] The system may further comprise a mesh screen covering the second ends of the micropores or beads disposed within the individual micropores within the product capture housing.

[0050] In a further embodiment of the system for identifying multiple nucleic acid molecules in a sample of the present invention, the product capture subunit comprises an array of multiple individual microwells, each having an open end and a closed end.

[0051] In another embodiment of the system for identifying multiple nucleic acid molecules in a sample of the present invention, in which the space defined by the cartridge further includes multiple secondary reaction chambers and multiple mixing chambers, the product capture housing includes multiple fluid flow paths to allow materials to pass from the multiple mixing chambers through the row of product capture subunits and into contact with the array of micropores or microwells within those subunits.

[0052] The multiple fluid channels may be located above and / or below the solid support.

[0053] In another embodiment of the system for identifying multiple nucleic acid molecules in a sample of the present invention, the system may further include one or more valves for selecting whether to introduce reagents or reactants into or out of the cartridge through the inlet.

[0054] In further embodiments of the system for identifying multiple nucleic acid molecules in a sample of the present invention, the system further comprises one or more valves for selecting whether to direct reagents or reactants into or out of the product capture housing through an outlet and / or a location within the product capture housing spaced from the outlet.

[0055] In yet another embodiment of the system for identifying multiple nucleic acid molecules in a sample of the present invention, the system further comprises one or more heating elements in the cartridge proximate to the primary reaction chamber and / or the product capture housing.

[0056] In another embodiment of the system for identifying multiple nucleic acid molecules in a sample of the present invention, the space defined by the cartridge further includes one or more initial reaction chambers into which materials are discharged from the inlet, and from which the materials are discharged into multiple primary reaction chambers. The system may further include one or more heating elements in the cartridge adjacent to the initial reaction chambers.

[0057] In another embodiment of the system for identifying multiple nucleic acid molecules in a sample of the present invention, when the space defined by the cartridge further includes multiple secondary reaction chambers and multiple mixing chambers, the system may further include one or more heating elements within the cartridge adjacent to one of the secondary reaction chambers and / or one or more of the mixing chambers.

[0058] Another aspect of the present invention relates to a system for identifying multiple nucleic acid molecules in a sample. The system includes a cartridge including an inlet, an outlet, and an array of micropores or microwells, the cartridge being fluidically connected to the inlet and the outlet. The cartridge defines a volume including multiple primary reaction chambers in fluid communication with the inlet, which receive material from the inlet and produce a primary reaction-chamber product from the material. The volume also includes multiple secondary reaction chambers, one or more of which are fluidly connected to one of the multiple primary reaction chambers and receive material from one of the multiple primary reaction chambers and produce a secondary reaction-chamber product. At least some of the multiple primary and secondary reaction chambers are configured to hold troughs of fluid within the multiple primary and secondary reaction chambers, facilitating mixing of the sample, reagents, and / or product reactants for subsequent reactions to produce chamber or array products. The space also includes a plurality of mixing chambers, each fluidly coupled to one of the plurality of secondary reaction chambers, for receiving material from one of the plurality of secondary reaction chambers and discharging material into the product capture housing, such that each row of individual product capture subunits is fluidly coupled to one of the one or more mixing chambers and receives material from one of the one or more mixing chambers. The space also includes a product capture housing containing a solid support with a plurality of individual rows of multiple product capture subunits, each individual product capture subunit comprising an array of a plurality of individual hydrophilic micropores or microwells separated by a hydrophobic surface, where the secondary reaction products are further reacted to produce array products. The array products are detected in the micropores or microwells, where one or more rows of individual product capture subunits receive material that has passed through one of the plurality of primary reaction chambers.

[0059] Another aspect of the present invention relates to a system for identifying multiple nucleic acid molecules in a sample. The system includes an inlet, a second injection location, an outlet, and a cartridge fluidically coupled to the inlet, the second injection location, and the outlet. The cartridge defines a space containing a product capture housing containing a solid support with multiple individual rows of product capture subunits, each of which contains an array of multiple individual micropores. The product capture housing includes multiple fluid flow paths located above and below the solid support so that substances can pass from the inlet and / or the second injection location through the rows of product capture subunits, contact the array of micropores within the subunits, and to the outlet. One or more valves are used to select whether reagents or reactants are directed into or expelled from the product capture housing through the inlet, the outlet, and / or a second injection location separate from the outlet within the product capture housing.

[0060] A further aspect of the invention relates to a method for preparing a system for identifying a plurality of nucleic acid molecules in a sample, the method comprising providing a system of the invention and adding universal tags or capture oligonucleotide primers or probes to micropores or microwells of product capture subunits on a solid support within a product capture housing, such that the universal tags or capture oligonucleotide primers or probes are retained within the micropores or microwells.

[0061] The method for preparing a system for identifying multiple nucleic acid molecules in a sample of the present invention may further include filling one or more primary reaction chambers with primary reaction oligonucleotide probes or primers, each having a first portion containing a nucleotide sequence complementary to a portion of the target nucleic acid in the sample. According to this embodiment, the primary reaction oligonucleotide probes or primers may further contain a second portion containing a nucleotide sequence identical to or complementary to a portion of the universal tag or capture oligonucleotide primer retained in the micropore or microwell.

[0062] A further aspect of the present invention relates to a method for preparing a system for identifying multiple nucleic acid molecules in a sample, the method comprising providing the system of the present invention, the system comprising an inlet and a cartridge, the cartridge defining a space comprising: a plurality of primary reaction chambers in fluid communication with the inlet, each of which receives material from the inlet and produces primary reaction chamber products from the material; a product capture housing containing a solid support having a plurality of individual rows of multiple product capture subunits, each of which comprises an array of a plurality of individual hydrophilic micropores or microwells separated by a hydrophobic surface, wherein the primary reaction products are further reacted to produce array products that are detected in the micropores or microwells; and one or more rows of individual product capture subunits receiving material that has passed through one of the plurality of primary reaction chambers. The product capture housing includes a product capture housing; a plurality of secondary reaction chambers, one or more of which are fluidly coupled to one of the plurality of primary reaction chambers and receive a substance from one of the plurality of primary reaction chambers; and a plurality of mixing chambers, each of which is fluidly coupled to one of the plurality of secondary reaction chambers, receive a substance from one of the plurality of secondary reaction chambers, and discharge a substance into the product capture housing, such that each row of individual product capture subunits is fluidly coupled to one of the one or more mixing chambers and receive a substance from one of the one or more mixing chambers. The method further includes adding a universal tag or capture oligonucleotide primer or probe to a micropore or microwell of a product capture subunit on a solid support within the product capture housing, such that the universal tag or capture oligonucleotide primer or probe is retained in the micropore or microwell.

[0063] The method may further include loading one or more primary and / or secondary reaction chambers with primary or secondary reaction oligonucleotide probes or primers, each having a first portion comprising a nucleotide sequence complementary to a portion of a target nucleic acid in the sample. According to this embodiment, the primary or secondary reaction oligonucleotide probes or primers may further comprise a second portion comprising a nucleotide sequence identical to or complementary to a portion of the universal tag or capture oligonucleotide primer retained in the micropore or microwell.

[0064] In one embodiment of the method for preparing a system for identifying multiple nucleic acid molecules in a sample of the present invention, the product capture subunit comprises an array of individual micropores, each having opposing first and second open ends, the first end having a larger diameter and the second end having a smaller diameter than the first end, a first passage in fluid communication with the first end of the micropore, and a second passage in fluid communication with the second end of the micropore, wherein the universal tag or capture oligonucleotide primer or probe is added to the micropore by a method comprising the following steps in the indicated order: passing a hydrophobic liquid through the second passage while the universal tag or capture oligonucleotide primer or probe passes through the first passage and enters the micropore from the first open end; passing a hydrophobic liquid through the first passage while the hydrophobic liquid passes through the second passage; passing a volatile solvent through the first passage while the hydrophobic liquid passes through the second passage; passing heat, the hydrophobic liquid, the volatile solvent, and air through the second passage while air passes through the first passage.

[0065] In another embodiment of the method for preparing a system for identifying a plurality of nucleic acid molecules in a sample of the present invention, the product capture subunit comprises an array of individual micropores, each having opposite first and second open ends, the first end having a larger diameter and the second end having a smaller diameter than the diameter of the first end, a first passageway in fluid communication with the first ends of the micropores, and a second passageway in fluid communication with the second passageway and separated from the second ends of the micropores by a mesh screen covering the second end, wherein the detection or capture oligonucleotide primers or probes are in the order shown below: The micropore is loaded by a method comprising the steps of: passing a universal tag or capture oligonucleotide primer or probe through a first passage and entering the micropore from a first open end; passing a hydrophobic liquid through the first passage and expelling the universal tag or capture oligonucleotide primer or probe from the first passage; passing the hydrophobic liquid through the first passage while passing the hydrophobic liquid through a second passage; passing a volatile solvent through the first passage while passing the hydrophobic liquid through the second passage; passing air through the first passage while simultaneously passing heat, the hydrophobic liquid, the volatile solvent, and air through the second passage.

[0066] Another embodiment of the present invention relates to a process for identifying multiple nucleic acid molecules in a sample using the system of the present invention, wherein the process, following filling of one or more primary reaction chambers and / or one or more secondary reaction chambers (if present), includes conducting the primary and / or secondary reactions in the system and detecting the presence of target nucleic acid molecules in the sample in the microwells or micropores based on the performance of the primary and / or secondary reactions.

[0067] Another embodiment of the present invention relates to a process for identifying multiple nucleic acid molecules in a sample using the system of the present invention, wherein after performing primary and / or secondary reactions, the products of such reactions are amplified in microwells or micropores under conditions where a polymerase, exonuclease, endonuclease, or ribonuclease cleaves one or more probes containing a quencher group and a fluorescent group in a target-specific manner, liberating the fluorescent group and generating a signal if the target nucleic acid molecule is present in the sample.

[0068] Another embodiment of the present invention relates to a process for identifying multiple nucleic acid molecules in a sample using the system of the present invention. The process for performing a primary reaction and / or a secondary reaction includes providing a sample containing multiple target nucleic acid molecules and contacting the sample with a set of primary oligonucleotide primers having first portions complementary to portions of the target nucleic acid molecules, or the complement of the target nucleic acid molecules, and a polymerase to form a first polymerase extension or chain reaction mixture. This mixture is subjected to a first polymerase extension or chain reaction in one or more initial reaction or primary reaction chambers to produce a first set of extension products or amplification products. The products are then contacted with a set of secondary oligonucleotide primers having first portions complementary to portions of the primary extension products or primary amplification products, and a polymerase to form a second polymerase chain reaction mixture. This second mixture is subjected to a second polymerase chain reaction in a primary or secondary reaction chamber to produce a second set of amplification products. Each secondary amplification product comprises a 5' second partial sequence, a target nucleotide sequence specific portion, or its complement, and a 3' second partial complementary sequence.

[0069] Another embodiment of the present invention relates to a process for identifying multiple nucleic acid molecules in a sample using the system of the present invention. The process for performing a primary reaction and / or a secondary reaction includes providing a sample containing multiple target nucleic acid molecules and contacting the sample with a set of primary oligonucleotide primers having portions complementary to portions of the target nucleic acid molecules or their extension products, and a polymerase to form a first polymerase extension or chain reaction mixture. This mixture is subjected to a first polymerase chain reaction in one or more initial or primary reaction chambers to produce a first set of extension products or amplification products. These products are then contacted with a set of oligonucleotide probes having first and second portions complementary to portions of the first set of amplification products, and a ligase to form a ligase detection reaction mixture. This second mixture is subjected to a ligase detection reaction in a primary or secondary reaction chamber to produce a set of ligation products. Each ligation product comprises a 5' second subsequence, a target nucleotide sequence-specific portion or its complement, and a 3' second subsequence.

[0070] Yet another embodiment of the present invention relates to a process for identifying multiple nucleic acid molecules in a sample using the system of the present invention. The process of filling one or more primary reaction chambers (if present) and running the primary and / or secondary reactions in the system is carried out by a process involving the following steps in the order shown: passing a hydrophobic liquid through the system through an inlet; passing primary reaction oligonucleotide probes or primers, and reverse transcription and / or polymerase chain reaction reagents, plus the hydrophobic liquid, through the system through an inlet; running a polymerase extension or chain reaction in the system, and then passing materials out of the system through the inlet; passing the hydrophobic liquid, polymerase chain reaction reagents or ligase detection reaction reagents, plus the hydrophobic liquid through the inlet, and running a polymerase chain reaction or ligase detection reaction in the system.

[0071] Another embodiment of the present invention relates to a process for identifying multiple nucleic acid molecules in a sample using a system of the present invention, in which the product capture subunit comprises an array of individual micropores. Each micropore has opposing first and second open ends, the first end having a larger diameter and the second end having a smaller diameter than the first end, a first passage in fluid communication with the first end of the micropore, and a second passage in fluid communication with the second end of the micropore. The above-described secondary reaction in the system is carried out by a process involving the following steps in the order shown: The products of a polymerase chain reaction or ligase detection reaction are delivered to a product capture housing through the first passage, while a hydrophobic liquid is passed through the second passage. The hydrophobic liquid is further passed through the first and second passages. The products of the polymerase chain reaction or ligase detection reaction are then subjected to a polymerase chain reaction with universal tag primers and probes within the micropores of the product capture subunit.

[0072] Another embodiment of the present invention relates to a process for identifying multiple nucleic acid molecules in a sample using a system of the present invention, in which the product capture subunit comprises an array of individual micropores. Each micropore has opposing first and second open ends, the first end having a larger diameter and the second end having a smaller diameter than the first end, a first passage in fluid communication with the first end of the micropore, and a second passage in fluid communication with and separated from the second end of the micropore by a mesh screen covering the second end of the micropore. The process for performing a secondary reaction in the system is carried out by the following steps in the order shown: The product of a polymerase chain reaction or ligase detection reaction is delivered to a product capture housing through the first passage; a hydrophobic liquid is passed through the first passage; and a hydrophobic liquid is further passed through the first and second passages. The product of the polymerase chain reaction or ligase detection reaction is subjected to a polymerase chain reaction with universal tag primers and probes within the micropores of the product capture subunit.

[0073] Another embodiment of the present invention relates to a process for using the system of the present invention to identify a plurality of nucleic acid molecules in a sample that include a target nucleotide sequence that differs from the nucleotide sequences of other nucleic acid molecules in the sample or in other samples by one or more nucleotides, one or more nucleotide insertions or deletions, one or more copy numbers, one or more transcript sequences, one or more translocations, and / or one or more methylated residues.

[0074] Another embodiment of the present invention relates to a process for identifying multiple nucleic acid molecules in a sample using a system of the present invention, wherein preparing the system comprises loading one or more primary reaction chambers with primary reaction oligonucleotide probes or primers, each having a first portion comprising a nucleotide sequence complementary to a portion of a target nucleic acid in the sample, and the process following loading the one or more primary reaction chambers further comprises running a primary reaction in the system and obtaining the nucleotide sequence of the target nucleic acid molecule in the sample following the process of running the primary reaction.

[0075] Another embodiment of the present invention relates to a process for identifying multiple nucleic acid molecules in a sample using the system of the present invention. The process includes providing a sample containing multiple target nucleic acid molecules and contacting the sample with a set of primary oligonucleotide primers having a first portion complementary to a portion of the target nucleic acid molecule and a second portion, and a polymerase to form a polymerase chain reaction mixture. This mixture is subjected to a polymerase chain reaction in a primary reaction chamber to produce a set of amplification products. The amplification products are delivered to a product capture housing containing a solid support with multiple individual rows of multiple capture subunits, each individual product capture subunit containing an array of multiple individual micropores containing immobilized capture probes complementary to the second portion. The target nucleic acid molecules are captured by the immobilized capture probes and copied. The nucleotide sequences of the immobilized target nucleic acid molecules are obtained by performing a sequencing reaction in the micropores.

[0076] According to this embodiment, the product capture subunit includes an array of a plurality of individual micropores, each having opposite first and second open ends, the first end having a larger diameter and the second end having a smaller diameter than the diameter of the first end. The process may further include a mesh screen covering the second ends of the micropores within the product capture housing.

[0077] In another embodiment, the process of identifying multiple nucleic acid molecules in a sample using the system of the present invention further comprises placing beads containing immobilized capture probes into individual micropores.

[0078] In a further embodiment, the process of identifying multiple nucleic acid molecules in a sample using the system of the present invention further comprises removing at least one second portion from the amplification product before the process of obtaining the nucleotide sequence and after subjecting the polymerase chain reaction mixture to polymerase chain reaction. The process of removing at least one second portion can be performed using uracil DNA glycosylase, apurinic / apyrimidinic endonuclease, endonuclease III, endonuclease IV, endonuclease V, alkyladenine DNA glycosylase, formamidopyrimidine DNA glycosylase, or 8-oxyguanine DNA glycosylase, or a combination thereof.

[0079] Another embodiment relates to a process for identifying multiple nucleic acid molecules in a sample using a system of the invention. The process includes providing a system of the invention and adding universal tags or capture oligonucleotide primers or probes to micropores or microwells of product capture subunits on a solid support within a product capture housing, where the universal tags or capture oligonucleotide primers or probes are retained within the micropores or microwells. The process further includes loading primary reaction oligonucleotide probes or primers, each having a first portion comprising a nucleotide sequence complementary to a portion of a target nucleic acid in the sample, into one or more primary reaction chambers and running a primary reaction in the system. The process further includes obtaining the nucleotide sequences of the target nucleic acid molecules in the sample following the process of running the primary reaction. The product capture subunit includes an array of individual micropores, each having an opposing first and second open end, the first end having a larger diameter and the second end having a smaller diameter than the first end, a first passage in fluid communication with the first end of the micropore, and a second passage in fluid communication with and separated from the second end of the micropore by a mesh screen covering the second end of the micropore, wherein the process for obtaining a nucleotide sequence is carried out by a process including the following steps in the order shown: Polymerase chain reaction products are delivered to a product capture housing through the first passage; a hydrophobic liquid is then passed through the first passage, thereby distributing the products into individual microwells; the hydrophobic liquid is then passed through the first and second passages; the products are amplified in a polymerase chain reaction and / or an isothermal reaction using capture oligonucleotide primers immobilized on the inner surfaces of the microwells under conditions that produce amplification products; a volatile solvent is then passed through the first passage while the hydrophobic liquid is passed through the second passage. The products of the polymerase chain reaction and / or isothermal reaction are denatured, unimmobilized nucleic acid molecules are washed away through the first passage, and a hydrophobic liquid is passed through the second passage, thereby isolating the products into individual microwells.A hydrophobic liquid, which is denser than water, is passed through the first passage, while a volatile solvent, air, and sequencing reagents are passed through the second passage.The sequencing reaction is then carried out in the product capture subunit.

[0080] The present invention also relates to a process for preparing a microtiter plate for identifying multiple nucleic acid molecules in a sample. This process includes providing a microtiter plate with a plurality of individual rows and columns of product capture subunits, each of which contains an array of a plurality of individual hydrophilic microwells separated by a hydrophobic surface. The microwells of the microtiter plate are filled with an aqueous liquid containing oligonucleotide primers and / or probes. The microtiter plate is centrifuged to distribute the aqueous liquid into the unfilled microwells of each individual product capture subunit in the microtiter plate. Thereafter, when the centrifugation is terminated, the aqueous liquid is no longer in contact with the hydrophobic surface. The aqueous liquid is evaporated, and the microwells are dried, leaving the oligonucleotide primers in the microwells.

[0081] Another embodiment of the present invention relates to a process for identifying multiple nucleic acid molecules in a sample using the process of the present invention for preparing dried oligonucleotide primers in microwells of a microtiter plate. This process involves adding an aqueous sample to the microtiter plate so that the hydrophobic liquid is positioned above the aqueous sample, followed by adding a hydrophobic liquid to the microtiter plate. The microtiter plate is centrifuged to disperse the aqueous liquid into unfilled microwells in the microtiter plate. The centrifugation is then terminated, leaving the aqueous liquid free from contact with the hydrophobic surface. An amplification reaction of nucleic acid molecules is performed under conditions in which a polymerase, exonuclease, endonuclease, or ribonuclease cleaves one or more probes containing a quencher group and a fluorescent group in a target-specific manner, liberating the fluorescent group and generating a signal if the target nucleic acid molecule is present in the sample.

[0082] According to this embodiment, a plurality of nucleic acid molecules are identified that include a target nucleotide sequence that differs from the nucleotide sequences of other nucleic acid molecules in the sample or in other samples by one or more nucleotides, one or more nucleotide insertions or deletions, one or more copy numbers, one or more transcript sequences, one or more translocations, and / or one or more methylated residues.

[0083] Another aspect of the present invention relates to a system for identifying multiple nucleic acid molecules in a sample. The system includes an inlet, an outlet, and a cartridge fluidly coupled to the inlet and the outlet. The cartridge defines a space containing a product capture housing containing a solid support with multiple individual rows of product capture subunits. Each individual product capture subunit includes an array of multiple individual hydrophilic micropores separated by a hydrophobic surface, each micropore having opposing first and second open ends, the first end having a larger diameter and the second end having a smaller diameter than the first end. The product capture housing includes multiple fluid flow paths located above and below the solid support so that substances can pass from the inlet through the row of product capture subunits, contact the array of micropores within the subunits, and then to the outlet.

[0084] In one embodiment, the system for identifying multiple nucleic acid molecules in a sample further includes one or more valves for selecting whether to direct reagents and / or reactants into or out of the product capture housing through an inlet or outlet.

[0085] In another embodiment for identifying multiple nucleic acid molecules in a sample, the system further comprises one or more heating elements in the cartridge adjacent to the product capture housing.

[0086] The present invention relates to a method for preparing a system for identifying multiple nucleic acid molecules in a sample. The method includes providing the system of the present invention, which includes an inlet, an outlet, and a cartridge fluidly connected to the inlet and outlet and defining a space, as described above. The method further includes adding capture oligonucleotide primers or probes to micropores of product capture subunits on a solid support within a product capture housing, and retaining the capture oligonucleotide primers or probes within the micropores or microwells. In one embodiment, the method further includes running a reaction in the system and detecting the presence of target nucleic acid molecules in the sample within the micropores based on the running of the reaction.

[0087] The present invention provides a series of devices, chambers, and assays for determining the cause of disease directly from a blood sample. Nucleic acids are purified from clinical samples, target regions are subjected to a series of amplification reactions, and targets are identified or enumerated using either real-time PCR or sequencing as readouts. A summary of the pressing clinical needs addressed by these devices is shown in Table 1.

[0088] (Table 1) Summary of clinical needs for determining the cause of disease directly from blood samples TIFF2024161098000002.tif254170TIFF2024161098000003.tif108170

[0089] One of the major challenges in detecting multiple unknown pathogens or mutations is amplifying all possible and targeted fragments while avoiding PCR dropouts in multiplex reactions. Multiplex PCR reactions can be difficult to optimize, and fragment dropout is a persistent problem. While initial PCR cycles in the 8-12 cycle range often maintain a relatively high copy number, more efficient fragments tend to over-amplify and dominate over less efficient fragments, resulting in fragment dropout in subsequent cycles. One solution to this problem is to perform an initial multiplex amplification with limited cycles, then split the product into 24-48 reaction chambers for subsequent amplification reactions at much lower complexity. Another solution is to dilute the initial amplification product and place it into subcompartments containing tens, hundreds, or even thousands of micropores or microwells. This allows a given micropore or microwell to be used for one to four qPCR or separate sequencing reactions, enabling accurate target enumeration or quantification while minimizing the risk of PCR dropouts.

[0090] One aspect of the present invention is a series of subcompartments, preferably arranged in rows and columns, each containing single, tens, hundreds, or thousands of micropores or microwells for subsequent qPCR, UniTaq, FRET, qLDR, or sequencing reactions and target identification. In preferred embodiments, the presence of the target is read by fluorescence. In some embodiments, the target is amplified and immobilized or bound to a solid support within the micropore or microwell. Such immobilization may be performed directly on the interior surface of the micropore or microwell, on dendrimeric primers immobilized on the surface of the micropore or microwell, or on microbeads pre-dispensed within the micropore or microwell before amplification or dispensed into the micropore or microwell after initial amplification. Once immobilized or bound to a solid support, the amplified target can be interrogated one or more times to determine the presence or absence of mutations, SNPs, or sequence variations within the target. This includes multiple rounds of ligation detection reaction (LDR), sequencing by synthesis, or sequencing by ligation.

[0091] The arrangement of subcompartments into rows and columns facilitates loading of such rows and columns with either universal or target-specific primers, enzymes, reagents, buffers, targets, or pre-amplified targets. Loading can be achieved by flowing liquid into a given row or column across all subcompartments via fluidic coupling or fluidically connected channels, or by precisely dispensing liquid into individual subcompartments using, for example, acoustic droplet ejection (ADE) technology. One manufacturer of ADE equipment is Labcyte (Sunnyvale, California).

[0092] In one embodiment of the present invention, this flexible design allows for the identification, genotyping, and / or quantification of viral, bacterial, protozoan, malarial, or other pathogenic nucleic acids, potentially representing 384, 768, or 1,536 targets, mutations, resistance genes, virulence genes, and / or strain or serovar variants. While detecting bacterial DNA directly from blood presents a particular challenge, typically yielding 1–2 colony-forming units per milliliter of blood, spatial multiplexing approaches may still allow for the identification of 32, 64, or 128 potential targets. Using the sequencing module described below, this design allows for the determination of approximately 150-base reads for 1,536 potential pathogen targets.

[0093] Another embodiment of this design uses spatial dilution (e.g., 48 bins) to accurately count copy numbers directly from plasma, enabling noninvasive prenatal testing for trisomy (NIPT). In each of the 48 bins, or 48 columns, the Watson strand must match the Crick strand (generated from one strand and a given fragment), providing an internal control for strand deletions or other errors. Multiple unique loci on chromosomes 2 (control), 13, 18, 21, X, and Y are used to establish copy number and identify trisomies or other chromosome copy changes. In one embodiment, 184 locus regions can be interrogated on both strands, but this can be increased to 368 or 736 locus regions.

[0094] Another embodiment of the design allows for single-molecule mutation detection by PCR-LDR-qPCR directly from plasma for up to 64 or 128 potential targets, with the added flexibility of assessing multiple mutations within a gene (i.e., mutations at K-ras codons 12 and 13) with a single signal. A similar level of flexibility can be applied when identifying and enumerating methylation of CpG sites in the promoter regions of selected genes. The ability to perform serial dilutions within the chamber allows for accurate enumeration of 384 RNA targets, including both rare and overexpressed lncRNAs, mRNAs, or splice variants, isolated from, for example, exosomes or platelets.

[0095] Another embodiment of the design allows for the determination of low-abundance mutations in 144 target regions, generating reads of approximately 150 bases on both strands, allowing each mutation to be accurately counted. Sequencing methylated regions allows for pre-enrichment of these regions, allowing for the interrogation of over 2,000 methylated CpG promoter regions, even when present at low abundance.

[0096] In one embodiment of the present invention (see FIG. 1A), subdivision Z is ii and rows B1 to B vThe micropores or microwells are fabricated in a solid support that can include composite materials, plastics, metals, glass, silicon, silicon nitride, or mixtures thereof. The dimensions of the micropores or microwells can range from 50 microns in diameter and about 50 microns to 400 microns deep, and can be open (i.e., micropores) or closed (i.e., microwells) at the bottom. The bottom of the 50 micron micropores can include another layer of 0.5 micron holes on a 200-400 nanometer thick silicon nitride layer, allowing the 50 micron micropores to be filled with liquid from the top, while the 0.5 micron holes at the bottom allow air to pass through. Microporous silicon nitride membranes can be fabricated by well-known methods, such as photolithographic patterning and reactive ion etching of a silicon nitride layer disposed on a silicon wafer substrate (DesOrmeaux JP et al., "Nanoporous Silicon Nitride Membranes Fabricated from Porous Nanocrystalline Silicon Templates," Nanoscale 6(18):10798-805 (2014), incorporated herein by reference in its entirety). In one embodiment, each subcompartment comprises 6 × 4 = 24 micropores or microwells, each 50 microns in diameter, formed in a square or hexagonal packing. Such an embodiment is optimal for subsequent qPCR, UniTaq, FRET, or qLDR detection.

[0097] Table 2. Various embodiments of microwells or micropores in cartridge or microtiter plate formats TIFF2024161098000004.tif209170

[0098] In another embodiment of the present invention (FIG. 1B), subdivision Z is comprised of columns A1-A i and rows B1 to B ix The micropores or microwells are fabricated in a solid support that can include composite materials, plastics, metals, glass, silicon, silicon nitride, or mixtures thereof. The dimensions of the micropores or microwells can range from 5 microns in diameter and about 5 microns to 40 microns deep, and can be open (i.e., micropores) or closed (i.e., microwells) at the bottom. The bottom of the 5 micron micropores can include another layer of 0.5 micron holes on a 200-400 nanometer thick silicon nitride layer, allowing the 5 micron micropores to be filled with liquid from the top, while the 0.5 micron holes at the bottom allow air to pass through. In one embodiment, each subcompartment contains 60 x 46 = 2,760 micropores or microwells, each 5 micron in diameter, formed by hexagonal packing. Such an embodiment is ideal for subsequent sequencing by synthesis or sequencing by ligation.

[0099] In another variation of the above embodiment, subcompartment Z is 400 microns wide by 600 microns long, with 100-micron-wide ridges X and Y between the subcompartments. The micropores or microwells may be 2.5 microns in diameter and approximately 2.5 to 20 microns deep, and may be open-bottomed (i.e., micropores) or closed-bottomed (i.e., microwells). The bottom of the 2.5 micron micropores may comprise a separate layer of 0.5-micron holes on top of a 200-400 nanometer thick silicon nitride, allowing liquid to fill the 2.5 micron micropores from the top while allowing air to pass through the 0.5-micron holes at the bottom to prevent liquid leakage. In one embodiment, each subcompartment contains 100 x 92 = 11,040 2.5-micron diameter micropores or microwells formed in a hexagonal packing pattern. Such an embodiment is ideal for subsequent sequencing by synthesis or sequencing by ligation.

[0100] In another embodiment of the present invention (FIG. 1C), subdivision Z is comprised of columns A1-A i and rows B1 to B ii The microwells are 800 microns wide by 1,200 microns long. Additional 200-micron-wide ridges, X and Y, are used between the subcompartments Z to provide spacing and additional structural support. These ridges may be designed with depressions or channels to allow fluid movement between the subcompartments. The microwells are fabricated from a solid support that may include composite materials, plastic, metal, glass, silicon, silicon nitride, or mixtures thereof. The microwell dimensions may range from 50 microns in diameter and approximately 50 microns to 400 microns deep. In one embodiment, each subcompartment contains 12 x 8 = 96 micropores or microwells, each 50 microns in diameter, formed in square or hexagonal packing. Such an embodiment is ideal for subsequent qPCR, UniTaq, FRET, or qLDR detection.

[0101] In another embodiment of the present invention (see FIG. 1D), subdivision Z isii and rows B1 to B v The micropores are located at the center of the subcompartment, measuring 400 microns wide by 600 microns long. Additional 100-micron-wide ridges, X and Y, are used between the subcompartments, Z, to provide spacing and additional structural support. Such ridges may be designed with depressions or channels to allow fluid movement between the subcompartments. The micropores are fabricated in a solid support that may include composite materials, plastic, metal, glass, silicon, silicon nitride, or mixtures thereof. The micropore dimensions may range from 5 microns in diameter and approximately 5 microns to 40 microns deep. The bottom of the 5-micron micropores may comprise another layer of 0.5-micron holes on a 200-400 nanometer-thick silicon nitride, allowing liquid to fill the 5-micron micropores from the top while allowing air to pass through the 0.5-micron holes at the bottom to prevent liquid leakage. In one embodiment, each subcompartment contains 60 x 46 = 2,760 5-micron diameter micropores or microwells formed by hexagonal packing. Such an embodiment is ideal for subsequent sequencing by synthesis or sequencing by ligation.

[0102] In another variation of the above embodiment, the subcompartments are 400 microns wide by 600 microns long, with 100-micron-wide ridges between them. The micropores or microwells may be 2.5 microns in diameter and approximately 2.5 microns to 20 microns deep. The bottom of the 2.5 micron micropores may comprise another layer of 0.5 micron holes on a 200-400 nanometer thick silicon nitride layer, allowing the 2.5 micron micropores to be filled from the top, while the 0.5 micron holes at the bottom allow air to pass through but prevent liquid from leaking. In one embodiment, each subcompartment contains 100 x 92 = 11,040 2.5-micron diameter micropores formed in a hexagonal packing pattern. Such an embodiment is ideal for subsequent sequencing by synthesis or sequencing by ligation.

[0103] The device is envisioned to comprise an array of micropores or microwells fluidically connected to a microfluidic channel that, in one embodiment, allows various reagents and enzymes to be delivered to a series of reaction chambers for pre-amplification reactions before transferring the products to the array of micropores or microwells for subsequent Taqman™ or sequencing readout.

[0104] The left (bottom) portion of Figure 2 is a schematic front view showing the fluidic connection of a microchannel to an array of 50-micron diameter microwells or micropores. In this portion of Figure 2, the microchannel resides in a space 6 defined by a cartridge 4 with an inlet 2 and an outlet 8. The right (top) portion of Figure 2 provides a detailed view of the components inside the cartridge and shows a schematic front view of an exemplary design of a pre-chamber that allows for fluidic transfer of liquids into a chamber containing thousands of microwells or micropores. This design represents a chamber configuration suitable for performing multiplex RT-PCR-nested PCR-UniTaq detection to identify low- and mid-abundance lncRNAs, mRNAs, and splice site variants isolated from CTCs or exosomes, as described below. In this illustration, the sample inlet is fluidically connected via inlet 12 to a large hexagonal chamber 10 (bottom), which is fluidically connected by conduit 14 to a first set of 12 diamond-shaped chambers 16 (every fourth chamber includes a large trough 18c, a medium trough 18b, and a small trough 18a), which is fluidically connected by conduit 20 to a second set of 24 diamond-shaped chambers 22 (every third chamber includes a large trough 24a and a small trough 24b), which is fluidically connected by conduit 26 to 24 elongated mixing chambers 28, which are fluidically connected by conduit 30 to subdivisions 32 (only two rows are shown at the top of the panel) containing microwells or micropores. A serpentine path may be designed to restrict fluid flow to ensure equal filling of all chambers at a given level. The illustration is not to scale and is for illustrative purposes.

[0105] The left (bottom) portion of Figure 3 is a schematic front view showing the fluidic connection of a microchannel to an array of 5-micron diameter micropores. In this portion of Figure 3, the microchannel resides in a space 106 defined by a cartridge 104 having an inlet 102 and an outlet 108. The right (top) portion of Figure 3 provides a detailed view of the components inside the cartridge and shows a schematic front view of another exemplary design of a pre-chamber that allows for fluidic transfer of liquids into a chamber containing millions of micropores suitable for Taqman™ or sequencing reactions. In this figure, the input sample is fluidly connected via inlet 112 to a large hexagonal chamber 110 (bottom), which is fluidly connected by conduit 114 to a first set of eight hexagonal chambers 116 (every fourth chamber contains a large trough 118a and a small trough 118b), which is fluidly connected by conduit 120 to a second set of sixteen hexagonal chambers 122 (every third chamber contains a large trough 124a and a small trough 124b), which is fluidly connected by conduit 126 to sixteen elongated mixing chambers 128, which are fluidly connected by conduit 130 to subcompartments 132 containing micropores (only two rows are shown at the top of the panel). The second set of 16 hexagonal chambers 122 are shown offset from one another slightly to allow for a larger liquid volume within each chamber while still maintaining a tight structure. Fluid paths may be designed to restrict fluid flow to ensure all chambers fill equally at a given level. The diagram is not to scale and is for illustrative purposes.

[0106] 4A-4C show a schematic front view (FIG. 4A), a schematic horizontal cross-section (FIG. 4B) along line BB in FIG. 4A, and a schematic vertical cross-section (FIG. 4C) along line CC in FIG. 4A of a 50-micron microwell within a subdivision 232 of a solid support, and a plate 204 that facilitates directing fluid flow. This view also applies to 5- or 2.5-micron micropores 202, although more micropores would be depicted within each chamber. The view is not to scale and is for illustrative purposes. This shows an example of a hexagonal spatial arrangement of wells. In this embodiment, the interior surfaces of the microwells have hydrophilic surfaces, while the exterior surfaces are hydrophobic, such that an aqueous liquid containing target or preamplified target and / or primers is flowed over the microwells (e.g., from bottom to top), filling each microwell. A hydrophobic liquid (i.e., mineral oil, silicone oil, fluorinated oil, or perfluorodecalin) is then poured over the wells, leaving aqueous liquid in the individual wells covered by the hydrophobic liquid, allowing subsequent enzymatic or amplification reactions to proceed independently in each isolated well.

[0107] In one embodiment of Figure 4, the surface of the plate is hydrophobic. In another embodiment, the surface of the plate is hydrophilic. In one embodiment, one or more valves controlling the inlet and outlet of the chamber are used to allow liquid to flow in from the bottom and out from the top. The flow of aqueous liquid from the bottom to the top into the microwells can be facilitated by applying positive pressure from the bottom, i.e., by pumping the liquid into a chamber open to the air at the top, and / or by applying negative pressure (i.e., syringe aspiration or partial vacuum). The flow rate can be adjusted using various combinations of pressures.

[0108] Figures 5A-5C show a schematic front view (Figure 5A), a schematic horizontal cross-section (Figure 5B) along line BB in Figure 5A, and a schematic vertical cross-section (Figure 5C) along line CC in Figure 5A of a 50 micron micropore 202 within a subcompartment 232 of a solid support. This is similar to Figures 4A-4C, but includes a front plate 204 and a back plate 206. In this view, the front of the chamber is the area between the front plate and the wide diameter side of the micropore, and the back of the chamber is the area between the back plate and the narrow diameter side of the micropore. The back plate can be pressed against a heating element to allow for temperature control, heating, and / or thermal cycling.

[0109] Figures 4A-4C and 5A-5C both show how ridges between subcompartments connect plates 204 and 206 to facilitate fluid flow direction and provide structural stability. This diagram also applies to 5 or 2.5 micron micropores, although more micropores would be depicted within each chamber. The diagrams are not to scale and are for illustrative purposes. In one embodiment, the vertical ridges are flush with the plate, while the horizontal ridges have depressions or channels that allow liquid to flow over the columns but prevent flow from one column to the next. Another embodiment, suitable for initial preloading of specific primers into rows, utilizes a temporary, supplemental plate with horizontal ridges that have ridges to block the channels between columns and provide additional height, allowing fluid to flow across the rows but preventing flow from one row to the next. After the rows are loaded with the desired primer set, the primers concentrate on the hydrophilic surfaces within the micropores as the liquid evaporates. Removing a plate can promote evaporation, and then replacing the last plate can allow flow from row to column, but not from one column to the next. Ridges on the back of the micropore-containing solid support are similarly structured and attached to a back plate so that liquid also flows column-wise on the back. The locations of channels or depressions in the lateral ridges can be offset to provide desired structural support. The exact dimensions of the ridges, depressions, or lateral ridge channels can be optimized to avoid dead spaces, i.e., spaces at the corners of a given chamber, where fluid flow is at a suboptimal limit.

[0110] 6A-6C, similar to FIGS. 5A-5C, show a schematic front view (FIG. 6A), a schematic horizontal cross-section (FIG. 6B) along line BB in FIG. 6A, and a schematic vertical cross-section (FIG. 6C) along line CC in FIG. 6A of a 50-, 5-, or 2.5-micron micropore 202 in a solid support 232. However, in this figure, it is shown how the bottom of the 50-, 5-, or 2.5-micron micropore comprises another layer 238 with 0.5-micron holes on a 200-400-nanometer-thick silicon nitride. This layer allows the 50-, 5-, or 2.5-micron micropore to be filled with liquid from the front, while allowing air to pass through the 0.5-micron pores on the back to prevent liquid leakage. In these figures, the front of the chamber is the region between the front plate 204 and the wide-diameter side of the micropore, and the back of the chamber is the region between the back plate 206 and the narrow-diameter side of the micropore. The back plate 206 can be pressed against a heating element to allow for temperature control, heating, and / or thermal cycling. In one embodiment, the surfaces of both plates 204 and 206 are hydrophobic. In another embodiment, the surface of one plate is hydrophilic and the other is hydrophobic. In another embodiment, the surfaces of both plates are hydrophilic. The figures are not to scale and are for illustrative purposes.

[0111] The following sections provide descriptions of different microfluidic chamber structures and provide examples of how the various chambers, microwells, and / or micropores can be filled with fluids suitable for subsequent nucleic acid amplification, detection, and / or sequencing reactions.

[0112] Figures 7A-7I show schematic front views of various pre-chamber designs that can perform various operations, including mixing different reagents, performing various amplification reactions, or storing portions of the amplification reactions for later use in subsequent reactions or for fluid transfer to chambers containing microwells or micropores. Fluid typically enters through the lower port and exits through the upper port. In some additional examples, multiple chambers of the same size and type are simultaneously filled. In these examples, the chambers are typically made of hydrophobic materials and the liquid is hydrophilic. In these examples, a small amount of low-density hydrophobic oil (i.e., mineral oil) can be used to seal the top of each chamber, allowing for thermal cycling without losing liquid moisture. In some cases, the hydrophobic liquid sealing the bottom of these chambers can be a denser liquid, such as a fluorinated oil, i.e., perfluorodecalin, while flushing aqueous liquid from the rear. Additionally, additives such as glycerol (which affects enzyme activity when used at greater than 10% v / v) or other compounds that enhance enzyme stability or enhance amplification of GC-rich targets, such as betaine, ectoine, hydroxyectoine, mannosylglycerate, mannosylglyceramide, diglycerol phosphate, or other sugars or sugar derivatives, can be used to adjust the density and viscosity of the aqueous layer. In Figure 7A, a small plug of mineral oil guides the aqueous reaction components as they are injected into the chamber. When the mineral oil reaches the top outlet and seals the chamber, the chamber is filled with aqueous liquid, and the bottom inlet is sealed with fluorinated oil. After thermal cycling (or other reactions), the liquid is drained, leaving a small amount of aqueous liquid retained in a shallow trough to the left of the inlet. When new reagents are introduced, they will mix with the amplification products of the previous reaction. Figure 7B is similar to Figure 7A, but a larger amount of product is retained in the trough. Figure 7C is similar to Figure 7A, but approximately half of the product is retained in the trough. FIG. 7D is a variation of FIG. 7A, in which several primer sets may be printed in a second trough on the right.Under these conditions, products from the first reaction in the lower chamber can be allowed to flow into this second chamber, filling the first (left) trough but not overflowing the second trough. When the liquid is drained, a small amount of aqueous liquid remains in the shallow trough to the left of the inlet. When new reagents are introduced, they mix with the amplification products from the previous reaction as well as the primers attached to the second trough. In this manner, a primary PCR can be followed by a secondary LDR or PCR reaction. Note that when the liquid from the secondary reaction is drained, products remain in both the left and right troughs. Figure 7E is a variation of Figure 7D, in which the first trough is larger to hold more of the first set of products. Figure 7F is similar to Figure 7C, but includes a second plastic part. This allows the second reaction fluid to move downward and ensure complete mixing with the remaining products from the first reaction. Figure 7G is similar to Figure 7A, but with reagents added from the side rather than the bottom. This allows some of the product from the first reaction to be retained in the chamber and later mixed with the second reaction. Figure 7H is similar to Figure 7G, but a larger amount of product is retained in the lower chamber. In Figure 7I, the chamber is similar to Figure 7H but with some additional sections. As mineral oil is forced up the inlet, some enters the two narrow hydrophobic tubes and the rest enters the side of the chamber. The aqueous liquid that follows does not enter the narrow hydrophobic opening but instead flows entirely into the reaction chamber, which has a small plug of mineral oil in front of it. When the mineral oil reaches the top outlet and the chamber seals, the chamber is filled with aqueous liquid, and the two narrow tubes are also filled with mineral oil, while the bottom inlet is sealed with fluorinated oil. After the reaction, the liquid is drained, and the mineral oil, followed by the air, is drained from the two narrow tubes. Any product produced in the chamber remains there. When enough aqueous fluid is added to push the liquid up past the top air opening, the product can flow into the next chamber.

[0113] Figures 8A-8C show schematic front views of various pre-chamber designs that allow fluid transfer of liquids in conduits 14, 20, 26, and 30 to chambers containing microwells or micropores. Figure 8A shows an example in which fluidically coupled primers and / or probes (gray circles 17) in eight chambers 16 exit the chambers and then pass via conduits 26 into more elongated chambers 28 and into microwell or micropore subdivisions 32, ultimately drying within the microwells or micropores and covalently binding to their internal surfaces. In one embodiment, rows are pre-loaded with one to four UniTaq primer sets (or alternatively, one to four universal tag primers with target-specific Taqman™ probes) during cartridge manufacture. In one embodiment, a temporary plate is used to provide a fluidic pathway across the entire column of microwells or micropores, but isolated from each other in each row. The gray circles at the top of the figure indicate potential locations where primer sets might be delivered or printed, for example, by acoustic droplet ejection, capillary, inkjet, or quill printing. After printing, the primer sets can be distributed to each row using microfluidic channels and dried within individual microwells or micropores. After the primer sets have been properly delivered and dried in place, the temporary plate is removed and replaced with a permanent cover, providing a fluid path over the rows of microwells or micropores while isolating each row from adjacent rows. Figure 8B shows an example of fluidic coupling between the 4+4 chambers 16 and 22, with troughs 18 and 24 and baffles 23, where reagents exit the chambers and flow into elongated chamber 28, conduit 30, and subcompartment 32. In this illustration, gray circles 25 represent specific primers suitable for DNA amplification reactions using polymerase and / or ligase. The left side of the elongated chamber is coated with or made of extremely hydrophobic plastic, while the right side is slightly hydrophobic or somewhat hydrophilic.As the small plug of mineral oil is forced out of the initial chamber, it naturally migrates leftward, forcing the aqueous reactants following it directly into the row of microwells or micropores (top of the diagram). Therefore, if the microwells or micropores are optimally filled by first exposing them to aqueous liquid (thereby avoiding trapped air bubbles impeding the fluid movement), this mechanism clears the mineral oil from its path. Figure 8C is a similar schematic diagram to Figure 8B, showing the fluid-coupled four chambers 16 with troughs 18 and baffles 19, where reagents exit the chambers and then flow into elongated chamber 28 and then into subcompartments 32. In this diagram, gray circles 17 represent specific primers suitable for DNA amplification reactions using polymerase and / or ligase. Figure 8C also shows an additional plastic ridge or divider 29, which serves to separate the hydrophobic oil from the aqueous solution as it is forced up through the chambers, preventing it from mixing with the aqueous solution.

[0114] 9A-9B show schematic side views of an embodiment for filling micropores, as illustrated in FIGS. 5B and 6B. In these figures, the interior of the micropores 202 are hydrophilic, while the other surfaces are hydrophobic. In one embodiment, different primers and probes are printed, for example, by acoustic droplet ejection, capillary, inkjet, or quill printing (see FIG. 8A) into a prechamber suitable for fluid transfer to the array of micropores. In FIGS. 9A and 9B, a front plate 204 is shown above a channel 240. FIG. 9A shows micropores 202 in a solid support 232, open on both the top and bottom. Primers (and probes) are fluidically introduced into the micropores through the upper channel 240, while oil (preferably denser than the aqueous solution) is introduced through the lower channel 242, which is formed with the back plate 206. The oil (which is less dense) then sweeps the aqueous solution away from the upper channel 240, fluidically separating the primers / probes. If the primers are covalently immobilized on the surface, this chemistry can occur when both the upper and lower channels 240 and 242 are filled with oil. Alternatively, the primers can be immobilized by capture; for example, biotinylated primers can be captured by a streptavidin-coated surface. If the primer-probe mixture is subsequently dried, it can be formulated in a volatile salt such as ammonium acetate, or a stabilizing buffer containing betaine, ectoine, hydroxyectoine, mannosylglycerate, mannosylglylamido, diglycerol phosphate, or other sugars or sugar derivatives can be added. The upper oil can then be flushed away with a volatile organic solvent (i.e., hexanol) that is immiscible with the aqueous solution in the micropores. Flushing the volatile organic solvent with air and evaporating the water under mild heat allows the desired primers and probes to dry and remain on the interior surface of the micropores. The lower oil can also be flushed away with the volatile organic solvent, followed by air, to dry the array chamber. Alternatively, if the primer is immobilized on the inner surface of the micropores 202, excess primer is washed away, followed by the addition of any volatile organic (ie, ethanol) and drying.Figure 9B shows micropore 202 in solid support 232, open to the upper channel 240 (formed by plate 202), with another layer 238 of 0.5 micron holes on a 200-400 nanometer thick silicon nitride. This layer allows liquid to fill the 50, 5, or 2.5 micron micropores from the front via channel 240, while allowing air to pass through the 0.5 micron holes in lower channel 242 (formed by back plate 206). In this illustration, oil has been added to lower channel 242 to allow for later heating of the reactants in micropore 202, if needed during the optional primer immobilization step. Other embodiments, such as adding primers / probes without immobilization, may or may not use oil in the lower and / or upper chambers.

[0115] Figure 10 shows a schematic front view of an embodiment in which reaction chambers are filled prior to filling the microwells or micropores of subcompartments 32. This configuration includes two sets of reaction chambers 16 and 22, each with troughs 18 and 24, fed by conduits 14 and 20. The second set is pre-spotted with appropriate ligation probe oligonucleotides (gray circles 25). The left side shows a schematic of a portion of the microchannels and chambers for the initial multiplex PCR pre-amplification and subsequent ligase detection reaction (LDR) prior to Taqman™ readout on the micropore array. In the next panel, a light oil cap is introduced into the lower conduit 14, followed by an aqueous liquid containing target, PCR primers, and PCR reagents. Heavy oil is then used to fluidically transfer this aqueous reaction mixture into the first set of reaction chambers 16. After the PCR thermal cycling step, the oil and most of the aqueous reactants are expelled by conduit 14, while a small amount of PCR product is retained in the troughs 18 of each of the first two chambers 16. The chambers are again filled with light oil via conduit 14, followed by LDR reagents and enzymes, and the aqueous reaction mixture is then fluidically transferred using heavy oil to a second set of reaction chambers 22 (where it mixes with the pre-spotted LDR primers). From reaction chamber 22, conduit 26 flows into reaction chamber 28 (divided by plastic ridge or divider 29) and then into microwell or micropore subdivision 32 via conduit 30. After the LDR thermal cycling step, the reagents are again drained, but the LDR product is retained in trough 24. This product is now suitable for fluidic mixing with a PCR master mix and transfer to the micropore array for subsequent Taqman™ reactions, as described in Figures 11A-11B.

[0116] 11A-11B show schematic side views of an embodiment for filling micropores 202, as illustrated in FIGS. 5C and 6C, to perform real-time PCR reactions, such as Taqman™ or UniTaq reactions. In FIGS. 11A-11B, a front plate 204 is shown to the left of flow channel 240, and a back plate 206 is shown to the right of flow channel 242. The diagram begins with micropores 202 in solid support 232 pre-filled and dried with one to four UniTaq primer sets (or alternatively, one to four universal tag primers with target-specific Taqman™ probes). In one embodiment, the inner surface of micropore 202 has a hydrophilic surface, while the outer front surface 244 and back surface 246 are hydrophobic, so that when an aqueous liquid containing a target or preamplified target and / or primers is flowed over the micropores (e.g., from the bottom through channel 242, from the front side 244 with the larger pore diameter, through channel 240, and to the top), the aqueous liquid fills each micropore. In one embodiment, one or more valves controlling the inflow and outflow of the chambers are used to allow the liquid to flow in from the bottom of channel 240 or 242 and out from the top of channel 240 or 242. In one embodiment, the inflow and outflow of fluids before and after the chambers are regulated or controlled by individual valves or by the application of individual pressures. The flow of aqueous liquid from the bottom to the top of the micropores can be facilitated by applying positive pressure from the bottom via channel 240 or 242, i.e., by pumping the liquid into a chamber whose top is open to the air, and / or by applying negative pressure (i.e., syringe aspiration or partial vacuum). The flow rate can be adjusted using various combinations of pressure from the top, bottom, front, or back. For illustrative purposes, consider the task of filling micropore 202 with an aqueous liquid suitable for subsequent personalized amplification within micropore 202. In Figure 11A, all surfaces, including front 244 and back 246, except for the inner surface of micropore 202, are hydrophobic.When aqueous fluid is injected from the lower front surface using positive pressure, the fluid enters the micropores 202 through channels 240 at the front surface 244 and then flows through channels 242, forcing air out the back surface 246, forming a meniscus at the back surface of the micropores 202. To generate sufficient pressure to force the liquid out the back surface of the initially filled micropores, a hydrophobic liquid is forced from the lower back surface 246 into channels 242, covering the aqueous meniscus at the back surface of the micropores 202 immediately after it forms, without increasing in weight as the aqueous liquid rises at the front surface. The optimal pressure difference can be determined experimentally. This difference is believed to be a function of the viscosity, density, and volumetric differences of the liquid, as well as the hydrophobicity of the outer surface of the microporous solid support. When an aqueous liquid is injected into the micropores 202 from the front surface 244 through the channels 240, the inflow of a hydrophobic liquid (i.e., heavy oil) from the front surface 244 through the channels 240 pushes the aqueous liquid out of the non-productive volume and into the micropores 202, simultaneously coating each individual micropore 202 on the front surface 244 with oil. This fills each micropore with aqueous liquid, and the front surface 244 and back surface 246 are sealed with hydrophobic liquid. Each micropore 202 is fluidically isolated and suitable for subsequent independent amplification and thermal cycling reactions. In Figure 11B, all surfaces are hydrophobic except for the inner surfaces of the micropores 202 and the silicon nitride 238 with 0.5 micron holes. When aqueous fluid is injected using positive pressure from the lower front surface 244 via the channels 240, the fluid enters the micropores 202 from the front surface 244 and pushes air out the back surface 246 via the channels 242, preventing the liquid from passing through the 0.5 micron silicon nitride pores. When the aqueous liquid fills the micropores 202 from the front surface 244, oil flows in from the front surface 244 through the channels 240, forcing the aqueous liquid out of the non-productive volume and into the micropores 202, simultaneously coating each individual micropore 202 on the front surface 244 with oil. The back surface of the chamber may be filled with humidified air for heating, ultimately heating the aqueous liquid within the micropores. Alternatively, oil may be filled into the back surface 246 of the chamber via the channels 242, as shown. This fills each micropore 202 with aqueous liquid, sealing the front and back surfaces with oil.Each micropore is fluidically isolated and suitable for subsequent independent amplification and thermocycling reactions.

[0117] FIG. 12 shows a schematic side view of an embodiment for filling micropores, such as those illustrated in FIGS. 6A-6C, to perform sequencing reactions. In FIG. 12, the front plate 204 is shown to the left of the channel 240, and the back plate 206 is shown to the right of the channel 242. In this example, all surfaces, including the front surface 244 and back surface 246, are hydrophobic, except for the inner surface of the micropore 202 and the silicon nitride 238 with 0.5 micron holes. When aqueous fluid is injected using positive pressure from the lower front surface 244 via the channel 240, the fluid enters the micropores 202 from the front surface 244, pushing air out the back surface 246 via the channel 242 without passing through the 0.5 micron silicon nitride holes. When the aqueous fluid fills the micropores 202 from the front surface 244, oil flows in from the front surface 244, forcing the aqueous fluid out of the non-productive volume and into the micropores, simultaneously coating each individual micropore on the front surface 244 with oil. The back surface 246 of the channel 242 is also filled with oil. This fills each micropore 202 with an aqueous liquid, and the front surface 244 and back surface 246 are sealed with oil. Each micropore 202 is fluidically isolated and suitable for subsequent independent thermocycling reactions to amplify template strands and anchor them to a solid support on the micropore's interior surface. Oil is forced through the channel 240 and out the front surface 244, denaturing the opposite strand products and washing away other products and primers. A heavy oil plug is used to block the bottom of the front surface 244 in the channel 240, while simultaneously rinsing the back surface 246 through the channel 242 and optionally air-drying, delivering the target strands clonally propagated and anchored in the micropores 202 suitable for sequencing to the array. The flow of aqueous liquid into the micropores from the bottom of the back surface 246 to the top front surface 244 and back surface 246 can be facilitated by applying positive pressure from below, i.e., by pumping the liquid through channels 240 or 242 that are open to air at the top, and / or by applying negative pressure (i.e., syringe aspiration or partial vacuum). The flow rate can be adjusted using various combinations of pressure or by restricting the opening at the top back surface to ensure that most of the sequencing reagent volume enters at the bottom back surface 246, passes through the micropores 202, and exits at the top front surface 244.An additional advantage of using a silicon nitride layer 238 on the back side of the micropores 202 is that aqueous liquid added from the front side 244 does not disrupt the air interface on the back side 246, but passes freely through the 0.5 micron holes to the front side 244 of the micropores 202 when the back side 246 of the channel 242 is filled with aqueous liquid. This allows for greater flexibility in reagent addition and influx or efflux of various reagents in subsequent sequencing reactions.

[0118] Figures 13A-13B show schematic front views of a chamber format using the microwells or micropores described in Figures 1 and 6A-6C. Figure 13A shows a schematic of a microwell format in which the subcompartments 332 within cartridge 304 defining space 306 are 800 microns wide by 1200 microns long (shown as rectangular cross-sections) and contain 96 50 micron diameter microwells. Additional 200 micron wide ridges 350 are used between subcompartments 332 to provide spacing and additional structural support between the subcompartments. These are represented as "white" areas between the rows and columns of the rectangular subcompartments 332. For simplicity, this schematic shows 32 columns by 32 rows. Other embodiments include 48 columns by 48 rows and 64 columns by 64 rows. Figure 13B shows a schematic of a microfluidic chamber for sequencing on an array of micropores in a microtiter plate format. The schematic shows 32 double columns by 32 double rows within cartridge 404 defining space 406, with a close-up showing only two double columns and one double row of subdivisions 432 (separated by white ridges 450), each containing 2,072 micropores. Other embodiments include 48 double columns by 48 double rows, 64 double columns by 64 double rows, and still other embodiments include 96 columns by 96 rows, and 128 columns by 128 rows. In one embodiment, the micropore-containing chambers are fed through an inlet using acoustic droplet ejection to flow reagents, enzymes, targets, or pre-amplified targets into all of the chambers in the array through a series of individual openings that can be fluidically closed or fluidically opened. When acoustic droplet ejection is used, fluid flow from inlet 402 to the individual openings can be facilitated by applying negative pressure (i.e., vacuum) from the opposite side and / or by feeding droplets in conduit 455 into a series of hydrophilic input chambers 452 and conduits 454 and transitions 456, which then feed subcompartments 432 containing an array of micropores. In this schematic, each individual opening is connected to a hydrophilic input chamber 452, which feeds two rows of subcompartments 432 containing micropores.Additionally, the chambers are also fluidly connected to allow reagents to flow into all of the chambers through one inlet and out a single waste or outlet 408 on the other side. Once the hydrophilic input chambers 452 are properly filled with reagents, enzymes, targets, or pre-amplified targets, these openings are closed and oil or other reagents are then added through one inlet, allowing the input solution to fluidly move into the micropores for further reaction.

[0119] Alternative microwell or micropore configurations can also be considered. OpenArray technology is available from ThermoFisher (Carlsbad, CA). This technology uses a slide-sized microscope metal plate with 3,072 through-holes that can be configured in a variety of different ways. For example, the plate can be divided into 48 subarrays with 64 through-holes or micropores (each subarray is spaced the same as a traditional 384-well microtiter plate). In the current configuration, each through-hole is 300 microns in diameter and 300 microns deep. The through-holes are hydrophilic or have a hydrophilic coating, while the front and back surfaces of the plate have a hydrophobic coating. This allows aqueous reagents to be retained in the through-holes by surface tension. After appropriate amplification and detection primers are loaded into the through-holes, these primers can be dried on the inner surface of the through-hole. Subsequent addition of sample, enzyme, and appropriate buffer dissolves the primers, while the reaction mixture is sealed in place within each through-hole by applying a hydrophobic liquid (i.e., mineral oil) on either side. Scaling this technology by fabricating through-holes with a diameter of 60 microns would allow for approximately 1,225 through-holes per subarray, for a total of 58,800 through-holes or micropores per slide-sized microscope plate. Another system developed by ThermoFisher is the QuantStudio 3D Digital PCR 20K chip, which consists of a silicon substrate etched with 20,000 microwells, each 60 microns in diameter. Primers, reagents, and enzymes are added, and the plate is sealed to dispense the liquid into the microwells and run the reaction. The chip's limitations include the ability to run only a single reaction. Another system developed by Formulatrix (Bedford, MA) is known as the Constellation Digital PCR System. In this system, a standard microtiter plate is divided into 24 chambers containing 32,000 microwells (approximately 50 microns in diameter) or 96 chambers containing 8,000 microwells.This design also supports the use of 24 chambers containing 200,000 microwells (approximately 20 microns in diameter), 96 chambers containing 50,000 microwells, or 384 chambers containing 12,500 microwells. Each chamber contains an input well fluidically connected to an input channel, which is in turn fluidically connected to numerous connecting channels comprised of individual compartments, all of which are fluidically connected to an output channel with a vent or air hole. The bottom of the channels is made of transparent plastic suitable for sealing the plate. Primers, reagents, and enzymes are added to the input wells, which are pumped through the input and connecting channels, with excess moving to the output channels and vent. A roller is then used to apply pressure to the bottom seal, blocking the channels, thereby turning each compartment into an isolated microwell suitable for thermal cycling and digital PCR readout. This system can be modified to create a temporary seal on the bottom plastic only, either by using pressure to temporarily block the connecting channels and create compartments (microwells) only during the amplification reaction, or by using a temporary sealant that can be later dissolved. After the target is amplified and immobilized in the individual compartments (microwells) in the subsequent sequencing reaction, the bottom plastic seal is released, allowing unreacted reagents and non-covalently immobilized products to be denatured and washed away. The resulting clonally amplified single-stranded target is suitable for subsequent sequencing-by-synthesis reactions, as described below or known in the art.

[0120] Figure 14 shows a schematic side view of a microtiter plate format 500 using microwells 302 in the solid support 332 shown in Figure 13A, suitable for pre-filling with appropriate primers and probes. Step A shows a side view of one chamber in a hydrophobic plate. This includes a 50-micron hydrophilic well with raised ridges 350 on both sides. In step B, the plate is inverted and filled with one to four UniTaq primer sets (or alternatively, one to four universal tag primer sets equipped with mutation- or methylation-specific Taqman™ probes) using acoustic droplet ejection. In step C, the plate is centrifuged, dispersing the aqueous liquid into the empty microwells. In contrast, step D shows droplets formed on the microwells after centrifugation, such that the aqueous solution avoids the hydrophobic surface. In step E, the aqueous solution is evaporated, leaving the wells with dried primer / probe sets (shown in step F).

[0121] Figure 15 shows a schematic side view of a microtiter plate format using microwells 302 and raised areas 350 in solid support 332 as shown in panel A of Figure 13, optionally pre-filled with appropriate Taqman™ or UniTaq primers and probes (as illustrated). Step A shows a side view of one chamber in a hydrophobic plate. This contains a 50-micron hydrophilic well with raised areas on both sides. In step B, the plate is inverted and filled with reagents appropriate for real-time amplification (i.e., Taqman™ reaction) and target DNA using acoustic droplet ejection. PCR primers and Taqman™ probe(s) are pre-added to the chamber, sometimes dried (as illustrated in Figure 14), or added along with the target, enzyme, and reagents. In step C, the aqueous layer is covered with hydrophobic mineral oil. In step D, the plate is transferred to a swinging bucket rotor and centrifuged. A concentrated aqueous liquid disperses into the empty microwells. In step E, the plate is moved to a thermocycler, where the droplets are covered with mineral oil and separated into individual microwells suitable for amplification.

[0122] One aspect of the present invention is directed to a method for identifying, in a sample, a plurality of nucleic acid molecules that include a target nucleotide sequence that differs from the nucleotide sequences of other nucleic acid molecules in the sample or other samples by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues. The method includes providing a sample potentially containing one or more nucleic acid molecules that include a target nucleotide sequence that differs from the nucleotide sequences of other nucleic acid molecules by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues. One or more primary oligonucleotide primer sets are provided, each primary oligonucleotide primer set including (a) a first primary oligonucleotide primer that includes a nucleotide sequence complementary to a sequence adjacent to the target nucleotide sequence, and (b) a second primary oligonucleotide primer that includes a nucleotide sequence complementary to a portion of an extension product formed from the first primary oligonucleotide primer. The sample to be contacted is blended with one or more primary oligonucleotide primer sets, a deoxynucleotide mix, and a DNA polymerase to form a polymerase chain reaction mixture, and the polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles, including denaturation, hybridization, and extension, to form a primary extension product containing the target nucleotide sequence or its complement. The initial PCR products are distributed among 24, 36, or 48 primary PCR reaction chambers. The method further includes blending the primary extension products with a polymerase and one or more secondary oligonucleotide primer sets to form a secondary polymerase reaction mixture. Each secondary oligonucleotide primer set includes (a) a first secondary oligonucleotide primer comprising a 5' primer-specific portion and a nucleotide sequence complementary to a sequence adjacent to and / or including the target nucleotide sequence, and (b) a second secondary oligonucleotide primer comprising a 5' primer-specific portion and a nucleotide sequence complementary to a portion of the extension product formed from the first secondary oligonucleotide primer.The contacted sample is blended with one or more secondary oligonucleotide primer sets, a deoxynucleotide mix, and a DNA polymerase to form a polymerase chain reaction mixture, and the polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles, including denaturation, hybridization, and extension, to form primary extension products containing the target nucleotide sequence or its complement. The secondary extension product sequences in the sample are detected and identified to identify the presence of one or more nucleic acid molecules containing the target nucleotide sequence that differs from the nucleotide sequences of other nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues.

[0123] 16 to 18 show various embodiments of this aspect of the present invention.

[0124] Figure 16 (Steps A-F) illustrates an exemplary PCR-PCR-qPCR for unknown pathogen identification. The method begins with the isolation of pathogen genomic DNA, as shown in Step A. If the pathogen is an RNA virus, an initial reverse transcriptase step is used to generate cDNA. As shown in Figure 16 (Step B), the sample is subjected to an amplification reaction, e.g., polymerase chain reaction (PCR), in the initial reaction chamber to amplify the target-containing region of interest. A multiplex PCR amplification reaction is performed using locus-specific primers and a deoxynucleotide mix. In one embodiment, amplification is performed with limited cycles (12-20 cycles) to maintain the relative ratios of the different amplicons generated. In another embodiment, each primer contains an identical tail of 8-11 bases at its 5' end to prevent amplification of primer dimers. The initial PCR products are distributed among 24, 36, or 48 primary PCR reaction chambers.

[0125] As shown in step C of Figure 16, target-specific oligonucleotide secondary primers are hybridized to the primary amplification product, and a polymerase (filled diamond) is used to amplify the target-containing region of interest. Another layer of specificity can be incorporated into the method by including a 3' cleavage blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the secondary primer, as shown in step C of this figure. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, leaving a 3' OH group that is highly amenable to polymerase extension (Figure 16, step C). The first secondary oligonucleotide primer contains a 5' primer-specific portion (Ai), and the second secondary oligonucleotide primer contains a 5' primer-specific portion (Ci) that allows for subsequent amplification of the secondary amplification product. After the secondary amplification reaction, the extension products from each primary PCR reaction chamber are distributed into 384 or 768 microwells or micropores containing one or more tag-specific primer pairs (each pair consisting of corresponding primers Ai and Ci) for PCR amplification and detection. As shown in steps E and F of Figure 16, detection of PCR products can be performed using a conventional TaqMan™ detection assay (see U.S. Patent No. 6,270,967 to Whitcombe et al. and U.S. Patent No. 7,601,821 to Anderson et al., incorporated herein by reference in their entireties). In TaqMan™-based detection, an oligonucleotide probe spanning the target region is used for amplification and detection in conjunction with a primer suitable for hybridization to a primer-specific portion of the secondary PCR product. The TaqMan™ probe contains a fluorescent reporter group (F1) at one end and a quencher molecule (Q) at the other end, which are in close proximity to each other in the intact probe such that the quencher molecule quenches the fluorescence of the reporter group. During amplification, the TaqMan™ probe and upstream primer hybridize to complementary regions of the ligation product. The 5' to 3' nuclease activity of the polymerase extends the hybridized primer, liberating the fluorescent group on the TaqMan™ probe and generating a detectable signal (Figure 16, Step F).

[0126] Figure 17 (Steps A-F) illustrates an exemplary PCR-PCR-qPCR method for identifying unknown pathogens. The method begins with the isolation of pathogen genomic DNA, as shown in Step A. If the pathogen is an RNA virus, an initial reverse transcriptase step is used to generate cDNA. As shown in Figure 17 (Step B), the sample is subjected to an amplification reaction, e.g., polymerase chain reaction (PCR), in the initial reaction chamber to amplify the target-containing region of interest. A multiplex PCR amplification reaction is performed using locus-specific primers and a deoxynucleotide mix. In one embodiment, amplification is performed with limited cycles (12-20 cycles) to maintain the relative ratios of the different amplicons generated. In another embodiment, each primer contains an identical tail of 8-11 bases at its 5' end to prevent amplification of primer dimers. The initial PCR products are distributed among 24, 36, or 48 primary PCR reaction chambers.

[0127] The UniTaq system is fully described in U.S. Patent Application Publication No. 2011 / 0212846 to Spier, which is incorporated herein by reference in its entirety. The UniTaq system involves the use of three unique "tag" sequences, with at least one unique tag sequence (Ai) present in a first oligonucleotide primer and the second and third unique tag moieties (Bi and Ci) present in a second oligonucleotide primer sequence, as shown in step C of Figure 17. When the oligonucleotide primers of a primer set are PCR amplified, the resulting extension product will contain the Ai sequence-target-specific sequence-Bi' sequence-Ci' sequence. An important aspect of the UniTaq approach is that it allows for highly multiplexed detection of nucleic acids, since both secondary oligonucleotide primers of a PCR primer set must be an exact match to generate a positive signal. For example, as described herein, this is achieved by requiring hybridization of two moieties, i.e., two tags, to each other.

[0128] As shown in step C of Figure 17, target-specific oligonucleotide secondary primers are hybridized to the primary amplification product, and a polymerase (filled diamond) is used to amplify the target-containing region of interest. Another layer of specificity can be incorporated into the method by including a 3' cleavage blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the secondary primer, as shown in step C of this figure. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, leaving a 3' OH group that is highly amenable to polymerase extension (Figure 17, step C). The first secondary oligonucleotide primer contains a 5' primer-specific portion (Ai), and the second secondary oligonucleotide primer contains 5' primer-specific portions (Bi, Ci) that allow for subsequent amplification and detection of the secondary amplification product. After the secondary amplification reaction, the extension products from each primary PCR reaction chamber are distributed into 384 or 768 microwells or micropores containing one or more tag-specific primer pairs, each consisting of a corresponding primer (F1-Bi-Q-Ai and Ci). For detection, the secondary PCR product, which contains Ai (first primer-specific portion), Bi' (UniTaq detection portion), and Ci' (second primer-specific portion), is primed on both strands using a first oligonucleotide primer with the same nucleotide sequence as Ai and a second oligonucleotide primer (i.e., Ci) complementary to Ci'. The first oligonucleotide primer also contains a UniTaq detection probe (Bi) bearing a detectable label F1 at one end and a quencher molecule (Q) at the other end (F1-Bi-Q-Ai). Optionally, a polymerase blocking unit, such as HEG, THF, Sp-18, ZEN, or any other blocker known in the art sufficient to stop polymerase extension, is placed proximal to the quencher molecule. PCR amplification results in the formation of a double-stranded product, as shown in step F of Figure 17. In this example, the polymerase blocking unit prevents the polymerase from copying the 5' portion (Bi) of the first universal primer, thereby preventing the bottom strand of the product from forming a hairpin when it becomes single-stranded.When such a hairpin is formed, the 3' end of the stem anneals to the amplicon, and polymerase extension of this 3' end terminates the PCR reaction.

[0129] When the double-stranded PCR product is denatured and then the temperature is reduced, the upper strand of the product forms a hairpin with a stem between the 5' portion of the first oligonucleotide primer (Bi) and the portion Bi' at the opposite end of the strand (Figure 17, step G). Also during this step, a second oligonucleotide primer anneals to the 5' primer-specific portion of the product (Ci'), forming the hairpin. When the second universal primer is extended in step G, the 5' nuclease activity of the polymerase cleaves the detectable label D1 or quencher molecule from the 5' end of the amplicon, thereby increasing the distance between the label and the quencher molecule and allowing detection of the label.

[0130] Figure 18 (Steps A-F) illustrates an exemplary PCR-PCR-qPCR (UniRq) method for unknown pathogen identification. The method begins with the isolation of pathogen genomic DNA, as shown in Step A. If the pathogen is an RNA virus, an initial reverse transcriptase step is used to generate cDNA. As shown in Figure 18 (Step B), the sample is subjected to an amplification reaction, e.g., polymerase chain reaction (PCR), in the initial reaction chamber to amplify the target-containing region of interest. A multiplex PCR amplification reaction is performed using locus-specific primers and a deoxynucleotide mix. In one embodiment, amplification is performed with limited cycles (12-20 cycles) to maintain the relative ratios of the different amplicons generated. In another embodiment, each primer contains an identical tail of 8-11 bases at its 5' end to prevent amplification of primer dimers. The initial PCR products are distributed among 24, 36, or 48 primary PCR reaction chambers.

[0131] The split probe system is fully described in U.S. Patent No. 9,598,728 to Barany et al., which is incorporated herein by reference in its entirety. Here, a split probe system designed for PCR amplification involves the use of four unique "tag" sequences, where the unique tag sequence (Ai) and split portions of the second and third unique tag portions (Bi', ti') are present in a first secondary oligonucleotide primer, and the other split portions of the second and third unique tag portions (tj and Bj) and the fourth unique tag sequence (Ci) are present in a second secondary oligonucleotide primer sequence, as shown in step C of Figure 18. When the oligonucleotide primers of a primer set are PCR amplified, the resulting extension product will contain the Ai sequence - Bi' and ti' sequences - target-specific sequence - ti', Bj' sequences - Ci' sequence. An important aspect of the split probe approach is that it allows for highly multiplexed detection of nucleic acids, since both secondary oligonucleotide primers of a PCR primer set must be correct to obtain a positive signal. For example, as described herein, this is accomplished by requiring hybridization of two moieties, ie, two tags, to one another.

[0132] As shown in step C of Figure 18, target-specific oligonucleotide secondary primers are hybridized to the primary amplification product, and a polymerase (filled diamond) is used to amplify the target-containing region of interest. Another layer of specificity can be incorporated into the method by including a 3' cleavage blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the secondary primer, as shown in step C of this figure. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, leaving a 3' OH group that is highly amenable to polymerase extension (Figure 18, step C). The first secondary oligonucleotide primer contains a 5' primer-specific portion (Ai, Bi', ti'), and the second secondary oligonucleotide primer contains a 5' primer-specific portion (tj, Bj, Ci) that allows for subsequent amplification and detection of the secondary amplification product. After the secondary amplification reaction, the extension products from each primary PCR reaction chamber are distributed to 384 or 768 microwells or micropores containing one or more tag-specific primer pairs, each consisting of corresponding primers (F1-r-Bj, Bi-Q-Ai, and Ci). For detection, the secondary PCR product, which contains Ai (first primer-specific portion), Bi' (split UniTaq detection portion), ti' (region complementary to the target sequence), the target sequence containing the internal ti, tj sequence, tj' (region complementary to the target sequence), Bi' (split UniTaq detection portion), and Ci' (second primer-specific portion), is primed on both strands using a first oligonucleotide primer with the same nucleotide sequence as Ai and a second oligonucleotide primer (i.e., Ci) complementary to Ci'. The first oligonucleotide primer also contains a UniTaq detection probe (Bj, Bi, with an internal ribose base) with a detectable label F1 at one end and a quencher molecule (Q) at the other end (F1-r-Bj, Bi-Q-Ai). Optionally, a polymerase blocking unit, such as HEG, THF, Sp-18, ZEN, or any other blocker known in the art that is sufficient to stop polymerase extension, is placed proximal to the quencher molecule. PCR amplification results in the formation of a double-stranded product, as shown in step F of Figure 18.In this example, the polymerase blocking unit prevents the polymerase from copying the 5' portion (Bj, Bi) of the first universal primer, preventing the bottom strand of the product from forming a hairpin when it becomes single-stranded. If such a hairpin is formed, it will anneal to the amplicon at the 3' end of the stem, and polymerase extension of this 3' end will terminate the PCR reaction.

[0133] When the double-stranded PCR product is denatured and then cooled, the upper strand of the product forms four hairpins between the pathogen-specific sequences (ti and ti'; tj and tj'), Bi and Bi', and Bj and Bj'. The ribose base of the Bj sequence then becomes double-stranded, allowing RNase H2 to liberate the fluorescent group F1 from the product, increasing the distance between the label and the quencher, thereby enabling detection (Figure 18, step G). One advantage of the split-probe design is that spurious products resulting from primer dimer formation, i.e., (F1-r-Bj, Bi-Q-Ai-Bi'-ti'-primer dimer-tj', Bj'-Ci'), do not produce false-positive signals. This is because the hairpins between ti and ti'; tj and tj' do not form, leaving only the stems of Bi and Bi'. As a result, the r-Bj and Bj' sequences do not form stems at the hybridization temperature used in the amplification reaction.

[0134] Another aspect of the present invention is directed to a method for identifying, in a sample, a plurality of nucleic acid molecules that include a target nucleotide sequence that differs from the nucleotide sequences of other nucleic acid molecules in the sample or other samples by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues. The method includes providing a sample potentially containing one or more nucleic acid molecules that include a target nucleotide sequence that differs from the nucleotide sequences of other nucleic acid molecules by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues. One or more primary oligonucleotide primer sets are provided, each primary oligonucleotide primer set including (a) a first primary oligonucleotide primer that includes a nucleotide sequence complementary to a sequence adjacent to the target nucleotide sequence, and (b) a second primary oligonucleotide primer that includes a nucleotide sequence complementary to a portion of an extension product formed from the first primary oligonucleotide primer. The sample to be contacted is blended with one or more primary oligonucleotide primer sets, a deoxynucleotide mix, and a DNA polymerase to form a polymerase chain reaction mixture in an initial reaction chamber. The polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles, including denaturation, hybridization, and extension, to form a primary extension product containing the target nucleotide sequence or its complement. The initial PCR products are distributed among 24, 36, or 48 primary LDR reaction chambers. The method further includes blending the primary extension product with a ligase and one or more oligonucleotide probe sets to form a ligation reaction mixture. Each oligonucleotide probe set includes (a) a first oligonucleotide probe having a target nucleotide sequence-specific portion and (b) a second oligonucleotide probe having a target nucleotide sequence-specific portion, where the first and second oligonucleotide probes of the probe set are configured to hybridize adjacent to each other in a base-specific manner to a complementary target nucleotide sequence in the primary extension product having a junction therebetween.The first and second oligonucleotide probes of one or more oligonucleotide probe sets are ligated together to form ligation product sequences in a ligation reaction mixture, and the ligation product sequences in the sample are detected and identified to identify the presence of one or more nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues.

[0135] 19-23 show various embodiments of this aspect of the invention.

[0136] Figure 19 (Steps A-F) illustrates an exemplary PCR-LDR-qPCR (Taqman™) method for unknown pathogen identification. The method begins with the isolation of pathogen genomic DNA, as shown in Step A. If the pathogen is an RNA virus, an initial reverse transcriptase step is used to generate cDNA. As shown in Figure 19 (Step B), the sample is subjected to an amplification reaction, e.g., polymerase chain reaction (PCR), in the initial PCR reaction chamber to amplify the target-containing region of interest. A multiplex PCR amplification reaction is performed using locus-specific primers and a deoxynucleotide mix. In one embodiment, amplification is performed with limited cycles (12-20 cycles) to maintain the relative ratios of the different amplicons generated. In another embodiment, 20-40 cycles are used to amplify the region of interest. In another embodiment, each primer contains an identical tail of 8-11 bases at the 5' end to prevent amplification of primer dimers. The initial PCR products are distributed among 24, 36, or 48 primary LDR reaction chambers (Step C).

[0137] As shown in step D of Figure 19, target-specific oligonucleotide probes hybridize to the amplification products, and when the two oligonucleotides hybridize to their complementary sequences, they are covalently linked together and sealed by a ligase (black circle). The upstream oligonucleotide probe contains a 5' primer-specific portion (Ai), and the downstream oligonucleotide probe contains a 3' primer-specific portion (Ci') that allows for subsequent amplification of the ligation product. After ligation, the ligation products from each primary LDR reaction chamber are distributed into 384 or 768 microwells or micropores containing one or more tag-specific primer pairs (each pair consisting of corresponding primers Ai and Ci) for PCR amplification and detection. As shown in steps E and F of Figure 19, detection of the ligation products can be performed using a conventional TaqMan™ detection assay (see U.S. Patent No. 6,270,967 to Whitcombe et al. and U.S. Patent No. 7,601,821 to Anderson et al., both of which are incorporated by reference in their entireties). In TaqMan™-based detection, an oligonucleotide probe spanning the ligation junction is used for amplification and detection in conjunction with a primer suitable for hybridization to the primer-specific portion of the ligation product. The TaqMan™ probe contains a fluorescent reporter group (F1) at one end and a quencher molecule (Q) at the other end, which are in close proximity to each other in the intact probe such that the quencher molecule quenches the fluorescence of the reporter group. During amplification, the TaqMan™ probe and upstream primer hybridize to complementary regions of the ligation product. The 5'->3' nuclease activity of the polymerase extends the hybridized primer, liberating the fluorescent group on the TaqMan™ probe and generating a detectable signal (Figure 19, Step F).

[0138] Figure 20 illustrates another exemplary PCR-LDR-qPCR (UniTaq) method for unknown pathogen identification. This method begins with the isolation of pathogen genomic DNA, as shown in step A. If the pathogen is an RNA virus, an initial reverse transcriptase step is used to generate cDNA. As shown in Figure 20 (step B), the sample is subjected to an amplification reaction, such as polymerase chain reaction (PCR), in the initial reaction chamber to amplify the target-containing region of interest. In this embodiment, the ligation probe is designed to contain UniTaq primer and tag sequences for easy detection. In another embodiment, each primer contains an identical tail of 8 to 11 bases at its 5' end to prevent amplification of primer dimers. The initial PCR products are distributed among 24, 36, or 48 primary LDR reaction chambers (step C).

[0139] The UniTaq system is fully described in U.S. Patent Application Publication No. 2011 / 0212846 to Spier, which is incorporated herein by reference in its entirety. The UniTaq system involves the use of three unique "tag" sequences, with at least one of the unique tag sequences (Ai) present in a first oligonucleotide probe and the second and third unique tag moieties (Bi and Ci) present in a second oligonucleotide probe sequence, as shown in step D of Figure 20. Upon ligation of the oligonucleotide probes of a probe set, the resulting ligation product will contain the Ai sequence-target-specific sequence-Bi' sequence-Ci' sequence. An important aspect of the UniTaq approach is that it allows for highly multiplexed detection of nucleic acids, since both oligonucleotide probes of a ligation probe set must be correct to obtain a positive signal. For example, as described herein, this is achieved by requiring hybridization of two moieties, i.e., two tags, to each other.

[0140] After ligation, the ligation products from each primary LDR reaction chamber are distributed into 384 or 768 microwells or micropores containing the appropriate UniTaq primer pair (Figure 20, Step E). For detection, the ligation products, which contain Ai (first primer-specific portion), Bi' (UniTaq detection portion), and Ci' (second primer-specific portion), are primed on both strands using a first oligonucleotide primer with the same nucleotide sequence as Ai and a second oligonucleotide primer (i.e., Ci) complementary to Ci'. The first oligonucleotide primer also contains a UniTaq detection probe (Bi) bearing a detectable label F1 at one end and a quencher molecule (Q) at the other end (F1-Bi-Q-Ai). Optionally, a polymerase blocking unit, such as HEG, THF, Sp-18, ZEN, or any other blocker known in the art that is sufficient to terminate polymerase extension, is placed proximal to the quencher molecule. PCR amplification results in the formation of a double-stranded product, shown in step F of Figure 20. In this example, the polymerase blocking unit prevents the polymerase from copying the 5' portion (Bi) of the first universal primer, thereby preventing the bottom strand of the product from forming a hairpin when it becomes single-stranded. If such a hairpin is formed, it will anneal to the amplicon at the 3' end of the stem, and polymerase extension of this 3' end will terminate the PCR reaction.

[0141] When the double-stranded PCR product is denatured and then the temperature is reduced, the upper strand of the product forms a hairpin with a stem between the 5' portion (Bi) of the first oligonucleotide primer and the portion Bi' at the opposite end of the strand (Figure 20, step G). Also during this step, the second oligonucleotide primer anneals to the 5' primer-specific portion (Ci') of the product, forming the hairpin. When the second universal primer is extended in step G, the 5' nuclease activity of the polymerase cleaves the detectable label D1 or quencher molecule from the 5' end of the amplicon, thereby increasing the distance between the label and the quencher molecule and allowing detection of the label.

[0142] Figure 21 illustrates another exemplary PCR-LDR-qPCR (UniSpTq) method for unknown pathogen identification. This method begins with the isolation of pathogen genomic DNA, as shown in step A. If the pathogen is an RNA virus, an initial reverse transcriptase step is used to generate cDNA. As shown in Figure 20 (step B), the sample is subjected to an amplification reaction, such as polymerase chain reaction (PCR), in the initial reaction chamber to amplify the target-containing region of interest. In this embodiment, the ligation probe is designed to contain a split probe and tag sequence for ease of detection. In another embodiment, each primer contains an identical tail of 8 to 11 bases at its 5' end to prevent amplification of primer dimers. The initial PCR products are distributed among 24, 36, or 48 primary LDR reaction chambers (step C).

[0143] The split probe system is fully described in U.S. Patent No. 9,598,728 to Barany et al., which is incorporated herein by reference in its entirety. The split probe system involves the use of four unique "tag" sequences, where the unique tag sequence (Ai) and the second and third unique tag portions (Bi', Zi) of the split portion are present in a first oligonucleotide probe, and the second and third unique tag portions (Zj', Bj') of the other split portion and the fourth unique tag sequence (Ci') are present in a second oligonucleotide probe sequence, as shown in step D of Figure 21. Upon ligation of the oligonucleotide probes of a probe set, the resulting ligation product contains the sequence Ai sequence-Bi' and Zi sequence-target-specific sequence-Zi', Bj' sequence-Ci' sequence. An important aspect of the split probe approach is that it allows for highly multiplexed detection of nucleic acids, since both oligonucleotide probes of a ligation probe set must be correct to obtain a positive signal. For example, as described herein, this is accomplished by requiring hybridization of two moieties, ie, two tags, to one another.

[0144] After ligation, the ligation products from each primary LDR reaction chamber are distributed into 384 or 768 microwells or micropores containing the appropriate UniTaq primer pair (Figure 21, step E). During detection, the ligation products, which contain Ai (first primer-specific portion), Bi' (first split-probe detection portion), Bj' (second split-probe detection portion), and Ci' (second primer-specific portion), are primed on both strands using a first oligonucleotide primer with the same nucleotide sequence as Ai and a second oligonucleotide primer (i.e., Ci) complementary to Ci'. The first oligonucleotide primer also contains a UniTaq detection probe (Bj,Bi) bearing a detectable label F1 at one end and a quencher molecule (Q) at the other end (F1-Bj,Bi-Q-Ai). Optionally, a polymerase blocking unit, such as HEG, THF, Sp-18, ZEN, or any other blocker known in the art sufficient to stop polymerase extension, is placed proximal to the quencher molecule. PCR amplification results in the formation of a double-stranded product, as shown in step F of Figure 21. In this example, the polymerase blocking unit prevents the polymerase from copying the 5' portion (Bj, Bi) of the first universal primer, thereby preventing the bottom strand of the product from forming a hairpin when it becomes single-stranded. When such a hairpin is formed, it anneals to the amplicon at the 3' end of the stem, thereby terminating the PCR reaction with polymerase extension of this 3' end.

[0145] When the double-stranded PCR product is denatured and then the temperature is reduced, the top strand of the product forms three hairpins between Bi and Bi', zi and zi', and Bj and Bj' (Figure 21, step G). Also during this step, a second oligonucleotide primer anneals to the 5' primer-specific portion (Ci') of the product, forming a hairpin. When the second universal primer is extended in step G, the 5' nuclease activity of the polymerase cleaves the detectable label D1 or quencher molecule from the 5' end of the amplicon, thereby increasing the distance between the label and the quencher molecule and allowing detection of the label. As soon as the polymerase passes through Bj', the short zi-zi' stem breaks, and the polymerase continues extension, generating a dsDNA product.

[0146] Both the UniTaq probe and split probe approaches offer the advantage of allowing standard primer / probe sets to be printed into the appropriate micropore or microwell. The Ci primer, whether ligated to form a product or left unligated, creates a copy of the downstream LDR probe. If that extension product forms a primer dimer with the upstream probe / primer using the UniTaq probe design in the absence of target, such a product (F1-Bi-Q-Ai-partial target-Bi'-Ci') will form a Bi and Bi' hairpin at the hybridization temperature, resulting in a false-positive signal. One advantage of the split probe design is that such a spurious product (F1-Bj, Bi-Q-Ai-partial target-zi', Bj'-Ci') will not produce a false-positive signal. This is because the Bi and Bi'; zi and zi' hairpins are not formed, leaving only the Bj and Bj' sequences, which consequently do not form a stem at the hybridization temperature used in the amplification reaction.

[0147] The ligation products can also be used to generate a signal directly in a process called PCR-qLDR, as illustrated below in Figures 22 and 23. One such approach is described in WO / 2016 / 057832 by Barany et al., the entire disclosure of which is incorporated herein by reference, and which uses a ligation detection probe that generates a FRET or fluorescent signal after ligation.

[0148] In one embodiment, a first ligation probe includes a 3' target-specific region and a 5' tail sequence with a donor or acceptor moiety, and a second ligation probe of the probe set includes a 5' target-specific region and a 3' tail sequence, each with an acceptor or donor moiety. The 5' and 3' tail sequences of the ligation probes of the probe set are complementary to each other, and when the acceptor and donor groups are brought into close proximity, a detectable signal is generated via Förster resonance energy transfer (FRET). After ligation, unligated oligonucleotide probes are washed away, and the ligation product is denatured from the immobilized amplification product. Upon denaturation, the complementary 5' and 3' tail sequences of the ligation product hybridize to each other, bringing the donor and acceptor groups into close proximity, generating a detectable FRET signal.

[0149] In another embodiment, the upstream probe may include a fluorescent reporter group at the 5' end, followed by a tail sequence portion, a quencher group (e.g., ZEN), and a target-specific portion. In the single-stranded form, the fluorescent group is quenched by the ZEN group. When the upstream and downstream ligation probes are ligated and the resulting ligation product is denatured, the complementary 5' and 3' tail portions of the ligation product hybridize to form a short double-stranded portion. Under these conditions, the reporter group is not quenched by the quencher group, resulting in a detectable signal. This is called a hybridization dequenching probe (HuQP).

[0150] The PCR-free qLDR method is called "Multiple Ligase Reactions and Probe Cleavages for SNP Detection" (Kim, "PCR-Free Multiple Ligase Reactions and Probe Cleavages for the SNP Detection of KRAS Mutation with Attomole Sensitivity," Analyst 141(16):6381-6386 (2016), incorporated herein by reference in its entirety). In this method, two primers hybridize and ligate to the target if perfect complementarity with the target exists at the junction. The two primers also contain non-complementary sequences at their 3' and 5' ends that are not ligated. After ligation, the ligation product (LP) forms a complex with a strand displacement hairpin (SDH), because the melting temperature (Tm) of the LP is higher for the SDH than for the target. Furthermore, the released target can be reused for a new ligation with the two primers. The addition of SDH to the ligase reaction allows for the isothermal ligation of two primers to each single target using multiple enzymes. To generate detectable signals, a modified cycling probe assay using gold nanoparticles (AuNPs) is performed after SNP-specific ligation. In the cycling probe assay, a target-binding chimeric probe containing a fluorescent donor and quencher at each end is digested by RNase H. RNase H cleaves the RNA phosphodiester bond only when present in an RNA-DNA heteroduplex. Heteroduplex DNA is not digested, nor are single- or double-stranded RNA or DNA. The cycling probe assay is designed to take advantage of this property of RNase H. Once the RNA is degraded and the target DNA strand is released, another intact RNA molecule can hybridize with the DNA, resulting in linear signal amplification.

[0151] Figure 22 shows another exemplary PCR-qLDR (UniLDq) method for unknown pathogen identification. This method begins with the isolation of pathogen genomic DNA, as shown in step A. If the pathogen is an RNA virus, an initial reverse transcriptase step is used to generate cDNA. As shown in Figure 22 (step B), the sample is subjected to an amplification reaction, such as polymerase chain reaction (PCR), in the initial reaction chamber to amplify the target-containing region of interest. In this embodiment, the ligation probe is designed to contain a tag sequence for ease of detection. In another embodiment, each primer contains an identical tail of 8 to 11 bases at its 5' end to prevent amplification of primer dimers. The initial PCR product is then distributed into 384 or 768 microwells or micropores (step C).

[0152] The pathogen-specific ligation oligonucleotides contain tags (Bi'-ti'-upstream target sequence; downstream target sequence-tj'-Bj') for subsequent detection. The ti' and tj' sequences are complementary to the target sequences ti and tj at the ligation junction. Blocking the LNA or PNA wild-type probe prevents ligation to the wild-type sequence when detecting a specific SNP or mutation. Another layer of specificity can be incorporated into the method by including a 3' cleavage blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe, as shown in step D of this figure. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, leaving a 3' OH group for ligation (Figure 22, step D). Once the target-specific oligonucleotide probe hybridizes to the amplification product and the 3' OH group is released in the optional RNase H step, the two oligonucleotides are covalently linked together and closed by a ligase (black circle) when hybridized to their complementary sequences (Figure 22, Step E).

[0153] In the presence of probes (F1-r-Bj, Bi-Q), after the denaturation step, the temperature is lowered, forming four double-stranded stems between the probes and the pathogen-specific sequences (ti and ti'; tj and tj'), Bi and Bi', and Bj and Bj'. The ribose bases of the Bj sequences then become double-stranded, allowing RNase H2 to release the fluorescent group and generate a signal (Figure 22, step F). The cleaved probes dissociate from the product, allowing new probes to hybridize and generate additional signal. Unligated LDR primers do not necessarily form hairpins, so RNase H2 will not generate a signal. In one embodiment of this approach, after the PCR reaction, the products are dispensed into microwells or micropores that already contain the universal probe(s) in addition to the target-specific LDR primers.

[0154] Figure 23 shows another exemplary PCR-qLDR (TsLDG) method for unknown pathogen identification. This method begins with the isolation of pathogen genomic DNA, as shown in step A. If the pathogen is an RNA virus, an initial reverse transcriptase step is used to generate cDNA. As shown in Figure 23 (step B), the sample is subjected to an amplification reaction, such as polymerase chain reaction (PCR), in the initial reaction chamber to amplify the target-containing region of interest. In this embodiment, the ligation probe is designed to contain a tag sequence for ease of detection. In another embodiment, each primer contains an identical tail of 8 to 11 bases at its 5' end to prevent amplification of primer dimers. The initial PCR product is then distributed into 384 or 768 microwells or micropores (step C).

[0155] The pathogen-specific ligation oligonucleotides carry tags (Bi' - upstream target sequence; downstream target sequence - tj') for subsequent detection. The tj' sequence is complementary to the tj sequence of the target at the ligation junction. When detecting a specific SNP or mutation, blocking the LNA or PNA wild-type probe prevents ligation to the wild-type sequence. As shown in step D of this figure, another layer of specificity can be incorporated into the method by including a 3' cleavage blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, leaving a 3' OH group for ligation (Figure 23, step D). Once the target-specific oligonucleotide probe hybridizes to the amplification product and the 3' OH group is released in the optional RNase H step, the two oligonucleotides are covalently linked together and closed by a ligase (black circle) when hybridized to complementary sequences (Figure 23, Step E).

[0156] In the presence of probe (F1-r-pathogen sequence-Bi-Q), after a denaturation step, the temperature is lowered, forming two double-stranded stems between the pathogen-specific sequences (ti, tj and ti', tj') and between Bi and Bi'. This causes the ribose bases of the pathogen sequence to become double-stranded, allowing RNase H2 to release the fluorescent group and generate a signal. The cleaved probe dissociates from the product, allowing a new probe to hybridize and generate further signal. Since neither of the unligated LDR primers form a stem, RNase H2 will not generate a signal. In one embodiment of this approach, after the PCR reaction, the product is dispensed into microwells or micropores that already contain pathogen sequence-specific probe(s) in addition to target-specific LDR primers.

[0157] qLDR with a cleavable probe (cP) generates a greater degree of signal than LDR with either a FRET probe or a hybridization dequenching (HuQP) probe. The latter generates a linear signal that is a function of cycles. That is, when LDR is performed for "X" cycles, the amount of fluorescent signal generated, "F," is proportional to X (i.e., F = f(X)). When using a cleavable probe similar to those in Figures 22 and 23, the amount of signal generated is a function of both the number of times the probe is cleaved during one LDR cycle, "C," and the number of cycles, X (i.e., F = f(X)(X-1)C). In practical terms, performing 50 cycles of LDR-FRET or LDR-HuQP results in a 50-fold dynamic range of signal change, while performing 50 cycles of LDR-cP results in a 1,225-fold dynamic range of signal change.

[0158] Figure 24 is a schematic front view of a portion of an exemplary design for pre-chamber loading, enabling fluid transfer of liquids into chambers containing microwells or micropores. This design shows a chamber structure and microwells or micropores suitable for performing multiplex PCR-nested PCR-UniTaq detection (or multiplex PCR-nested PCR-real-time PCR using target-specific Taqman™ probes) for the identification and quantification of unknown pathogens. In Figure 24, the input sample is fluidically connected to a large hexagonal chamber 16 (containing a trough 18; lower, initial reaction chamber), which is fluidically connected by conduit 20 to a hexagonal chamber 22 (a primary PCR reaction chamber containing a large trough 24 and baffles 23), which is fluidically connected by conduit 26 to an elongated mixing chamber 28, which is fluidically connected by conduit 30 to a chamber containing microwell or micropore subdivisions 32 (only four rows are shown at the top of the panel). The figure is not to scale and is for illustrative purposes. During cartridge manufacturing, rows of subcompartments are pre-loaded with one to four UniTaq primer sets (or alternatively, one to four universal tag primers with target-specific Taqman™ probes). The primary PCR reaction chambers leading to the rows of microwells or micropores are pre-loaded with nested PCR primer sets bearing either UniTaq or universal tag sequences at their 5' ends. The gray circles 25 on the right side of the diagram indicate potential locations where probe sets might be delivered or printed, for example, by acoustic droplet ejection, capillary, inkjet, or quill printing. This example shows four subcompartments, each containing 24 microwells or micropores, planned in 24 columns and 32 rows, corresponding to 768 targets. In this example, nine cycles of initial multiplex PCR amplification (or reverse transcription PCR for RNA targets) are performed to generate up to 512 copies of each original target in the initial reaction chamber.New PCR reagents are added as needed, and the initial multiplex reaction mixture is distributed into the primary PCR reaction chambers (pre-filled with the nested PCR primers described above). In this case, the average number of copies of each original pathogen distributed in each primary PCR reaction chamber is 20. Optionally, primers containing RNA bases and 3' blocking groups are unblocked by RNase H2 only upon binding to the correct target, providing additional specificity and avoiding spurious products. Target-specific primers with UniTaq or universal tags are used in 16-, 32-, or 64-mer primer sets to perform five cycles of nested PCR, producing an average of 640 copies of each pathogen-specific target per primary PCR reaction chamber. New PCR reagents are added as needed, mixed with the nested PCR products in each primary PCR reaction chamber, and distributed into the mixing chamber and then into the micropores of each column. The universal or UniTaq primers in each subcompartment of each row amplify only the products from each column with the correct tag. The Poisson distribution of micropores will count pathogen-specific targets that are initially present in low abundance, while the Ct values ​​across the micropores of each subcompartment will provide approximate copy information of pathogen-specific targets that are initially present in high abundance.

[0159] The cartridge design of Figure 24 can also be used to perform multiplex PCR-LDR-UniTaq detection (or multiplex PCR-LDR-real-time PCR using target-specific Taqman™ probes) for the identification and quantification of unknown pathogens. During cartridge manufacture, the primary LDR reaction chamber leading to the row of microwells or micropores is pre-filled with an LDR' probe set bearing either UniTaq or universal tag sequences at the unligated 5' (upstream) and 3' (downstream) ends. The gray circles 25 on the right side of the figure indicate potential locations where the probe set might be delivered or printed, for example, by acoustic droplet ejection, capillary, inkjet, or quill printing. The probes are dried, and the cover portion of the cartridge is attached, sealing the probe set in place. During cartridge use, reactants fluidly travel from the cartridge's initial reaction chamber through the primary LDR reaction chamber, the mixing chamber, and finally to the row of microwells or micropores, each row separated from its neighbors. This example shows a planned 768-count subdivision with four 24 columns and eight 32 rows, each containing 24 microwells or micropores. In this example, 30 cycles of initial multiplex PCR amplification (or reverse transcription PCR for RNA targets) are performed to amplify the original targets in the initial reaction chamber. The polymerase is inactivated (e.g., by heat killing or protease digestion), and the multiplexed products are diluted 10-fold into a ligase reaction mix containing ligase, ATP, or NAD and distributed to the primary LDR reaction chamber (pre-filled with the LDR probes described above). Optionally, PCR primers and / or LDR upstream probes, containing RNA bases and a 3' blocking group, are unblocked by RNase H2 only upon binding to the correct target, providing additional specificity and avoiding spurious products. UniTaq or universal tagged allele-specific probes are used in 16-, 32-, or 64-pair primer sets to perform 20 cycles of LDR.Fresh PCR reagents are added, mixed with the LDR products from each primary LDR reaction chamber, and distributed to the mixing chamber and then to the micropores in each column. Universal or UniTaq primers in each subcompartment of each row amplify only products from each column with the correct tag. The Ct values ​​across the 24 micropores in each subcompartment provide approximate copy numbers of pathogen-specific targets initially present at high abundance.

[0160] In an alternative embodiment using 48 columns and 48 rows of subdivisions, each containing 96 microwells, equivalent to 2,304 microwells, one to four UniTaq primer sets (or alternatively, one to four universal tag primers with target-specific Taqman™ probes) are delivered directly to the appropriate subdivisions in each row by acoustic droplet ejection, capillary, inkjet, or quill printing, and then dried within the individual microwells. Ten cycles of initial multiplex PCR amplification (or reverse transcription PCR for RNA targets) are performed to generate up to 1,024 copies of each original target in the initial reaction chamber or well. Tandem PCR primers are used, if desired. Fresh PCR reagents are added, and the initial multiplex reaction is distributed into 48 wells or primary PCR reaction chambers (nested PCR primers are added using acoustic droplet ejection). In this case, the average number of copies of each original pathogen distributed per well or primary PCR reaction chamber is 20. In some cases, primers containing RNA bases and a 3' blocking group are unblocked by RNase H2 only upon binding to the correct target, providing additional specificity and avoiding spurious products. Using target-specific primers with UniTaq or universal tags in 24- or 48-mer sets, three to four cycles of nested PCR produce an average of 160 to 320 copies of each pathogen-specific target per well or primary PCR reaction chamber. Fresh PCR reagents are added and mixed with the nested PCR products in each well or primary PCR reaction chamber, and the products from each well or primary PCR reaction chamber are distributed into two sets of 24 or four sets of 12 microwells, each containing 96 microwells. When two sets are used, the second set is a 100 / 1 dilution of the first set. When four sets are used, each set is a 20 / 1 dilution of the previous set. This allows for comprehensive coverage of pathogens present across a large scale. On average, each initial subdivision will contain 12 copies of each original pathogen, and a given microwell will contain 1 or 0 copies of the original pathogen. If more pathogens are present, each subdivision will contain additional copies.The universal or UniTaq primers in each subdivision of each row will only amplify products from each column with the correct tag. The Poisson distribution of 96 microwells will initially count pathogen-specific targets present at low abundance, while the Ct values ​​across the microwells of a subdivision will provide approximate copy information for pathogen-specific targets present at high abundance initially.

[0161] 25A-25B show schematic side views of the cartridge 4, valves, and reagent configuration for identifying and quantifying unknown pathogens using multiplex PCR-nested PCR-real-time PCR with UniTaq or target-specific Taqman™ probes; identifying and quantifying low levels of unknown mutations in plasma using multiplex PCR-LDR-real-time PCR with UniTaq or mutation-specific Taqman™ probes; and identifying and quantifying low levels of methylation and unknown mutations in plasma using multiplex PCR-LDR-real-time PCR with UniTaq or target-specific Taqman™ probes. Figure 25A is a schematic front view showing the fluidic connection of the microchannel to an array of 50-micron diameter microwells or micropores. For simplicity, the figure illustrates one main chamber, including one initial multiplex reaction chamber 10, 16 primary multiplex PCR reaction chambers 16 with troughs 18, 16 secondary multiplex reaction chambers 22 with troughs 24 and baffles 23, 16 narrow mixing chambers 28, and 16 rows of subcompartments 32 consisting of thousands of micropores or microwells. As shown, these are connected together by conduits 14, 20, 26, and 30. Fluid enters cartridge 4 through inlet 2 and exits through outlet 8. However, other chamber configurations can be used; for example, the multiplex PCR-nested PCR-real-time PCR for pathogen detection described in Figure 24 does not require a secondary multiplex reaction chamber. Figure 25B shows a fluidic system for multiplex PCR-nested PCR-real-time PCR using UniTaq or target-specific Taqman™ probes, using a micropore plate system (typically depicted in Figures 11-12) consisting of thousands of micropores 202. The micropore plate allows fluid communication from both sides of the pores: the first side (top of the plate, shown on the left side of the plate) communicates with valves 1, 2, and 3, while the second side (bottom of the plate, shown on the right side of the plate) communicates with valves 4 and 5.Valve 1 dispenses lysis / protease buffer, enzymes, wash buffer, elution buffer, buffer, EtOH, light oil, and heavy oil, as needed, through the initial multiplex reaction chamber, the primary PCR reaction chamber, and additional chambers across the first side of the micropore plate to waste via valve 3. Additionally, valve 1 can select a waste port that can be used to empty the first side of the micropore plate, other chambers, PCR reaction chambers, and initial multiplex reaction chambers by introducing air in the reverse direction through valve 3. Valve 1 can also select valve 2. Valve 2 dispenses initial multiplex PCR primers, master PCR mix, initial reverse transcription primers, master reverse transcription mix, wash solution, EtOH, and air through the initial multiplex reaction chamber, the PCR reaction chamber, and additional chambers across the first side of the micropore plate to waste via valve 3. Valve 4 dispenses air, light oil, heavy oil, and waste across the second side of the micropore plate to waste via valve 5. Additionally, valve 1 can select a waste port that can be used to empty the second side of the micropore plate by introducing air in the reverse direction through valve 5.

[0162] Table 3. Reagent composition for multiplex PCR, nested PCR, and real-time PCR. TIFF2024161098000005.tif71170

[0163] Figure 25B shows several heating elements 1-4 designed to individually heat / cool the initial multiplex reaction chamber 10, 24-48 primary multiplex PCR reaction chambers 16, 24-48 secondary multiplex reaction chambers 22, and main chamber 28, which includes 24-48 rows, and subdivisions 32 of thousands of micropores or microwells 202. Temperature control, heating, and / or thermal cycling can be achieved by pressing the back plate 206 (opposite the front plate 204) or one or more flat surfaces 244 and 246 of the micropore or microwell channel(s) 240 and 242, and the reaction chambers, against these heating elements. As shown in Figure 25, the two heating elements on the back side of the 24-48 primary multiplex PCR reaction chambers 16 and 24-48 secondary multiplex reaction chambers 22 are designed as two rectangular (horizontal) strips that control all of the primary chambers independently of all of the secondary chambers. For example, alternative configurations may be used, such as those with independent heating elements for each primary chamber, including the initial reaction chamber 10, primary chamber 16, secondary chamber 22, mixing chamber 28, and the main chamber containing thousands of microwell or micropore subdivisions; with additional rows or rows of chambers (i.e., primary, secondary, tertiary, etc.) with heating elements; and / or with 24-48 spatial multiplexes arranged in geometries other than rows or columns. For example, a plate may contain 24 individual input wells, each fluidly connected to an individual primary multiplex PCR reaction chamber 16, an individual secondary multiplex reaction chamber 22, an individual mixing chamber 28, and an individual chamber containing hundreds to thousands of micropore or microwell subdivisions. Samples may be injected into the 24 individual input wells by acoustic droplet ejection or other fluidics means after undergoing any initial multiplex reactions.

[0164] Figure 26 (Steps A-F) illustrates an exemplary PCR-PCR-qPCR method for identifying unknown bacterial pathogens directly from blood. The method begins with the isolation of pathogen genomic DNA, as shown in Step A. Pre-capture of bacteria directly from blood, i.e., using aptamers or antibodies, facilitates detection. The challenge is amplifying only the rare bacterial DNA from the large amount of excess WBC DNA. As shown in Figure 26 (Step B), the sample is distributed into 24, 36, or 48 primary PCR reaction chambers, each of which is subjected to an amplification reaction, e.g., polymerase chain reaction (PCR), to amplify the target-containing region of interest. Multiplex PCR amplification reactions are performed using target-specific primers and deoxynucleotide mixes. Optionally, a strand-displacing polymerase is used with tandem or multiple primers for each target. In one embodiment, amplification is performed with limited cycles (12-20 cycles). In another embodiment, each primer contains an identical 8-11 base tail at its 5' end to prevent amplification of primer dimers.

[0165] As shown in step C of Figure 26, target-specific oligonucleotide secondary primers are hybridized to the primary amplification product in the secondary PCR reaction chamber, and a polymerase (filled diamond) is used to amplify the target-containing region of interest. Another layer of specificity can be incorporated into the method by including a 3' cleavage blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the secondary primer, as shown in step C of this figure. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, leaving a 3' OH group suitable for polymerase extension (Figure 26, step C). Following nested primer amplification, the product from each secondary PCR reaction chamber is distributed into 384 or 768 microwells or micropores. PCR products can be detected using the corresponding primer pairs Ai and Ci and TaqMan™ probes spanning the ligation junctions, as described above in Figure 16 (see Figure 26, steps D-F), or by other suitable means known in the art.

[0166] Figure 27 (steps A-F) shows another exemplary PCR-PCR-qPCR method for identifying unknown bacterial pathogens directly from blood. This method begins with the initial distribution of target nucleic acids into primary PCR reaction chambers and multiplex PCR amplification, similar to that shown in Figure 26, except using UniTaq readout.

[0167] As shown in step C of Figure 27, target-specific oligonucleotide secondary primers are hybridized to the primary amplification product in the secondary PCR reaction chamber, and a polymerase (filled diamond) is used to amplify the target-containing region of interest. Another layer of specificity can be incorporated into the method by including a 3' cleavage blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the secondary primer, as shown in step C of this figure. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, leaving a 3' OH group suitable for polymerase extension (Figure 27, step C). Following nested primer amplification, the products from each secondary PCR reaction chamber are distributed into 384 or 768 microwells or micropores. PCR products are amplified using UniTaq-specific primers (i.e., F1-Bi-Q-Ai, Ci) and detected as described above in Figure 17 (see Figure 27, steps D-G) or using other suitable means known in the art.

[0168] The cartridge design in Figure 24 can also be used to perform multiplex PCR-nested PCR-UniTaq detection of unknown bacterial pathogens directly from blood. (Alternatively, multiplex PCR-nested PCR-real-time PCR using target-specific Taqman™ probes.) During cartridge manufacturing, the rows are pre-loaded with one to four UniTaq primer sets (or alternatively, one to four universal tag primers with target-specific Taqman™ probes). During cartridge use, reactants fluidly move from the cartridge's initial reaction chamber through the primary PCR reaction chamber, the mixing chamber, and finally to a row of microwells or micropores, each separated from adjacent rows. This illustration shows 24 columns and 32 rows, equivalent to 768 subdivisions, each containing 24 microwells or micropores. Samples are distributed across 24 columns, and initial multiplex PCR amplification is performed for 20 cycles using a strand-displacing polymerase and a set of large tandem primers or multiple primer sets with 10-12 bp tails to generate 1,000,000 copies of each original target (if present). Nested primers containing RNA bases and 3' blocking groups are unblocked by RNase H2 only upon binding to the correct target, providing additional specificity and avoiding spurious products. Target-specific primers with UniTaq or universal tags are used in 16-, 32-, or 64-mer primer sets to perform 10 cycles of nested PCR in each primary PCR reaction chamber. Fresh PCR reagents are added as needed, mixed with the nested PCR products in each primary PCR reaction chamber, and then dispensed from the mixing chamber into the microwells or micropores of each column. The universal or UniTaq primers in each subcompartment of each row amplify only the products from each column with the correct tag. Target preamplification and the use of a tail to prevent primer dimerization allows for the identification of bacterial pathogens at the single-cell level.

[0169] In an alternative embodiment using 48 columns and 48 rows of subdivisions, each containing 96 microwells, equivalent to 2,304 microwells, one to four UniTaq primer sets (or alternatively, one to four universal tag primers with target-specific Taqman™ probes) are delivered directly to the appropriate subdivisions in each row by acoustic droplet ejection, capillary, inkjet, or quill printing, and then dried within the individual microwells. The initial sample is distributed among the 48 wells. Nine cycles of multiplex PCR are performed in the wells or initial reaction chambers, yielding up to 512 copies of each original pathogen (if present). Tandem or more PCR primer sets are used. Additionally, all PCR primers preferably contain the same 10-12 base 5' tail sequence to suppress primer dimer amplification. On average, each initial subdivision yields 10 copies of each original pathogen, with one or zero copies per given microwell. If more pathogens are present, additional copies will be obtained in each subdivision. The universal or UniTaq primers in each subdivision of each row will only amplify products from each column with the correct tag. The Poisson distribution of 96 microwells will initially count pathogen-specific targets present in low abundance, while the Ct values ​​across the microwells will provide approximate copy information for pathogen-specific targets present in high abundance initially.

[0170] Figure 28 shows another exemplary PCR-LDR-qPCR reaction (optionally including carryover prevention) for detecting low-level mutations. Genomic or cfDNA is isolated (Figure 28, step A) and distributed into 24, 36, 48, or 64 wells or primary PCR reaction chambers, followed by PCR. The isolated DNA sample is optionally treated with UDG to digest dU-containing nucleic acid molecules that may be present in the sample (Figure 28, step B). A locus-specific upstream primer, a locus-specific downstream primer, a blocking LNA or PNA probe containing the wild-type sequence, and a deoxynucleotide mix optionally containing dUTP are used to selectively amplify the region of interest. In this embodiment, another layer of selectivity can be incorporated into the method by including a 3' cleavage blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the upstream primer. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, leaving a 3'OH group several bases upstream of the mutation, suitable for polymerase extension (Figure 28, Step B). A blocking LNA or PNA probe containing a wild-type sequence that partially overlaps with the upstream PCR primer has a competitive preference for binding to the wild-type sequence but not to the mutant DNA, thereby suppressing amplification of the wild-type DNA during each round of PCR. As shown in Figure 28, Step C, the amplification product may contain dU, but post-treatment with UDG or a similar enzyme can prevent carryover. The products from each primary PCR reaction chamber are distributed to the corresponding secondary LDR reaction chamber.

[0171] As shown in step D of Figure 28, a target-specific oligonucleotide probe hybridizes to the amplification product, and when the two oligonucleotides hybridize to their complementary sequences, they are covalently linked together and closed by a ligase (black circle). In this embodiment, the upstream oligonucleotide probe, which has a sequence specific for detecting the mutation of interest, further includes a 5' primer-specific portion (Ai) that facilitates subsequent detection of the ligation product. Again, the presence of a blocking LNA or PNA probe containing the wild-type sequence prevents ligation to the wild-type target sequence, which may be present after enrichment of the mutant sequence, during the PCR amplification step. The downstream oligonucleotide probe, which has a sequence common to both the mutant and wild-type sequences, includes a 3' primer-specific portion (Ci'), which, together with the 5' primer-specific portion (Ai) of the upstream probe, which has a sequence specific for detecting the mutation, allows for subsequent amplification and detection of only the mutant ligation product. As shown in step D of this figure, another layer of specificity can be incorporated into the method by including a 3' cleavage blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe. Upon target-specific hybridization, the RNA base is removed by RNase H (asterisk), leaving a 3' OH group that is suitable for ligation (Figure 28, step D). Following ligation, the products of each secondary LDR reaction chamber are distributed into 384 or 768 microwells or micropores. As described above in Figure 19 (see Figure 28, steps E-G), ligation products can be detected using the corresponding primer pair Ai and Ci and a TaqMan™ probe spanning the ligation junction, or by other suitable means known in the art.

[0172] Figure 29 shows another exemplary PCR-LDR-qPCR reaction (optionally including carryover prevention) for detecting low-level mutations. Genomic or cfDNA is isolated (Figure 29, Step A) and distributed into 24, 36, 48, or 64 wells or primary PCR reaction chambers, followed by PCR. The isolated DNA sample is optionally treated with UDG to digest dU-containing nucleic acid molecules that may be present in the sample (Figure 29, Step B). An upstream locus-specific primer is designed several bases upstream of the mutation and contains a 3' cleavage blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r). Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, freeing a 3' OH group suitable for polymerase extension (Figure 29, Step B). A blocking LNA or PNA probe containing a wild-type sequence that partially overlaps with the upstream PCR primer has a competitive preference for binding to the wild-type sequence, but not as strongly as to the mutant DNA, thereby suppressing amplification of the wild-type DNA during each round of PCR. As shown in step C of Figure 29, the amplification product may contain dU, but post-treatment with UDG or a similar enzyme can prevent carryover. The products from each primary PCR reaction chamber are distributed to the corresponding secondary LDR reaction chamber.

[0173] As shown in step D of Figure 29, a target-specific oligonucleotide probe hybridizes to the amplification product, and when the two oligonucleotides hybridize to their complementary sequences, they are covalently linked together and closed by a ligase (black circle). In this embodiment, the upstream oligonucleotide probe, which has a sequence specific for detecting the mutation of interest, further includes a 5' primer-specific portion (Ai) that facilitates subsequent detection of the ligation product. Again, the presence of a blocking LNA or PNA probe containing the wild-type sequence suppresses ligation to the wild-type target sequence, which may be present after enrichment for the mutant sequence, during the PCR amplification step. The downstream oligonucleotide probe, which has a sequence common to both the mutant and wild-type sequences, includes a 3' primer-specific portion (Bi'-Ci'), which, together with the 5' primer-specific portion (Ai) of the upstream probe, which has a sequence specific for detecting the mutation, allows for subsequent amplification and detection of only the mutant ligation product. As shown in step D of this figure, another layer of specificity can be incorporated into the method by including a 3' cleavage blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe. Upon target-specific hybridization, the RNA base is removed by RNase H (asterisk), leaving a 3' OH group that is suitable for ligation (Figure 29, step D). Following ligation, the products of each secondary LDR reaction chamber are distributed into 384 or 768 microwells or micropores. The ligation products are amplified using UniTaq-specific primers (i.e., F1-Bi-Q-Ai, Ci) and detected as described above in Figure 20 (see Figure 29, steps E-H) or using other suitable means known in the art.

[0174] Figure 30 is a schematic front view of a portion of an exemplary design of a pre-chamber loading system that allows fluid transfer of liquid into a chamber containing microwells or micropores. This design shows a chamber structure and microwells or micropores suitable for performing multiplex PCR-LDR-UniTaq detection to identify and quantitate low levels of unknown mutations in plasma. (Alternatively, multiplex PCR-LDR-real-time PCR using mutation-specific Taqman™ probes is used.) In Figure 30, the input sample is fluidly connected to the initial reaction chamber 10 (bottom) via inlet 12 and mixed with appropriate reagents. The initial reaction chambers 10 (bottom) are fluidly connected by conduits 14 to a first set of hexagonal chambers 16 (primary PCR reaction chambers, including small troughs 18), which are fluidly connected by conduits 20 to a second set of hexagonal chambers 22 (secondary PCR reaction chambers, including large troughs 24 and baffles 23), which are fluidly connected by conduits 26 to an elongated mixing chamber 28, which is fluidly connected by conduits 30 to chambers containing microwell or micropore subdivisions 32 (only four rows are shown at the top of the panel). The diagram is not to scale and is for illustrative purposes. During cartridge manufacture, the rows are pre-filled with one to four UniTaq primer sets (or alternatively, one to four universal tag primers with target-specific Taqman™ probes). During cartridge manufacture, the secondary LDR reaction chambers 22 leading to the array of microwell or micropore subcompartments are optionally pre-filled with LDR' probe sets bearing either UniTaq or universal tag sequences at the unligated 5' (upstream) and 3' (downstream) ends. The gray circles 25 on the left side of the diagram indicate potential locations where the probe sets may be delivered or printed, for example, by acoustic droplet ejection, capillary, inkjet, or quill printing. The probes are dried and the cartridge cover portion is attached, sealing the probe sets in place.Alternatively, if the same LDR primer set is used in each pre-chamber, it can be added after the PCR step, eliminating the need to print it on the cartridge first. During cartridge use, reactants flow from the cartridge's initial reaction chamber 10 through the primary PCR reaction chamber 16, the secondary LDR reaction chamber 22, and finally to the mixing chamber 28, where each row of microwell or micropore subdivisions 32 is separated from its neighbors. This illustration shows four subdivisions, arranged in 24 columns and 32 rows, equivalent to 768, each containing 24 microwells or micropores. In this example, an initial multiplex PCR amplification is repeated in each of the initial primary PCR reaction chambers 16 for 10-40 cycles in the presence of PNAs or LNAs that suppress amplification of wild-type sequences but not mutant sequences. In another embodiment, to minimize fragment dropout during multiplex PCR, an initial "pre-amplification" multiplex PCR is performed for 8-20 cycles in the initial reaction chambers 10. The products are then distributed to the primary PCR reaction chambers 16. In one variation, each primary reaction chamber contains one to four PCR primer sets with PNA or LNA that suppress amplification of the wild-type sequence, and single or multiplex PCR is performed for an additional 10 to 30 cycles, allowing one to four fragments containing potential mutations to be amplified in a single primary reaction chamber. In another variation, six sets of four primary reaction chambers contain four to 16 PCR primer sets with PNA or LNA that suppress amplification of the wild-type sequence, and multiplex PCR is performed for an additional 10 to 30 cycles, allowing four to 16 fragments containing potential mutations to be amplified in a single primary reaction chamber. The polymerase is inactivated (e.g., by heat killing or protease digestion), and the multiplex product from each chamber is diluted 10-fold into a ligase reaction mix containing ligase, ATP, or NAD and distributed to the corresponding secondary LDR reaction chamber 22 (pre-filled with the LDR probes described above).PCR primers and / or LDR upstream probes, optionally containing RNA bases and a 3' blocking group, are unblocked by RNase H2 only upon binding to the correct target, providing additional specificity and avoiding spurious products. 20 cycles of LDR are performed using 16-, 32-, or 64-pair primer sets of allele-specific probes with UniTaq or universal tags. LDR primers can be designed to generate the same signal in the same subcompartment for different mutations in the same gene. Fresh PCR reagents are added and mixed with the LDR products in each secondary PCR reaction chamber 22, then distributed through the mixing chamber 28 to the micropores in each column. Universal or UniTaq primers in each subcompartment in each row amplify only the products from each column with the correct tag. The presence or absence of specific mutations in each column allows the number of low-level mutations in the plasma to be counted.

[0175] In an alternative embodiment using 48 columns and 48 rows of subdivisions, each containing 96 microwells, equivalent to 2,304 microwells, one to four UniTaq primer sets (or alternatively, one to four universal tag primers with target-specific Taqman™ probes) are delivered directly to the appropriate subdivisions in each row by acoustic droplet ejection, capillary, inkjet, or quill printing, and then dried within the individual microwells. The initial sample is distributed into 48 wells or primary PCR reaction chambers 16. The highest level of DNA in plasma is 10,000 genome equivalents. On average, each target is 200 copies per primary PCR reaction chamber 16, with a maximum of one mutation. To reduce amplification of wild-type DNA, 10 to 40 cycles of locus-specific PCR are performed using blocking PNA or LNA. Optional: Use dUTP during the PCR reaction (pre-treat with UDG to prevent carryover contamination from the initial sample). Optionally, PCR primers and / or LDR upstream probes containing RNA bases and a 3' blocking group are unblocked by RNase H2 only upon binding to the correct target, providing additional specificity and avoiding spurious products. Additionally, all downstream PCR primers preferably contain the same 5' tail sequence of 8-11 bases to suppress amplification of primer dimers. In another embodiment, to minimize fragment dropout during multiplex PCR, an initial "preamplification" multiplex PCR is performed for 8-20 cycles in the initial wells or reaction chambers. This product is then distributed among 48 wells or primary PCR reaction chambers 16. In one variation, each of the 48 wells or primary reaction chambers contains one to four PCR primer sets with PNA or LNA to suppress amplification of the wild-type sequence. Single or multiplex PCR is then performed for an additional 10-30 cycles, allowing one to four fragments containing potential mutations to be amplified in a single well or primary reaction chamber.In another variation, 12 sets of four primary reaction chambers contain 4 to 16 PCR primer sets with PNA or LNA that suppress amplification of wild-type sequences, and multiplex PCR is performed for an additional 10 to 30 cycles to amplify 4 to 16 fragments containing potential mutations in a single well or primary reaction chamber. The products from each well are diluted with LDR primers and buffer. 20 cycles of LDR are performed using UniTaq-tailed allele-specific primers in 16-, 32-, or 64-pair primer sets in the wells or secondary LDR reaction chambers 22. LDR primers can be designed to generate the same signal in the same subcompartment for different mutations in the same gene. LDR reactions can be performed in the same reaction chamber or in two separate reaction chambers, which are then recombined. UniTaq master mix and UDG are added, and the products from each well or secondary LDR reaction chamber 22 are distributed to 48 subcompartments, each containing 96 micropores. The subcompartments are pre-spotted with appropriate UniTaq primers and / or probes (see Figures 28 and 29). One, two, or four potential products are PCR-amplified within each micropore using the pre-spotted primer sets, and the Ct values ​​within each micropore of each subcompartment are measured. The UniTaq primers use one, two, or four different fluorescent dyes.

[0176] The cartridge and valve configuration of Figure 25 can also be used to identify and quantify low levels of unknown mutations in plasma using multiplex PCR-LDR-real-time PCR with UniTaq or mutation-specific Taqman™ probes. This figure also shows a fluidic system for multiplex PCR-LDR-real-time PCR with UniTaq or mutation-specific Taqman™ probes using a micropore plate composed of thousands of micropores. The micropore plate allows fluid communication from both sides of the pores. The first side (top of the plate, shown on the left side of the plate) communicates with valves 1, 2, and 3, while the second side (bottom of the plate, shown on the right side of the plate) communicates with valves 4 and 5. Valve 1 dispenses lysis / protease buffer, enzymes, wash buffer, elution buffer, buffer, EtOH, light oil, and heavy oil, as needed, through 24-48 initial multiplex PCR reaction chambers, 24-48 LDR reaction chambers, and additional chambers across the first side of the micropore plate, and then to waste via valve 3. Additionally, valve 1 can select a waste port that can be used to empty the first side of the micropore plate, other chambers, LDR reaction chambers, and initial multiplex PCR reaction chambers by introducing air in the reverse direction through valve 3. Valve 1 can also select valve 2. Valve 2 dispenses initial multiplex PCR primers, optional LDR primers, master PCR mix, master LDR mix, master UDG mix, buffer, wash solution, EtOH, and air through 24-48 initial multiplex PCR reaction chambers, 24-48 LDR reaction chambers, and additional chambers across the first side of the micropore plate, and then to waste via valve 3. Valve 4 dispenses air, light oil, heavy oil, and waste across the second side of the micropore plate to waste via valve 5. Additionally, valve 1 can select a waste port that can be used to empty the second side of the micropore plate by introducing air in the reverse direction through valve 5.

[0177] Table 4: Reagent composition for multiplex PCR-LDR-real-time PCR TIFF2024161098000006.tif76170

[0178] Figure 25B shows several heating elements designed to individually heat / cool the initial multiplex reaction chamber 10, 24-48 primary multiplex PCR reaction chambers 16, 24-48 secondary multiplex reaction chambers 22, and the main chambers containing thousands of micropore or microwell subdivisions in 24-48 rows. Temperature control, heating, and / or thermal cycling can be achieved by pressing the back plate, or one or more flat surfaces of the micropore or microwell chambers, and the reaction chambers, against these heating elements. As shown in Figure 25, the two heating elements on the back side of the 24-48 primary multiplex PCR reaction chambers 10 and 24-48 secondary multiplex reaction chambers 22 are designed as two rectangular (horizontal) strips that control all of the primary chambers independently of all of the secondary chambers. Alternative configurations can also be used, such as splitting the initial multiplex PCR into two steps: (i) linear extension of one of the multiplex primers, with or without a blocking primer to suppress extension of wild-type DNA, and (ii) addition of a complementary primer to restrict or extend PCR amplification of the initial extension product. Such a configuration would require (i) 24-48 primary multiplex polymerase extension reaction chambers, (ii) 24-48 secondary multiplex reaction chambers, (iii) 24-48 tertiary multiplex reaction chambers, and (iv) at least four independently controlled heating elements behind the primary chamber containing thousands of micropores or microwells in 24-48 rows.

[0179] Figure 31 shows another exemplary PCR-LDR-qPCR reaction (optionally including carryover prevention) for detecting methylation. Genomic or cfDNA is isolated (Figure 31, step A) and treated with Bsh1236I (CG^CG) in the initial reaction chamber to completely digest unmethylated DNA. The isolated DNA sample is optionally treated with UDG to digest dU-containing nucleic acid molecules that may be present in the sample (Figure 31, step B). The enzyme-treated DNA is then treated with bisulfite to convert C to U instead of 5meC, making the strands non-complementary. The bisulfite-treated DNA is then distributed into 24, 36, 48, or 64 wells or primary PCR reaction chambers, and locus-specific regions containing the methylated CpGs of interest are amplified using PCR (Figure 31, step B). In this embodiment, another layer of selectivity can be incorporated into the method by including a 3' cleavage blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the upstream primer. Upon target-specific hybridization, the RNA base is removed by RNase H (asterisk), several bases upstream of the mutation, freeing a 3' OH group suitable for polymerase extension (Figure 31, Step B). To prevent primer dimerization, the downstream primer contains an identical tail of 8-11 bases at its 5' end. As shown in Figure 31, Step C, the amplification product may contain dU, but post-treatment with UDG or a similar enzyme can prevent carryover.

[0180] As shown in step D of Figure 31, a target-specific oligonucleotide probe hybridizes to the amplification product, and once the two oligonucleotides hybridize to complementary sequences in the secondary LDR reaction chamber, they are covalently linked together and closed by a ligase (black circle). In this embodiment, the upstream oligonucleotide probe, which has a sequence specific for detecting the methylation status of the CpG of interest, further includes a 5' primer-specific portion (Ai) that facilitates subsequent detection of the ligation product. The downstream oligonucleotide probe includes a 3' primer-specific portion (Ci'), which, together with the 5' primer-specific portion (Ai) of the upstream probe, which has a sequence specific for mutation detection, enables subsequent amplification and detection of only the methylation-specific ligation product. Another layer of specificity can be incorporated into the method by including a 3' cleavage blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe, as shown in step D of this figure. Upon target-specific hybridization, the RNA bases are removed by RNase H (asterisk), leaving 3'OH groups that are excellent for ligation (Figure 31, Step D). Following ligation, the ligation products can be detected using the corresponding primer pair Ai and Ci and a TaqMan™ probe spanning the ligation junction, as described above in Figure 19 (see Figure 31, Steps E-G), or using other suitable means known in the art.

[0181] Figure 32 shows another exemplary PCR-LDR-qPCR reaction (optionally including carryover prevention) for detecting methylation, using initial steps similar to steps A-C of Figure 31. As shown in step D of Figure 32, a target-specific oligonucleotide probe hybridizes to the amplification product, and when the two oligonucleotides hybridize to their complementary sequences, they are covalently linked together and closed by a ligase (black circle). In this embodiment, the upstream oligonucleotide probe, which has a sequence specific for detecting the methylation status of the CpG of interest, further includes a 5' primer-specific portion (Ai) that facilitates subsequent detection of the ligation product. The downstream oligonucleotide probe includes a 3' primer-specific portion (Bi'-Ci'), which, together with the 5' primer-specific portion (Ai) of the upstream probe, which has a sequence specific for mutation detection, allows for subsequent amplification and detection of only the methylation-specific ligation product. As shown in step D of this figure, another layer of specificity can be incorporated into the method by including a 3' cleavage blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe. Upon target-specific hybridization, the RNA base is removed by RNase H (asterisk), leaving a 3' OH group that is excellent for ligation (Figure 32, step D). Following ligation, the ligation product is amplified using UniTaq-specific primers (i.e., F1-Bi-Q-Ai, Ci) and detected as described above in Figure 20 (see Figure 32, steps E-H) or using other suitable means known in the art.

[0182] The cartridge design of Figure 30 can also be used to perform multiplex PCR-LDR-UniTaq detection to identify and quantitate low-level methylation and unknown mutations in plasma (alternatively, using multiplex PCR-LDR-real-time PCR with mutation- or methylation-specific Taqman™ probes).

[0183] The cartridge and valve configuration of Figure 25 can also be used to identify and quantify low-level methylation and unknown mutations in plasma using multiplex PCR-LDR-real-time PCR with UniTaq or target-specific Taqman™ probes. This figure shows a fluidic system for multiplex PCR-LDR-real-time PCR with UniTaq or target-specific Taqman™ probes using a micropore plate composed of thousands of micropores. The micropore plate allows fluid communication from both sides of the pores. The first side (top of the plate, shown on the left side of the plate) communicates with valves 1, 2, and 3, while the second side (bottom of the plate, shown on the right side of the plate) communicates with valves 4 and 5. Valve 1 dispenses lysis / protease buffer, enzymes, wash buffer, elution buffer, buffer, EtOH, light oil, and heavy oil as needed through the bisulfite reaction chamber, 24-48 initial multiplex PCR reaction chambers, 24-48 LDR reaction chambers, and additional chambers across the first side of the micropore plate, and then to waste via valve 3. Additionally, valve 1 can select a waste port that can be used to empty the first side of the micropore plate, other chambers, the LDR reaction chambers, the initial multiplex PCR reaction chambers, and the bisulfite reaction chambers, by introducing air in the reverse direction through valve 3. Valve 1 can also select valve 2. Valve 2 dispenses the initial multiplex PCR primers for methylated targets, initial multiplex PCR primers for mutant targets, any LDR primers, master PCR mix, master LDR mix, master UDG mix, Bsh1236I, bisulfite, buffer, wash solution, EtOH, and air through the bisulfite reaction chambers, 24-48 initial multiplex PCR reaction chambers, 24-48 LDR reaction chambers, and additional chambers across the first side of the micropore plate to waste via valve 3. Valve 4 dispenses air, light oil, heavy oil, and waste across the second side of the micropore plate to waste via valve 5.Additionally, valve 1 can select a waste port that can be used to empty the second side of the micropore plate by introducing air in the reverse direction through valve 5.

[0184] Table 5. Reagent composition for multiplex PCR-LDR-real-time PCR (with bisulfite) TIFF2024161098000007.tif81146

[0185] Figure 25B shows several heating elements designed to individually heat / cool the primary chambers, which contain the initial multiplex reaction chamber 10, 24-48 primary multiplex PCR reaction chambers 16, 24-48 secondary multiplex reaction chambers 22, and thousands of micropore or microwell subdivisions in 24-48 rows. Temperature control, heating, and / or thermal cycling can be achieved by pressing the back plate, or one or more flat surfaces of the micropore or microwell chambers, and the reaction chambers, against these heating elements. As shown in Figure 25, the two heating elements on the back side of the 24-48 primary multiplex PCR reaction chambers 10 and 24-48 secondary multiplex reaction chambers 22 are designed as two rectangular (horizontal) strips, controlling all primary chambers independently of all secondary chambers. Alternative configurations can also be used. For example, instead of the Bsh1236I selection process, methylated DNA may be enriched using methyl-specific binding proteins or antibodies for methylated DNA. This step can be performed within the cartridge or before the methyl-enriched DNA is placed in the cartridge. The initial multiplex PCR after bisulfite treatment can be divided into two steps: (i) linear extension of one of the multiplex primers, with or without a blocking primer to inhibit extension of unmethylated DNA, and (ii) the addition of a complementary primer to restrict or extend PCR amplification of the initial extension product. Such a configuration requires (i) 24-48 primary multiplex polymerase extension reaction chambers, (ii) 24-48 secondary multiplex reaction chambers, (iii) 24-48 tertiary multiplex reaction chambers, and (iv) at least four independently controlled heating elements behind the main chamber, which contains thousands of micropores or microwells in 24-48 rows.

[0186] Figure 33 shows an RT-PCR-PCR-qPCR reaction for detecting low levels of alternatively spliced ​​transcripts. Step A in Figure 33 shows the wild-type transcript containing exon 3a (top) and the low-level alternatively spliced ​​transcript containing exon 3b (bottom). The method involves isolating mRNA and generating cDNA copies with reverse transcriptase in the initial reaction chamber using a 3' transcript-specific primer (i.e., exon 4). PCR amplification is performed with limited cycles (i.e., 7 cycles) to activate Taq polymerase and maintain the relative ratios of different amplicons (Figure 33, step B). In one embodiment, the initial multiplex reaction is distributed into six primary PCR reaction chambers. In this case, the average number of copies of each original transcript distributed to each primary PCR reaction chamber is 20.

[0187] As shown in step C of Figure 33, target-specific oligonucleotide secondary primers are hybridized to the primary amplification product in the primary PCR reaction chamber, and a polymerase (filled diamond) is used to PCR amplify the target-containing region of interest (i.e., 10 cycles). In this embodiment, primers specific for the alternative splice variant (i.e., exon 3b) but not the wild-type variant (i.e., exon 3a) are utilized to generate only amplification products corresponding to the alternative splice variant. The products from each of the six chambers are separately diluted into four smaller secondary reaction / dilution chambers (24 chambers total). Following nested primer amplification, the PCR products from each secondary reaction / dilution chamber are separately diluted and distributed into 384 or 768 micropores. Products are amplified using UniTaq-specific primers (i.e., F1-Bi-Q-Ai, Ci) and detected as described above in Figure 20 (see Figure 33, steps D-F) or by other suitable means known in the art.

[0188] Figure 34 shows a schematic front view of a portion of an exemplary design for pre-chamber filling, which allows fluid transfer into a chamber containing microwells or micropores. This design shows a chamber structure and microwells or micropores suitable for performing multiplex RT-PCR-nested PCR-UniTaq detection to enumerate both rare and overexpressed lncRNAs, mRNAs, or splice variants. (Alternatively, multiplex RT-PCR-nested PCR-real-time PCR using target-specific Taqman™ probes.) In Figure 34, the input sample is fluidically connected to the initial reaction chamber 10 (bottom) via inlet 12. The initial reaction chamber 10 is fluidically connected by conduit 14 to a hexagonal chamber 16 (with a large trough 18, constituting the primary PCR reaction chamber). The primary PCR reaction chambers 16 are fluidly connected by conduits 20 to a second set of hexagonal chambers 22 (four chambers to which each of the first chambers is connected, each comprising a large trough 24a, a medium trough 24c, a small trough 24b, and a very small trough 24d, constituting the secondary reaction / dilution chambers 22). The hexagonal chambers 22 are fluidly connected by conduits 26 to an elongated mixing chamber 28, which is fluidly connected by conduits 30 to a subdivision 32 of chambers containing microwells or micropores (only four rows are shown at the top of the panel). The diagram is not to scale and is for illustrative purposes. During cartridge manufacture, the rows are pre-filled with one to four UniTaq primer sets (or alternatively, one to four universal tag primers with target-specific Taqman™ probes). During cartridge manufacture, the chambers leading to the rows of microwells or micropores are pre-filled with nested PCR primer sets bearing either UniTaq or universal tag sequences at their 5' ends. The grey circles 17 on the left side of the diagram indicate the potential locations where the primer sets may be delivered or printed, for example, by acoustic droplet ejection, capillary, inkjet, or quill printing. The primers are allowed to dry and the cover portion of the cartridge is attached, sealing the probe set in place.During cartridge use, reactants move fluidically from the cartridge's initial chamber to the final row of microwells or micropores, each separated from adjacent rows. This illustration shows four subcompartments organized in 24 columns and 32 rows, equivalent to 768 locations, with each subcompartment containing 24 microwells or micropores. In this example, seven cycles of multiplex reverse transcription PCR are performed in the initial reaction chamber to amplify the original target. The initial multiplex products are then distributed to the primary PCR reaction chambers. In this case, an average of 20 copies of each original transcript are distributed to each primary PCR reaction chamber. Ten cycles of nested PCR are performed using target-specific primers with UniTaq or universal tags, in 16-, 32-, or 64-mer primer sets. Each primary PCR reaction chamber is designed to retain a constant ratio of liquid volume after draining. Three cycles of filling and draining are performed to separately dilute the products. The products from each primary PCR reaction chamber are then distributed to the secondary reaction / dilution chambers. Each secondary reaction / dilution chamber is designed to retain a constant ratio of liquid volume after draining. Three cycles of filling and draining are performed to separately dilute the products. Nested PCR products are distributed to the mixing chamber and then to each column's micropores. Universal or UniTaq primers in each row amplify only products from each column with the correct tag. Poisson distribution within the micropores allows counting of low-, medium-, and high-copy lncRNAs, mRNAs, or splice variants.

[0189] In an alternative embodiment using 48 columns and 48 rows of subdivisions, each containing 96 microwells, equivalent to 2,304 microwells, one to four UniTaq primer sets (or alternatively, one to four universal tag primers with target-specific Taqman™ probes) are delivered directly to the appropriate subdivisions in each row by acoustic droplet ejection, capillary, inkjet, or quill printing, and then dried within the individual microwells. Ten cycles of multiplex RT-PCR are performed in the initial reaction chambers or wells, yielding up to 1,024 copies of each original RNA molecule. Optionally, "tandem" PCR primers are used. Additionally, all PCR primers may contain the same 5' tail sequence, preferably 10-11 bases, to suppress primer dimer amplification. The initial multiplex product is distributed into 48 wells, or primary PCR reaction chambers. Each well receives an average of 20 copies of each original RNA target. Using 24- or 48-mer primer sets with UniTaq tails, three to four cycles of nested PCR can yield up to 160–320 copies of each original pathogen. The products from each well are distributed across two sets of 24 or four sets of 12 subcompartments, each containing 96 micropores. If two sets are used, the second set is a 100 / 1 dilution of the first set. If four sets are used, each set is a 20 / 1 dilution of the previous set. This allows for a large-scale coverage of the RNA molecules present. On average, each initial subcompartment yields 12 copies of each original RNA molecule, with one or zero copies of the original RNA per given micropore. If more RNAs are present, additional copies are obtained per subcompartment. One, two, or four potential products are PCR-amplified within each micropore using pre-spotted UniTaq primer sets, and the Ct values ​​within each micropore of each subcompartment are measured. The UniTaq primers use one, two, or four different fluorescent dyes.The Poisson distribution of 96 micropores across two or four dilution sets allows some enumeration of very low as well as very high RNA copy numbers in the sample.

[0190] Another embodiment of the present invention is a system for sequencing by synthesis or ligation of target molecules on a solid support. For example, as depicted in FIG. 1, one or more target molecules are amplified within a 5-micron diameter micropore. The target is amplified and immobilized or bound to a solid support within the micropore or microwell. Such immobilization may be performed directly on the interior surface of the micropore or microwell, on dendrimeric primers immobilized on the surface of the micropore or microwell, or on microbeads that are pre-dispensed within the micropore or microwell before amplification or dispensed into the micropore or microwell after amplification. Microbeads can be porous with a significant surface area, allowing for higher levels of amplification than can be achieved with the interior surface of a micropore alone. By immobilizing or binding to a solid support, the amplified target can be interrogated one or more times to determine the presence or absence of mutations, SNPs, or sequence variations within the target.

[0191] Standard approaches for detecting fluorescent products in sequencing-by-synthesis rely on amplifying only one target per well and generating sequencing reads using a single universal primer. One approach for amplifying a single target molecule, known as cluster amplification, involves immobilizing both forward and reverse primers on a solid support. However, this approach limits the total yield of strands within a cluster because extension products tend to rehybridize with each other rather than hybridize to new primers. An alternative approach is to amplify DNA on beads in aqueous droplets surrounded by oil. Here, we propose a simple method for performing amplification in solution, capturing the product on a solid support or immobilized primers, and further extending it to generate copies of the amplified product. This method allows for larger reactions, resulting in high yields of multiple amplification products that can be sequenced using selected target-specific primers or, alternatively, a set of different sequencing primers containing common and variable regions. With appropriate loading and primer selection, each round of sequencing allows for unique sequencing reads in approximately 30%-35% of the micropores.

[0192] Micropores and microwells are constructed with a hydrophilic surface on the inside and a hydrophobic surface on the outside. This structure is suitable for injecting sample fluids into different individual volumes of liquid, allowing for amplification without crosstalk between micropores or microwells. Furthermore, functionalizing the hydrophilic surface allows for binding / immobilization of primers within the micropores or microwells but not on the outside, thereby eliminating crosstalk.

[0193] When the solid support is composed of poly(methyl methacrylate) (also known as PMMA, Plexiglas, Lucite), cyclic olefin copolymer (COC), polyethylene, or polypropylene coating materials, one approach is to create micropores or microwells by UV laser ablation. Alternatively, micropores and microwells can be created by injection molding, imprinting, hot embossing, or etching, and then masking techniques can be used to expose specific surfaces to UV light.These processes generate carboxylate surfaces suitable for EDC / NHS-mediated covalent attachment of 5' amino-terminated oligonucleotides to fabricate microarrays (Situma et al., "Fabrication of DNA Microarrays onto Poly(methyl methacrylate) with Ultraviolet Patterning and Microfluidics for the Detection of Low-abundant Point Mutations," Anal Biochem 340(1):123-35 (2005); McCarley et al., "Resist-free Patterning of Surface Architectures in Polymer-based Microanalytical Devices," J Am Chem Soc. 127(3):842-3 (2005); Soper et al., "Fabrication of DNA Microarrays onto Polymer Substrates Using UV Modification Protocols With Integration Into Microfluidic Platforms for the Sensing of Low-abundant DNA Point Mutations," Methods 37(1):103-13 (2005); Wang et al. al., "Microarrays Assembled in Microfluidic Chips Fabricated From Poly(Methyl Methacrylate) for the Detection of Low-Abundant DNA Mutations," Anal Chem. 75(5):1130-40 (2003), each of which is incorporated herein by reference in its entirety.

[0194] In an alternative embodiment, covalently bonded polymer brushes are grown on the surface of PMMA via atom transfer radical polymerization (ATRP) (Balamurugan et al., “Aqueous-based Initiator Attachment and ATRP Grafting of Polymer Brushes from Poly(Methyl Methacrylate) Substrates,” Langmuir 28(40):14254-60 (2012), incorporated herein by reference in its entirety). This approach relies on the covalent anchoring of an ATRP initiator to the PMMA surface and subsequent surface-initiated aqueous ATRP of poly(N-isopropylacrylamide) PNIPAAm. Briefly, selected regions of PMMA are UV-modified to introduce carboxylic acid functional groups, which are then converted to amino groups by reaction with ethylenediamine in an EDC / NHS system. These amine-functionalized PMMA surfaces are then reacted with the activated ester of the ATRP initiator N-hydroxysuccinimidyl-2-bromo-2-methylpropionate. Atom transfer polymerization is performed in water to grow PNIPAAm brushes from a covalently attached initiator surface. This aqueous route to surface grafting of polymers is adaptable to a variety of substrates and water-soluble ATRP monomers.

[0195] In an alternative embodiment, the COC surface was photografted with poly(ethylene glycol) methacrylate (PEGMA) using a two-step sequential approach: first, covalently bond a surface initiator, followed by graft polymerization of PEGMA from these sites in a second step (Stachowiak et al., "Hydrophilic Surface Modification of Cyclic Olefin Copolymer Microfluidic Chips Using Sequential Photografting," J Sep Sci. 30(7):1088-93 (2007), incorporated herein by reference in its entirety). A similar approach was also used for low-density polyethylene films (Wang et al., "Surface Modification of Low-Density Polyethylene Films by UV-Induced Graft Copolymerization and Its Relevance to Photolamination," Langmuir 14(4):921-927 (1998), incorporated herein by reference in its entirety).

[0196] In an alternative embodiment, a hydrophilic coating is used to convert a hydrophobic surface to a hydrophilic one (Zilio et al., "Universal Hydrophilic Coating of Thermoplastic Polymers Currently Used in Microfluidics," Biomed Microdevice. 16(1):107-14 (2014), incorporated herein by reference in its entirety). In another variation, a photoreactive coating is used to spatially control the wettability of the device and create a hydrophilic surface (Abate et al., "Photoreactive Coating for High-Contrast Spatial Patterning of Microfluidic Device Wettability," Lab Chip 8(12):2157-60 (2008), incorporated herein by reference in its entirety).

[0197] As described in U.S. Patent Application Publication No. 2015 / 0099642 to Barany et al., which is incorporated herein by reference in its entirety, the surface of the solid support may include a layer of linker molecules that connect the oligonucleotides to the solid support, although it will be understood that the linker molecules are not a required element of the present invention. The linker molecules are preferably long enough to allow the polymer of the completed substrate to freely interact with molecules exposed on the substrate. To achieve sufficient exposure, the linker molecules should be 6-50 atoms long. Appropriate linker molecules can be selected based on their hydrophilicity / hydrophobicity properties. The linker molecules may be, for example, arylacetylenes, ethylene glycol oligomers containing 2-10 monomer units, diamines, dibasic acids, amino acids, or combinations thereof.

[0198] The linker molecules can be attached to the substrate via carbon-carbon bonds, for example, using a (poly)trifluorochloroethylene surface. The linker molecules can also be attached to the polymerized monolayer, optionally in an ordered array, i.e., as part of the head group. In an alternative embodiment, the linker molecules are adsorbed to the surface of the substrate.

[0199] The device of the present invention can contain various types of oligonucleotides depending on the application. In one embodiment of the present invention, the oligonucleotide of the device is a capture oligonucleotide probe, as described in U.S. Patent Nos. 6,852,487 and 7,455,965 to Barany et al., the entire contents of which are incorporated herein by reference. Thus, the present invention also encompasses a method for capturing multiple target nucleotide sequences on a solid support.

[0200] Other suitable methods of solid-phase amplification that can be performed using the devices of the present invention are described in U.S. Patent No. 6,017,738 to Morris et al., U.S. Patent No. 7,741,463 to Gormley et al., U.S. Patent No. 7,754,429 to Rigatti et al., and U.S. Patent No. 6,355,431 to Chee et al., as well as U.S. Patent Publication Nos. 2009 / 0226975 to Sabot et al., 2001 / 0036632 to Yu et al., 2008 / 0108149 to Sundararajan et al., and 2005 / 0053980 to Gunderson et al., which are incorporated herein by reference in their entireties. The devices of the present invention are also suitable for performing other multiplexed nucleic acid reactions, including, but not limited to, single-base or multi-base extension reactions, primer extension assays, solid-phase sequencing, solid-phase oligonucleotide ligation assays, paired-end reads, RNA sequencing, copy number analysis, ChIP sequencing, and other reactions described in U.S. Patent Application Publication No. 2010 / 0015626 to Oliphant et al., which is incorporated herein by reference in its entirety.

[0201] As described in U.S. Patent Application Publication No. 2015 / 0099642 to Barany et al., which is incorporated herein by reference in its entirety, one aspect of the present invention relates to methods for binding oligonucleotides within microwells or micropores on a solid support. The first of these methods includes providing a solid support having a bottom surface, a top surface, and a plurality of side surfaces extending between the bottom surface and the top surface. The bottom surface, top surface, and a plurality of side surfaces collectively form a plurality of microwells or micropores on the solid support. A mask is utilized to cover the bottom surface of the solid support, and when the masked device is exposed to an activating agent, the unmasked surface of the solid support is activated, while the masked surface of the solid support is inactivated. The oligonucleotides are bound within the microwells or micropores on the solid support by removing the mask from the solid support and contacting the exposed solid support with a plurality of ol...

Claims

1. 1. A process for preparing a microtiter plate for identifying a plurality of nucleic acid molecules in a sample, comprising: providing a microtiter plate comprising a plurality of product capture sub-compartments arranged in individual rows and columns, each individual product capture sub-compartment comprising an array of a plurality of individual hydrophilic micropores or microwells separated from each other by a hydrophobic surface; filling the set of micropores or microwells of the microtiter plate with a set of aqueous liquids containing oligonucleotide primers and / or probes; Centrifuging the microtiter plate to distribute the aqueous liquid into the unfilled micropores or microwells of each individual product capture subcompartment within the microtiter plate; terminating the centrifugation so that the aqueous liquid is not in contact with the hydrophobic surface; allowing the aqueous liquid to evaporate; drying the micropores or microwells so as to leave the oligonucleotide primers and / or probes in the micropores or microwells; The process comprising:

2. 13. A process for identifying a plurality of nucleic acid molecules in a sample using a microtiter plate prepared by the process of claim 1, comprising: depositing an aqueous sample into said microtiter plate; applying a hydrophobic liquid to the microtiter plate such that the hydrophobic liquid overlies the aqueous sample; Centrifuging the microtiter plate to distribute the aqueous sample into unfilled micropores or microwells within the microtiter plate; terminating the centrifugation so that the sample is not in contact with the hydrophobic surface; performing an amplification reaction of the nucleic acid molecule under conditions in which a polymerase, exonuclease, endonuclease, or ribonuclease cleaves in a target-specific manner one or more probes containing a quencher group and a fluorescent group, such that if the nucleic acid molecule is present in the sample, the fluorescent group is released and a signal is generated; The process comprising:

3. 3. The process of claim 2, wherein a plurality of nucleic acid molecules are identified in a sample that include a target nucleotide sequence that differs from the nucleotide sequence of other nucleic acid molecules in the sample or in other samples by one or more nucleotides, one or more insertions or deletions of nucleotides, one or more copy numbers, one or more transcript sequences, one or more translocations, and / or one or more methylated residues.

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