Kits and apparatuses

Abstract

Provided herein are kits and devices that can be used for improved polynucleotide detection.

Description

[0001] This invention relates to kits suitable for detecting the presence of a large number of diagnostic biomarkers, including those for identifying the presence of biomarkers for cancer, infectious diseases, and transplant rejection. They are also useful for companion diagnostic testing, in which a reliable and cost-effective set of biomarkers must be identified.

[0002] Polymerase chain reaction (PCR) is a well-known and powerful technique for amplifying DNA or RNA present in laboratory and diagnostic samples to a level that can be reliably detected and / or quantified. However, it is subject to several limitations when used to study analyte samples containing low levels of such molecules. First, while the technique can detect as few as a single target molecule, it is prone to false positives due to unwanted amplification of other nucleic acid sequences present in the sample. This makes the selection of oligonucleotide primers for initiating the reaction critical; this, in turn, makes designing primers with the desired level of specificity relatively complex. Consequently, many PCR-based assays currently on the market have limited specificity.

[0003] The second drawback is that multiplexing of PCR-based methods is limited in practice to a maximum of a few dozen target sequences (usually no more than 10) to avoid primer-primer interactions, resulting in a relatively narrow operating window.

[0004] Another problem is that target quantification is difficult because PCR reactions cycle exponentially; small changes in reaction efficiency have a huge impact on the amount of detectable substance produced. Therefore, even with proper control and calibration, quantification is usually limited to about three times the accuracy.

[0005] Finally, mutations in the target region studied by PCR amplification methods can have undesirable side effects. For example, there have been instances where FDA-approved tests had to be withdrawn due to a large number of false negatives caused by mutations in the genetic region targeted by the test primers in the target organism. Conversely, if a specific single nucleotide polymorphism (SNP) is the target of amplification, PCR methods often produce false positives when wild-type variants are present. Avoiding this requires very careful primer design and further limits the power of multiplexing. This is especially important when searching for groups of SNPs, as this is a common requirement for cancer testing / screening or companion diagnostics.

[0006] US2006 / 110765 A1 (Wang et al.) teaches enzymatic cleavage at mismatch sites, which is generally an inefficient and non-specific reaction. Furthermore, as disclosed in Wang et al., because cleavage is used at mismatch sites, off-target hybridization of the probe with similar sequences in the sample can lead to false positives. It is also impossible to distinguish between two different genetic variants at the same or adjacent sites, as they will both result in the cleavage and amplification of the same probe. Therefore, the teachings of Wang et al. lead to low sensitivity and low specificity in the reaction protocol. In contrast, the technical advantages of the method disclosed in this invention provide a rapid, efficient method with high specificity for dsDNA that can be effectively blocked by mismatches. Moreover, the method of this invention is highly specific to the targeted genetic variant and can distinguish between different variants at the same or adjacent sites.

[0007] US2009 / 239283 A1 (Liu et al.) teaches the use of a non-extending 3' end removed by pyrophosphate digestion, making genetic engineering of custom polymerases capable of removing 3' blocking modifications necessary. In contrast, this invention utilizes the inherent natural pyrophosphate digestion activity of existing polymerases and does not use 3' blocking modifications. The method disclosed in Liu et al. also relies on removing only the terminal base from a portion of the probe for subsequent amplification and is limited to this embodiment by using 3' blocking modifications. In contrast, the method disclosed in this invention achieves an embodiment where the reaction requires stepwise removal of more than one base from the probe to initiate the reaction, making it substantially more robust to transient off-target annealing of background DNA or other probes, which can lead to unnecessary removal of terminal bases. Summary of the Invention

[0008] We have now developed a kit and apparatus for a novel method that builds upon our experience using the pyrophosphorolysis method employed in our earlier patent (see PCT / GB2019 / 052017) to overcome many of these limitations. To this end, it utilizes the double-strand specificity of pyrophosphorolysis (a reaction that cannot be efficiently performed on single-stranded oligonucleotide substrates or double-stranded substrates containing blocking groups or nucleotide mismatches). Therefore, according to the invention, a kit is provided comprising:

[0009] (a) A single-stranded probe oligonucleotide A0, wherein the single-stranded probe oligonucleotide A0 is capable of forming a first intermediate product with a target polynucleotide sequence, wherein the intermediate product is at least partially double-stranded;

[0010] (b) Ligase;

[0011] (c) Pyrophosphate hydrolase, which is capable of digesting the first intermediate product from the end of A0 in the 3'-5' direction to produce a partially digested chain A1;

[0012] (d) at least one single-stranded primer oligonucleotide substantially complementary to a portion of A0;

[0013] (e) Amplification enzyme;

[0014] (f) Suitable buffer solution.

[0015] and equipment, the equipment including:

[0016] At least one fluid channel between a first region, a second region, and a third region, wherein the first region includes one or more orifices, each orifice comprising:

[0017] dNTP;

[0018] At least one single-stranded primer oligonucleotide;

[0019] Amplifying enzymes used to initially amplify DNA present in the sample; and

[0020] The second region includes one or more holes, each hole comprising:

[0021] Single-stranded probe oligonucleotide A0, which can form a first intermediate product with a target polynucleotide sequence, said intermediate product being at least partially double-stranded;

[0022] Pyrophosphate hydrolase, which is capable of digesting the first intermediate product from the end of A0 in the 3'-5' direction to produce a partially digested chain A1;

[0023] At least one pyrophosphate ion source;

[0024] Ligase;

[0025] and

[0026] The third region includes one or more holes, each hole containing:

[0027] dNTP;

[0028] Buffer solution;

[0029] Amplification enzyme;

[0030] Means for detecting signals originating from A1 or a portion thereof, or more than one copy of A1 or more than one copy of a portion thereof; and

[0031] The pores in the second region or the third region further include at least one single-stranded primer oligonucleotide substantially complementary to a portion of A0.

[0032] The analytes to which the methods of the present invention can be applied are nucleic acids that include the target polynucleotide sequence being sought, such as naturally occurring or synthetic DNA or RNA molecules. In one embodiment, the analyte is typically present in an aqueous solution containing the analyte and other biological material, and in another embodiment, the analyte will be present along with other background nucleic acid molecules that are not of interest for the purpose of testing. In some embodiments, the analyte will be present in a low amount relative to these other nucleic acid components. Preferably, for example, when the analyte is derived from a biological sample containing cellular material, sample preparation techniques such as filtration, centrifugation, chromatography, or electrophoresis will be used to remove some or all of these other nucleic acids and foreign biological material prior to performing step (b) of the method. Suitably, the analyte is derived from a biological sample taken from a mammalian subject (especially a human patient), such as blood, plasma, sputum, urine, skin, or biopsy. In one embodiment, the biological sample will be lysed to release the analyte by destroying any present cells. In other embodiments, the analyte may already be present in the sample itself in a free form; for example, cell-free DNA circulating in blood or plasma. Brief description of the attached diagram

[0034] Figure 1 : A scheme for a simplified method of detecting polynucleotide sequences.

[0035] Figure 2 : A graph comparing the fluorescence levels (representing the presence of a specific target analyte sequence) detected when the 5'-3' exonuclease digestion step occurs during the pre-amplification step and when it moves to the pyrosequencing / ligation step of the protocol (as in protocols 3-5). In this example, the 5'-3' exonuclease is Lambda.

[0036] Figure 3: The inventors have tested the method of Scheme 3 of the present invention using a series of different PPI enzymes. Figure 3(A) shows the detection using 1% MAF T790M of Mako, Klenow, and Bsu. Figure 3(B) shows the detection using 0.5% MAF T790M of Bst LF at different PPI concentration ranges. Even without extended optimization, all four enzymes performed very well.

[0037] Figure 4: Detection results of 1% MAF T790M using the method of Scheme 4 of the present invention with four different pyrophosphate hydrolases (PPL) Mako, Klenow, Bsu and Bst LF.

[0038] Figure 5The graph shows the detected fluorescence levels (representing the presence of a specific target analyte sequence) of 0.5%, 0.10%, and 0.05% MAF exon 19del_6223 detected according to schemes 1 and 4.

[0039] Figure 6 The inventors tested EGFR exon 20T790M at 0.10%, 0.50%, and 1% MAF according to scheme 4.

[0040] Figure 7 This illustrates the detection of 1% MAF EGFR exon 20T790M with and without exonuclease during the RCA step.

[0041] Figure 8 The inventors investigated the effect of the PPL:RCA mixing ratio on the signal intensity detected by 0.5% MAF EGFR exon 20T790M, and the results showed that... Figure 8 As can be seen, a 1:2 PPL:RCA mixing ratio results in the lowest signal strength, but at the earliest time point. Immediately following in time is a 1:4 PPL:RCA mixing ratio with a greater signal strength. At the latest time point, the 1:8 PPL:RCA mixing ratio exhibits the highest signal strength.

[0042] Figure 9 The results of the comparative experiments conducted according to scheme 4 using SybrGreenI (50℃ and 60℃) and Syto82 (50℃ and 60℃) are shown.

[0043] Figure 10 The inventors investigated RCA using two different enzymes, BST LF and BST 2.0WS, according to scheme 4.

[0044] Figure 11: The inventors investigated the effect of different PPL enzymes on the RCA reaction at different PPL:RCA reaction mixture ratios. The results are shown in Figure 11(A) 1:4 PPL:RCA and Figure 11(B) 1:8 PPL:RCA. Except for BST LF, all PPL enzymes affected the RCA reaction at a 1:4 PPL:RCA ratio. At a 1:8 PPL:RCA ratio, all enzymes except BST LF and Klenow affected the RCA reaction.

[0045] Figure 12 The fluorescence measurement results of Example 11 show that when both oligonucleotides 3 and 4 are present, the fluorescence signal appears faster in the reaction, indicating that the pyrophosphate hydrolysis and ligation of oligonucleotide 3 has already occurred in the first reaction mixture.

[0046] Figure 13: Detect mutations in T790M and C797S_2389 with a 1% allele fraction in the same reaction.

[0047] Figure 14 Three mutations (0.5% allele fraction) were detected simultaneously in one well: G719X_6239, G719X_6252, and G719X_6253.

[0048] Figure 15 : A schematic diagram of cyclizing A1 to form A2 against the analyte target sequence. A0 is progressively digested relative to the target in the 3'-5' direction from the 3' end of A0 to form a partially digested chain A1, as shown in steps (A) and (B). This progressive digestion exposes the target region complementary to the 5' end of A0 / A1, and the 5' end of A1 then hybridizes into this region, as shown in step (C). A1 is then joined together to form the cyclized A2, step (D).

[0049] Figure 16 The fluorescence measurement results of Example 14 show the results of an implementation scheme in which A0 undergoes pyrophosphorylation to form A1, and then A1 cyclizes to form A2 targeting the target sequence.

[0050] Figure 17 The single-stranded probe oligonucleotide A0 is annealed to the target polynucleotide sequence to produce a first intermediate product that is at least partially double-stranded, wherein the 3' end of A0 forms a double-stranded complex with the target polynucleotide sequence. In this simplified embodiment of the invention, two A0 molecules and one target polynucleotide sequence are present to illustrate how A0 that is not annealed to the target does not participate in further steps of the method. In this illustrative example, the 3' end of A0 is annealed to the target polynucleotide sequence, while the 5' end of A0 is not annealed to the target polynucleotide sequence. The 5' end of A0 includes a 5' chemically blocking group, a co-initiating sequence, and a barcode region.

[0051] The first intermediate of the partially double-stranded A0 is pyrolyzed from the 3' end in the 3'-5' direction in the presence of pyrophosphatase to produce partially digested chain A1, analyte, and undigested A0 molecules that have not annealed with the target.

[0052] Figure 18 A1 is annealed to a single-stranded trigger oligonucleotide B, and the A1 strand is extended against B in the 5'-3' direction to produce oligonucleotide A2. In this illustrative example, the trigger oligonucleotide B has a 5' chemical block. Any undigested A0 is annealed to the trigger oligonucleotide B, but it cannot be extended against B in the 5'-3' direction to produce the target sequence for the subsequent part of the method. In this example, A2 is initiated with at least one single-stranded primer oligonucleotide, and produces A2 or more than one copy of the region of A2.

[0053] Figure 19 A1 is annealed to splice oligonucleotide D and then cyclized by joining its 3' and 5' ends. The cyclized A2 is now initiated with at least one single-stranded primer oligonucleotide and produces A2 or more than one copy of the region of A2. In this illustrative example, splice oligonucleotide D cannot extend against A1 due to 3'-modification (in this example, chemical modification) or by nucleotide mismatch between the 3' end of D and the corresponding region of A2.

[0054] Figure 20 The 3' region of the splice oligonucleotide D is annealed to the 3' region of A1, while the 5' region of the splice oligonucleotide D is annealed to the 5' region of the ligation probe C. Therefore, the second intermediate A2 consists of A1, C, and optionally an intermediate region formed by A1 extending in the 5'-3' direction to attach to the 5' end of C. In this illustrative example, the ligation probe C has a 3' chemically blocking group so that 3'-5' exonucleases can be used to digest any unligated A1.

[0055] A2 is initiated by at least one single-stranded primer oligonucleotide and produces more than one copy of A2 or the region of A2.

[0056] Description of the implementation plan

[0057] In one aspect of the present invention, a method is provided for detecting a target polynucleotide sequence in a given nucleic acid analyte present in a sample, the method comprising the following steps:

[0058] (a) Introducing one or more nucleic acid analytes into a first reaction mixture, the first reaction mixture comprising:

[0059] i. Single-stranded probe oligonucleotide A0;

[0060] ii. Pyrophosphate hydrolase; and

[0061] iii. Ligase

[0062] A0 undergoes pyrophosphate decomposition from the 3' end in the 3'-5' direction to produce at least partially digested chain A1, and A1 undergoes linkage to form A2;

[0063] (b) Detect the signal from the product of the previous step, wherein the product is A2 or a portion thereof, or more than one copy of A2 or more than one copy of a portion thereof, and thereby infer the presence or absence of the polynucleotide target sequence in the analyte.

[0064] In some embodiments, the first reaction mixture further comprises a source of pyrophosphate ions.

[0065] In some embodiments, the first reaction mixture further comprises at least one single-stranded primer oligonucleotide substantially complementary to a portion of A0, and deoxyribonucleotide triphosphates (dNTPs).

[0066] In some implementations, dNTPs are optional.

[0067] In some embodiments, the first reaction mixture also contains an amplification enzyme.

[0068] In some embodiments, the product of step (a) is introduced into a second reaction mixture prior to step (b), the second reaction mixture comprising at least one single-stranded primer oligonucleotide and dTNP.

[0069] In some embodiments, the second reaction mixture also contains an amplification enzyme.

[0070] In some implementations, one or more nucleic acid analytes may be introduced simultaneously into the first reaction mixture and the second reaction mixture.

[0071] In some implementations, one or more nucleic acid analytes can be sequentially introduced into the first reaction mixture and the second reaction mixture.

[0072] In some implementations, the dNTP is a hot-start dNTP.

[0073] Hot-start dNTPs are dNTPs modified at the 3' end with a heat-sensitive protecting group. The presence of this modification prevents DNA polymerase nucleotide incorporation until the nucleotide protecting group is removed using a heat activation step.

[0074] In some embodiments, during step (a), the analyte is annealed to a single-stranded probe oligonucleotide A0 to produce a first intermediate that is at least partially double-stranded and wherein the 3' end of A0 forms a double-stranded complex with the analyte target sequence.

[0075] In some embodiments, during step (a), the first intermediate is pyrolyzed from the 3' end of A0 in the 3'-5' direction to produce partially digested chain A1 and analyte.

[0076] In some embodiments, the first reaction mixture further comprises a linker oligonucleotide C, and the partially digested chain A1 is linked at its 3' end to the 5' end of C to produce oligonucleotide A2.

[0077] In some implementations, the partially digested chain A1 is looped through the connection of its 3' end and 5' end.

[0078] In some implementations, the connection of A1 occurs when:

[0079] During step (a); or

[0080] During step (b); or

[0081] Between steps (a) and (b).

[0082] In one embodiment, A1 is circularized against the analyte target sequence. In this embodiment, the target region exposed by stepwise digestion of A1 from the 3' end of A0 in a 3'-5' orientation is complementary to the 5' end of A0 / A1. In this embodiment, a ligase can be used to ligate the 3' and 5' ends of A1 to form the circularized oligonucleotide A2. This is, for example... Figure 15 As shown in the diagram. In one embodiment, the 5' end of A0 / A1 is complementary to the target in a region 5-50 nucleotides long. In one embodiment, it is 5-25 nucleotides long. In one embodiment, it is 5-20 nucleotides long. In one embodiment, it is 5-15 nucleotides long. In one embodiment, it is 5-12 nucleotides long. In one embodiment, it is 5-10 nucleotides long.

[0083] In some embodiments, the first reaction mixture further comprises a 5'-3' exonuclease, and the 5' end of A0 is conferred resistance to digestion by the 5'-3' exonuclease.

[0084] In some implementations, the sample is further treated with a protease after the amplification of the given nucleic acid analyte and before the addition of the first reaction mixture (step (a)).

[0085] In some embodiments, the first reaction mixture further comprises phosphatase or phosphorylase.

[0086] In some implementations, the product of the previous step is treated with pyrophosphatase before or during step (b).

[0087] In some implementations, the product of the previous step is treated with an exonuclease before or during step (b).

[0088] In some implementations, oligonucleotide C also includes 3' modifications or internal modifications to protect it from digestion by 3'-5' exonucleases.

[0089] In some implementations, the oligonucleotide C also includes a 5' modification to protect it from digestion by 5'-3' exonucleases.

[0090] In some embodiments, the first or second reaction mixture further comprises splint oligonucleotide D.

[0091] In some embodiments, D includes an oligonucleotide region complementary to the 3' end of A1 and a region complementary to the 5' end of oligonucleotide C or the 5' end of A1.

[0092] In some implementations, D cannot be extended against A1 due to 3' modification or mismatch between the 3' end of D and the corresponding region of A1.

[0093] In some implementations, the enzyme that performs pyrophosphate hydrolysis of A0 to form a partially digested chain A1 also amplifies A2.

[0094] In some implementations, detection is achieved using one or more oligonucleotide fluorescent binding dyes or molecular probes.

[0095] In some implementations, the increase in signal caused by the generation of the A2 amplicon over time is used to infer the concentration of the target sequence in the analyte.

[0096] In some implementations, more than one probe A0 is used, each probe A0 is selective for different target sequences, and each probe A0 contains a recognition region. It is also characterized in that the amplicon derived from A2 contains this recognition region, and thus the target sequence present in the analyte is inferred by detecting the recognition region.

[0097] In some implementations, molecular probes or sequencing are used to detect the recognition region.

[0098] In some implementations, the final step of the method further includes the following steps:

[0099] i. Label the product of step (b) using one or more oligonucleotide fluorescent binding dyes or molecular probes;

[0100] ii. Measure the fluorescence signal of the product;

[0101] iii. Exposing the product to a set of denaturing conditions; and

[0102] Polynucleotide target sequences in analytes can be identified by monitoring changes in the fluorescence signal of the product during exposure to denaturing conditions.

[0103] In some implementations, one or more nucleic acid analytes are split into more than one reaction volume, each volume containing one or more probe oligonucleotides A0 introduced to detect different target sequences.

[0104] In some implementations, different probes A0 contain a common initiation site, allowing the use of a single primer or a set of primers to amplify the region of A2.

[0105] In some implementations, a method is provided for detecting a target polynucleotide sequence in a given nucleic acid analyte present in a sample, the method comprising the following steps:

[0106] (a) Amplify a given nucleic acid analyte present in the sample;

[0107] (b) Introducing the product of step (a) into a first reaction mixture, the mixture comprising:

[0108] i. Single-stranded probe oligonucleotide A0;

[0109] ii. Pyrophosphate hydrolase; and

[0110] iii. Ligase

[0111] A0 is pyrophosphated from the 3' end in the 3'-5' direction to produce at least partially digested chain A1 and A1 undergoes linkage to form A2;

[0112] (c) Detect the signal from the product of the previous step, wherein the product is A2 or a portion thereof, or more than one copy of A2 or more than one copy of a portion thereof, and thereby infer the presence or absence of a polynucleotide target sequence in the analyte.

[0113] According to the present invention, a method for detecting a target polynucleotide sequence in a given nucleic acid analyte is provided. Analytes to which various methods of the present invention can be applied can be prepared from the aforementioned biological sample by a series of preliminary steps aimed at amplifying the analyte and separating it from background genomic DNA, which is typically present in significant excess. This method is generally applicable to the production of single-stranded target analytes, and therefore the method is useful not only when integrated with or included as part of the method of the first aspect of the present invention. Therefore, a method is provided for preparing at least one single-stranded analyte of a nucleic acid containing a target polynucleotide region, characterized by the following steps: (1) generating an amplicon of the analyte by performing an amplification cycle on a biological sample containing the analyte and optionally background genomic DNA.

[0114] In some preferred embodiments, amplification is performed using polymerase chain reaction (PCR) in the presence of polymerase, nucleoside triphosphate, and at least one corresponding primer pair, wherein one primer includes a 5'-3' exonuclease blocking group, and (2) the product of step (1) is optionally digested with an exonuclease having 5'-3' exonuclease activity. In one embodiment, the method may further include (3) reacting the product of step (2) with a protease to destroy the polymerase, and then (4) inactivating the protease by heating the product of step (3) to a temperature exceeding 50°C.

[0115] In some preferred embodiments, steps (1) to (4) are performed prior to step (a) of the method of the first aspect of the invention to produce an integrated method for detecting target sequences derived from a biological sample. In another embodiment, the biological sample has undergone cell lysis prior to step (1).

[0116] In some embodiments of step (1), the nucleoside triphosphates are a mixture of four naturally occurring DNA-specific deoxynucleoside triphosphates. In a preferred embodiment, the mixture of deoxynucleoside triphosphates contains deoxyuridine triphosphate (dUTP) instead of deoxythymidine triphosphate (dTTP), and step (1) is also performed in the presence of dUTP-DNA glycosylase (UDG) to remove any contaminating amplicon from previous assays. In yet another embodiment, a high-fidelity polymerase is used in step (1), for example, a product marketed under a trade name... Or one of the polymerases sold by Q5. In yet another embodiment, the polymerase may be KAPA HiFi uracil + DNA polymerase.

[0117] High-fidelity DNA polymerases have several safeguards to prevent the creation and propagation of errors during DNA replication. During polymerization, these enzymes exhibit a distinct binding preference for correct and incorrect nucleoside triphosphates. If an incorrect nucleotide does bind to the polymerase's active site, incorporation is slowed due to the suboptimal structure of the active site complex. This lag increases the chance that the incorrect nucleotide dissociates before polymerase progression, allowing the process to restart with the correct nucleoside triphosphate. If an incorrect nucleotide is inserted, proofreading DNA polymerases have an additional line of defense. Interference caused by a mismatched base is detected, and the polymerase moves the 3' end of the growing DNA strand to the proofreading 3'→5' exonuclease domain. There, the incorrect nucleotide is removed by the 3'→5' exonuclease activity, and the strand is then moved back to the polymerase domain, where polymerization can continue.

[0118] In some embodiments, nucleoside triphosphates are mixtures of synthetic or modified deoxynucleoside triphosphates.

[0119] In some implementations, the nucleoside triphosphate is a mixture of four deoxynucleoside triphosphates and synthetic or modified deoxynucleotide triphosphates.

[0120] In some implementations, step (1) is performed using a limited number of primers and an excess of amplification cycles. In this way, a fixed amount of amplicon is produced regardless of the initial amount of analyte. Therefore, the need for analyte quantification before subsequent steps is avoided. In another implementation of step (1) (which has the advantage of eliminating the need for step (2)), amplification is performed in the presence of a primer pair, where one primer is in excess of the other, resulting in the production of a single-stranded amplicon once one primer is fully utilized.

[0121] In some preferred embodiments of step (2), the 5' primer is blocked by an exonuclease blocking group selected from: phosphate thioester bonds, inverted bases, DNA spacers, and other oligonucleotide modifications known in the art. In another embodiment, the other primer in the primer pair has a phosphate group at its 5' end.

[0122] In some embodiments, the protease used in step (3) is proteinase K, and step (4) is performed by heating to a temperature of 80°C to 100°C for up to 30 minutes. In one embodiment, the protease used in step (3) is proteinase K, and step (3) is performed by heating to a temperature of 55°C for 5 minutes, and step (4) is performed by heating to a temperature of 95°C for 10 minutes. In another embodiment, at some time points after step (2), the reaction medium is treated with a phosphatase or a phosphorylase to remove any residual nucleoside triphosphates that may be present.

[0123] In some embodiments, the target polynucleotide sequence in the analyte will be a gene or chromosomal region in the DNA or RNA of cancerous tumor cells, characterized by the presence of one or more mutations; for example, in the form of one or more single nucleotide polymorphisms (SNPs). Therefore, the present invention could be used to monitor and / or treat disease recurrence. Patients declared disease-free after treatment may be monitored over time to detect disease recurrence. This needs to be non-invasive and requires sensitive detection of the target sequence from a blood sample. Similarly, for some cancers, residual cancer cells remain in the patient's body after treatment. Using the present invention to monitor the levels of these cells (or cell-free DNA) present in the patient's blood allows for the detection of disease recurrence or failure of current treatment and the need to switch to alternatives.

[0124] In some implementations, detection of the target polynucleotide sequence will allow for repeated testing of patient samples during disease treatment, enabling early detection of emerging treatment resistance. For example, epidermal growth factor receptor (EGFR) inhibitors, such as gefitinib and erlotinib, are commonly used as first-line treatment for non-small cell lung cancer (NSCLC). During treatment, tumors often develop mutations in the EGFR gene (e.g., T790M, C797S), leading to treatment resistance. Early detection of these mutations allows patients to switch to alternative therapies.

[0125] In some embodiments, the target polynucleotide sequence in the analyte will be a gene or chromosomal region in fetal-derived DNA or RNA, characterized by the presence of one or more mutations; for example, in the form of one or more single nucleotide polymorphisms (SNPs). Therefore, compared to other available detection techniques, the present invention can be used to detect mutations with very low allele fractions at an earlier stage of pregnancy.

[0126] In another implementation, the target polynucleotide sequence may be a gene or genomic region derived from another healthy individual, but the genetic information obtained may help generate valuable companion diagnostic information in one or more defined populations of the human population that allows for medical or therapeutic conclusions.

[0127] In yet another implementation, the target polynucleotide sequence may be specific to an infectious disease or to resistance to treatment with certain therapies; for example, a polynucleotide sequence specific to a gene or chromosomal region of a bacterium or virus, or a mutation therein that confers resistance to the therapy.

[0128] In some implementations, the target polynucleotide sequence can be specific to the donor DNA. When a transplanted organ is rejected by a patient, DNA from that organ sheds into the patient's bloodstream. Early detection of this DNA would allow for early detection of rejection. This can be achieved using a custom set of donor-specific markers, or by using a set of variants known to be common in the population, some of which will be present in the donor and some in the recipient. Thus, a method of requesting protection enables routine monitoring of organ recipients over time.

[0129] In yet another embodiment, different versions of methods using different combinations of probes (see below) are used in parallel, allowing simultaneous screening of more than one target sequence of the analyte; for example, cancer sources, cancer indicators, or more than one source of infection. In this method, the amplification products obtained by applying the method in parallel are contacted with a detection set comprising one or more oligonucleotide-binding dyes or sequence-specific molecular probes such as molecular beacons, hairpin probes, etc. Therefore, in another aspect of the invention, the use of at least one probe and optionally a linking oligonucleotide combined with one or more chemical and biological probes selective for target polynucleotide sequences is provided, or the use of at least one probe and optionally a linking oligonucleotide combined with a probe region identified by sequencing.

[0130] In some embodiments, the single-stranded probe oligonucleotide A0 comprises a trigger region and a 3' end complementary to the target polynucleotide sequence to be detected. In this way, a first intermediate product, at least partially double-stranded, is generated. In one embodiment, this step is performed in the presence of excess A0 and in an aqueous medium containing the analyte and any other nucleic acid molecules.

[0131] During step (a), the double-stranded region of the first intermediate is pyrolyzed from the 3' end of its A0 chain in the 3'-5' direction. Thus, the A0 chain is progressively digested, producing a partially digested chain; hereinafter referred to as A1. The pyrolysis reaction will stop at any mismatch when the probe oligonucleotide incorrectly hybridizes to a non-target sequence, preventing further steps of the method from proceeding. In another embodiment, this digestion continues until A1 lacks sufficient complementarity to form a stable double-stranded structure with the analyte or the target region therein. At this point, the individual chains are then separated by unwinding, resulting in the single-stranded form of A1. Under typical pyrolysis conditions, this separation occurs when there are 6 to 20 complementary nucleotides between the analyte and A0.

[0132] In another implementation, digestion continues until A1 lacks sufficient complementarity with the analyte or the target region therein to allow pyrophosphatase binding or the pyrophosphatase reaction to continue. This typically occurs when 6 to 20 complementary nucleotides remain between the analyte and the probe. In some implementations, this occurs when 6 to 40 complementary nucleotides remain.

[0133] In another embodiment where a sandwich oligonucleotide D (see below) with complementarity to the 5' and 3' ends of A1 is used, digestion continues until the complementary length between A1 and the target decreases to a point where the oligonucleotide D is energy-favorable for displacing the analyte molecule from A1. This typically occurs when the complementary region between A1 and the analyte molecule is similar in length to or shorter than the complementary region between the oligonucleotide D and the 3' end of A1, but it can also occur when the complementarity between A1 and the analyte molecule is longer than the complementary region between the oligonucleotide D and the 3' end of A1, because of the favorable intramolecular hybridization of the oligonucleotide D, which may have already hybridized to the 5' end of A1.

[0134] In another implementation, analyte molecules are used as clamps for A1 linkage (see...). Figure 16 Digestion continues until the 5' end of A1 can hybridize with the analyte molecule, so that the 3' and 5' ends of A1 are adjacent and separated by only one nick. At the nick, they are ligated together by a ligase, and digestion can no longer proceed.

[0135] Suitablely, pyrophosphate hydrolysis is carried out in a reaction medium, at a temperature range of 20°C to 90°C, in the presence of at least one polymerase exhibiting pyrophosphate hydrolytic activity and a pyrophosphate ion source. Further information on pyrophosphate hydrolysis reactions applied to polynucleotide digestion can be found, for example, in J. Biol. Chem. 244 (1969)pp.3019-3028 or found in our earlier patent applications.

[0136] In some implementations, the pyrophosphate hydrolysis step is driven by the presence of an excess polypyrophosphate source, suitable sources including those compounds containing three or more phosphorus atoms.

[0137] In some embodiments, the first reaction mixture contains an excess of a focused phosphoric acid source.

[0138] In some embodiments, the pyrophosphate hydrolysis step is driven by the presence of an excess of modified pyrophosphate source. Suitable modified pyrophosphates include those with bridging oxygens replaced by other atoms or groups, or pyrophosphates (or focused pyrophosphates) with substituted or modified groups substituted on other oxygens. Those skilled in the art will understand that many such examples of modified pyrophosphates suitable for the present invention exist, and their non-limiting selections are:

[0139]

[0140] In some embodiments, the first reaction mixture contains an excess of a modified focused phosphate source.

[0141] In some preferred embodiments, the pyrophosphate ion source is PNP, PCP, or tripolyphosphate (PPPi).

[0142] In addition, but not limited to, examples of pyrophosphate ion sources used in pyrophosphate hydrolysis step (b) can be found in WO2014 / 165210 and WO00 / 49180.

[0143] In some implementations, the excess modified pyrophosphate source can be represented as YH, where Y corresponds to the general formula (XO)2P(=B)-(ZP(=B)(OX)). n - where n is an integer from 1 to 4; each Z- is independently selected from -O-, -NH- or -CH2-; each B is independently O or S; the X group is independently selected from -H, -Na, -K, alkyl, alkenyl or heterocyclic group, provided that when both Z and B correspond to -O-, and when n is 1, at least one X group is not H.

[0144] In some implementations, Y corresponds to the general formula (XO)2P(=B)-(ZP(=B)(OX)) n - where n is 1, 2, 3, or 4. In another embodiment, the Y group corresponds to the general formula (XO)2P(=O)-ZP(=O)(OH)-, wherein one of the X groups is -H. In yet another preferred embodiment, Y corresponds to the general formula (XO)2P(=O)-ZP(=O)(OX)-, wherein at least one of the X groups is selected from methyl, ethyl, allyl, or dimethylallyl.

[0145] In an alternative embodiment, Y corresponds to the general formula (HO)2P(=O)-ZP(=O)(OH)- (where Z is -NH- or -CH2- or (XO)2P(=O)-ZP(=O)(OX)-, where X groups are all -Na or -K, and Z is -NH- or -CH2-.

[0146] In other embodiments, Y corresponds to the general formula (HO)2P(=B)-OP(=B)(OH)-, wherein each B group is independently O or S, and at least one is S.

[0147] Specific examples of preferred embodiments of Y include those of formula (X1-O)(HO)P(=O)-ZP(=O)(O-X2), wherein Z is O, NH or CH2, and (a) X1 is γ,γ-dimethylallyl and X2 is -H; or (b) both X1 and X2 are methyl; or (c) both X1 and X2 are ethyl; or (d) X1 is methyl and X2 is ethyl, or vice versa.

[0148] In some embodiments, when detection is performed using a molecular probe, the probe oligonucleotide A0 is configured to include an oligonucleotide recognition region on the 5' side of a region complementary to the target sequence, and the molecular probe used is designed to anneal to this recognition region. In one embodiment, only the 3' region of A0 is annealed to the target; that is, any other region lacks sufficient complementarity with the analyte to ensure the stable duplex exists at the temperature required for the pyrophosphate digestion step. Here and throughout, the term "sufficient complementarity" means that, with respect to a given region being complementary to a given region on the analyte, the complementary region is longer than 10 nucleotides.

[0149] In another aspect of the method of the present invention, alternative embodiments are provided, wherein the phosphorylation step of any of the foregoing embodiments is replaced by an exonuclease digestion step using a double-stranded specific exonuclease. Those skilled in the art will understand that double-stranded specific exonucleases include those that read in the 3'-5' direction, such as ExoIII, and those that read in the 5'-3' direction, such as Lambda Exo, etc.

[0150] In some embodiments of the invention, the exonuclease digestion step utilizes a double-stranded specific 5'-3' exonuclease, the 5' end of A0 is complementary to the target analyte, and a common initiation sequence and blocking group are located on the 3' side of the region complementary to the target. In another embodiment, when detection is to be performed using a molecular probe, the probe oligonucleotide A0 is configured to include an oligonucleotide recognition region on the 3' side of the region complementary to the target sequence, and the molecular probe used is designed to anneal to this recognition region.

[0151] In embodiments of the invention where the exonuclease digestion step utilizes a double-strand-specific 5'-3' exonuclease, an exonuclease having 3' to 5' exonuclease activity may optionally be added to the first reaction mixture to digest any other nucleic acid molecules present, while keeping A0 and any material containing partially digested strand A1 intact. Suitablely, this resistance to exonucleases is achieved as described elsewhere in this application.

[0152] In a preferred embodiment of the invention, the 5' end of A0 or the internal site on the 5' side of the initiation region is conferred resistance to exonuclease digestion. In this way, and optionally after or simultaneously with the pyrophosphate digestion step, an exonuclease having 5'-3' exonuclease activity can be added to the reaction medium to digest any other nucleic acid molecules present, while keeping A0 and any material containing partially digested chain A1 intact. Suitably, this resistance to exonuclease digestion is achieved by introducing one or more blocking groups at the desired site of the oligonucleotide A0. In one embodiment, these blocking groups can be selected from phosphorothioate linkages and other backbone modifications commonly used in the art, C3 spacers, phosphate groups, modified bases, etc.

[0153] In some implementations, the identification region will comprise or be embedded within a barcoding region, which has a unique sequence suitable for indirect identification using sequence-specific molecular probes applied to the amplified component A2, or for direct identification via sequencing of these components. Examples of molecular probes that can be used include, but are not limited to, molecular beacons. probe, Probes, etc.

[0154] In all implementations, the A2 strand or its desired region is subjected to amplification, resulting in more than one copy, typically millions of copies. This is achieved by initiating the region of A2 and any subsequent amplicons derived from A2 with single-stranded primer oligonucleotides, provided, for example, in the form of forward / reverse or sense / antisense pairs, which can be annealed to complementary regions on the region of A2 and any subsequent amplicons derived from A2. The initiated strand then becomes the starting point for amplification. Amplification methods include, but are not limited to, thermal cycling and isothermal methods such as polymerase chain reaction, recombinase polymerase amplification, and rolling circle amplification; the last one applies when A2 is circularized. By any of these methods, numerous amplicon copies of the region of A2 and, in some cases, its sequence complements can be rapidly generated. The exact methods for performing any of these amplification methods are well known to those skilled in the art, and the exact conditions and temperature patterns employed are readily available in the general literature read by the reader. Specifically, in the case of polymerase chain reaction (PCR), this method typically involves using polymerase and a source of multiple mononucleotide triphosphates to extend primer oligonucleotides in the 5'-3' direction against the A2 chain until a complementary chain is produced; dehybridizing the resulting double-stranded product to regenerate the A2 chain and the complementary chain; re-initiating the A2 chain and any amplicons therewith, and then repeating these extension / dehybridization / re-initiation steps multiple times to establish the concentration of the A2 amplicons at a level that can be reliably detected.

[0155] In some embodiments, the first reaction mixture further comprises a linker oligonucleotide C, and the partially digested chain A1 is linked at its 3' end to the 5' end of C, while in another embodiment, A1 is cyclized by linking its 3' end and 5' end;

[0156] Oligonucleotide A2 is produced in each case.

[0157] In one implementation, the connection of A1 occurs when:

[0158] During step (a); or

[0159] During step (b); or

[0160] Between steps (a) and (b).

[0161] In one embodiment, A1 optionally extends in the 5'-3' direction prior to connection.

[0162] In some embodiments, this optional extension and ligation is performed against a target oligonucleotide, while in another embodiment, it is performed by adding another splice oligonucleotide D, to which A1 is annealed prior to extension and / or ligation. In some embodiments, D comprises an oligonucleotide region complementary to the 3' end of A1 and a region complementary to the 5' end of oligonucleotide C or the 5' end of A1. In another embodiment, D cannot be extended against A1 due to 3' end modification or nucleotide mismatch between the 3' end of D and the corresponding region of A1.

[0163] In some embodiments, the ligation probe C has at least a portion of the 5' terminal region of the splint oligonucleotide D or a 5' region complementary to the target oligonucleotide. This method forms a second intermediate product in which the A2 chain comprises A1, C, and optionally an intermediate region formed by extending A1 in a 5'-3' direction to attach to the 5' end of C. In such embodiments, the primers used in step (c) are selected (see below) to amplify at least one region of A2, including the site where A1-C ligation occurs. In this embodiment, it is advantageous to include a 3' blocking group on C so that a 3'-5' exonuclease can be used to digest any unligated A1 prior to amplification. Suitable polymerases that can be used to extend A1 prior to ligation include, but are not limited to, Hemo KlenTaq, Mako, and Stoffel fragments.

[0164] In some embodiments, the first reaction mixture further comprises a phosphatase or a phosphorylase to remove nucleoside triphosphates produced by the pyrophosphate reaction by hydrolysis, thereby ensuring that the pyrophosphate reaction can continue and does not out-competed with the forward polymerization reaction.

[0165] In some embodiments, before or during step (b), the product of the previous step is treated with pyrophosphatase to hydrolyze pyrophosphate ions, preventing further pyrophosphatase breakdown and promoting forward polymerization.

[0166] In some implementations, the product of the previous step is treated with an exonuclease before or during step (b).

[0167] In some embodiments, the enzyme that performs pyrophosphate hydrolysis of A0 to form a partially digested chain A1 also amplifies A2. Those skilled in the art will recognize that many such enzymes exist.

[0168] Amplicons are detected, and the information obtained is used to infer the presence of polynucleotide target sequences in the original analyte and / or their associated properties. For example, in this way, target sequences specific to cancerous tumor cells can be detected by referring to a specific SNP being sought. In another embodiment, target sequences specific to viral or bacterial genomes (including their novel mutations) can be detected. Many methods for detecting amplicons or recognition regions can be used, including, for example, oligonucleotide-binding dyes, sequence-specific molecular probes such as fluorescently labeled molecular beacons or hairpin probes. Optionally, direct sequencing of A2 amplicons can be performed using one of the direct sequencing methods employed or reported in the art. When using oligonucleotide-binding dyes, fluorescently labeled beacons, or probes, it is convenient to detect amplicons using an arrangement comprising a source that stimulates electromagnetic radiation (laser, LED, lamp, etc.) and a photodetector arranged to detect emitted fluorescence and generate a signal comprising a data stream, which can be analyzed by a microprocessor or computer using a specially designed algorithm.

[0169] In some embodiments, detection is achieved using one or more oligonucleotide fluorescent binding dyes or molecular probes. In such embodiments, the increase in signal over time resulting from the generation of the A2 amplicon is used to infer the concentration of the target sequence in the analyte. In one embodiment, the final step of the method further includes the following steps:

[0170] i. Label the product of step (b) using one or more oligonucleotide fluorescent binding dyes or molecular probes;

[0171] ii. Measure the fluorescence signal of the product;

[0172] iii. Exposing the product to a set of denaturing conditions; and

[0173] Polynucleotide target sequences in analytes can be identified by monitoring changes in the fluorescence signal of the product during exposure to denaturing conditions.

[0174] In some implementations, more than one probe A0 is used, each probe A0 being selective for different target sequences, and each probe A0 containing a recognition region. Furthermore, the amplicon of A2 contains this recognition region, and therefore the target sequence present in the analyte is inferred by detecting the recognition region. In such implementations, the recognition region is detected using molecular probes or by sequencing.

[0175] In some implementations, one or more nucleic acid analytes are split into more than one reaction volume, each volume containing one or more probe oligonucleotides A0 introduced to detect different target sequences.

[0176] In some implementations, different probes A0 are used, each containing one or more common initiation sites, allowing amplification to be performed using a single primer or a set of primers.

[0177] In another aspect of the invention, a method for identifying a target polynucleotide sequence in a given nucleic acid analyte is provided, characterized by the steps of any prior embodiment of the invention, wherein more than one copy of an A2 or A2 region is labeled using one or more oligonucleotide fluorescent binding dyes or molecular probes. These more than one copy of fluorescence signals are measured, and the more than one copy is exposed to a set of denaturing conditions. The target polynucleotide sequence is identified by monitoring changes in the fluorescence signals of the more than one copy during exposure to the denaturing conditions.

[0178] In some implementations, denaturing conditions can be provided by altering the temperature, for example, by raising the temperature to the point where the double strands begin to dissociate. Alternatively or additionally, denaturing conditions can also be provided by altering the pH to make the conditions acidic or basic, or by adding additives or reagents such as strong acids or bases, concentrated inorganic salts, or organic solvents such as alcohols.

[0179] In another aspect of the invention, the above-described method is provided for use in screening mammalian subjects, particularly human patients, for the presence of infectious diseases, cancer, or for generating companion diagnostic information.

[0180] In another aspect of the invention, a control probe is provided for the methods described above. Embodiments of the invention include those that elucidate the presence of one or more specific target sequences by generating fluorescent signals. In such embodiments, signal levels generated by non-target DNA present in the sample may inevitably exist. For a given sample, this background signal begins later than the “true” signal, but such a beginning may differ between samples. Therefore, accurate detection of the presence of one or more target sequences at low concentrations depends on knowing what signal is expected in the absence of the target sequence. For contrived samples, a reference is available, but this is not the case for truly “blind” samples from patients. A control probe (E0) is used to determine the expected background signal characteristics for each assay probe. The control probe targets a sequence in the sample that is not expected to be present, and the signal generated from that probe can then be used to infer the expected rate of signal generation from the sample in the absence of the target sequence.

[0181] Therefore, a method for detecting a target polynucleotide sequence in a given nucleic acid analyte according to any of the foregoing methods is provided, characterized by the following steps:

[0182] a. Using a separate aliquot of the sample or in the same aliquot and using a second detection channel, the steps of the method are subsequently or simultaneously repeated using a second single-stranded probe oligonucleotide E0, the second single-stranded probe oligonucleotide E0 having a 3' end region that is at least partially mismatched with the target sequence;

[0183] b. In the absence of any target analyte in the sample, infer the expected background signal generated from A0; and

[0184] c. By comparing the expected background signal inferred in (a) with the actual signal observed when the target analyte is present, the presence or absence of the polynucleotide target sequence in the analyte can be inferred.

[0185] In some embodiments, the control probe (E0) and A0 are added to different portions of the sample, while in another embodiment, E0 and A0 are added to the same portion of the sample and different detection channels (e.g., different color dyes) are used to measure their respective signals. The signal generated by E0 can then be used to infer and correct the background signal expected to be generated by A0 when the polynucleotide target sequence is not present in the sample. For example, background signal correction may include subtracting the signal observed from E0 from the signal observed from A0, or calibrating the signal observed from A0 by using calibration curves of the relative signals generated by A0 and E0 under different conditions.

[0186] In some implementations, an E0 can be used to calibrate all possible measurement probes.

[0187] In some implementations, a single E0 can be used to calibrate each amplicon of the sample DNA generated in the initial amplification step. Each amplicon may contain more than one mutation / target sequence of interest, but a single E0 is sufficient to calibrate all assay probes for a single amplicon.

[0188] In another implementation, a separate E0 can be used for each target sequence. For example, if a C>T mutation is being targeted, an E0 can be designed to target a C>G mutation at the same site that is unknown in the patient. The signal curves generated by the E0 under various conditions can be evaluated in a calibration reaction, and these data are used to infer the expected signal from a assay probe targeting a C>T variant when that variant is absent.

[0189] exist Figures 17 to 20 Some implementation schemes of the method of the present invention can be seen in the figure.

[0190] exist Figure 17In this invention, a single-stranded probe oligonucleotide A0 is annealed to a target polynucleotide sequence to produce a first intermediate that is at least partially double-stranded, wherein the 3' end of A0 forms a double-stranded complex with the target polynucleotide sequence. In this simplified embodiment of the invention, two A0 molecules and one target polynucleotide sequence are present to illustrate how A0 that is not annealed to the target does not participate in further steps of the method. In this illustrative example, the 3' end of A0 is annealed to the target polynucleotide sequence, while the 5' end of A0 is not annealed to the target polynucleotide sequence. The 5' end of A0 includes a 5' chemically blocking group, a common initiating sequence, and a barcode region.

[0191] The first intermediate of the partially double-stranded A0 undergoes pyrophosphate digestion from the 3' end of A0 in the 3'-5' direction in the presence of pyrophosphate hydrolase to produce partially digested chain A1, analyte, and undigested A0 molecules that have not annealed to the target.

[0192] exist Figure 18 In this example, A1 is annealed to a single-stranded trigger oligonucleotide B, and the A1 strand is extended against B in the 5'-3' direction to produce oligonucleotide A2. In this illustrative example, the trigger oligonucleotide B has a 5' chemical block. Any undigested A0 annealed to the trigger oligonucleotide B, however, cannot be extended against B in the 5'-3' direction to produce the target sequence for the subsequent part of the method. In this example, A2 is initiated with at least one single-stranded primer oligonucleotide, and produces A2 or more than one copy of the region of A2.

[0193] exist Figure 19 In this process, A1 is annealed to splice oligonucleotide D, which is then cyclized by joining its 3' and 5' ends. The cyclized A2 is now initiated with at least one single-stranded primer oligonucleotide, resulting in A2 or more than one copy of a region of A2. In this illustrative example, splice oligonucleotide D cannot extend against A1 due to 3'-modification (in this example, chemical modification) or by nucleotide mismatch between the 3' end of D and the corresponding region of A2.

[0194] exist Figure 20 In this process, the 3' region of the splice oligonucleotide D is annealed to the 3' region of A1, while the 5' region of the splice oligonucleotide D is annealed to the 5' region of the ligation probe C. Thus, a second intermediate product A2 is formed, comprising A1, C, and optionally an intermediate region formed by A1 extending in the 5'-3' direction to connect with the 5' end of C. In this illustrative example, the ligation probe C has a 3' chemically blocking group so that 3'-5' exonucleases can be used to digest any unligated A1.

[0195] A2 is initiated by at least one single-stranded primer oligonucleotide and produces more than one copy of A2 or the region of A2.

[0196] The specificity of the method of this invention can be improved by introducing blocking oligonucleotides. For example, blocking oligonucleotides can be introduced to hybridize with at least a portion of wild-type DNA, promoting A0 to anneal only with the target polynucleotide sequence and not the wild-type sequence. Optionally or additionally, blocking oligonucleotides can be used to improve the specificity of polymerase chain reaction (PCR) to prevent the amplification of any wild-type sequences present. A common technique is to design an oligonucleotide that anneals between PCR primers and cannot be replaced or digested by PCR polymerase. The oligonucleotide is designed to anneal with non-target (usually healthy) sequences while mismatching (typically differing by a single base) with the target (mutated) sequence. This mismatch results in different melting temperatures for the two sequences, and the oligonucleotide is designed to remain annealed with the non-target sequence at the PCR extension temperature while dissociating from the target sequence.

[0197] Blocked oligonucleotides can often be modified to prevent them from being digested by the exonuclease activity of PCR polymerase, or to increase the melting temperature difference between the target and non-target sequences.

[0198] Incorporating locked nucleic acids (LNAs) or other modifications that alter the melting temperature into blocked oligonucleotides can significantly increase the difference in melting temperature between the oligonucleotides and non-target sequences.

[0199] Therefore, embodiments of the present invention are provided, in which blocked oligonucleotides are used. Blocked oligonucleotides must be resistant to pyrophosphate hydrolysis (PPL) to ensure they are not digested or replaced. This can be achieved in a variety of different ways, such as by mismatch at the 3' end or by modification such as phosphate thioester bonds or spacer groups.

[0200] In such embodiments or aspects of the invention using blocking oligonucleotides, the method for detecting a target polynucleotide sequence in a given nucleic acid analyte is characterized in that, before or during the same step of annealing the analyte target sequence with a single-stranded probe oligonucleotide A0 to produce at least a partial double strand and wherein the 3' end of A0 forms a double-stranded complex with the analyte target sequence, the single-stranded blocking oligonucleotide is annealed with at least a subset of a non-target polynucleotide sequence.

[0201] In some embodiments, the blocked oligonucleotide becomes resistant to pyrophosphate hydrolysis through a mismatch at its 3' end. In another embodiment, the blocked oligonucleotide becomes resistant through the presence of a 3'-blocking group. In yet another embodiment, the blocked oligonucleotide becomes resistant through the presence of a spacer group or other internal modifications. In yet another embodiment, the blocked oligonucleotide becomes resistant to pyrophosphate hydrolysis by including modifications or modified nucleotide bases that increase the melting temperature.

[0202] The term "phosphatase" as used herein refers to any enzyme or functional fragment thereof capable of removing nucleoside triphosphates produced by the methods of this invention through hydrolysis. This includes any enzyme or functional fragment thereof capable of cleaving phosphate monoesters into phosphate ions and alcohols.

[0203] The term "pyrophosphatase" as used in this article refers to any enzyme or its functional fragment that has the ability to catalyze the conversion of one pyrophosphate ion into two phosphate ions.

[0204] This also includes inorganic pyrophosphatases and inorganic diphosphatases. A non-limiting example is thermostable inorganic pyrophosphatase (TIPP).

[0205] In some implementations, modifications to any of the previously described implementations are provided, wherein the use of pyrophosphatase is optional.

[0206] In some embodiments of the present invention, a kit is provided for a method of detecting a target polynucleotide sequence in a given nucleic acid analyte present in a sample, comprising:

[0207] (a) A single-stranded probe oligonucleotide A0, which is capable of forming a first intermediate product with a target polynucleotide sequence, said intermediate product being at least partially double-stranded;

[0208] (b) Ligase;

[0209] (c) Pyrophosphate hydrolase, which is capable of digesting the first intermediate product from the end of A0 in the 3'-5' direction to produce a partially digested chain A1;

[0210] (d) at least one single-stranded primer oligonucleotide substantially complementary to a portion of A0;

[0211] (e) amplification enzyme; and

[0212] (f) Suitable buffer solution.

[0213] In one implementation, the 3' end of A0 is completely complementary to the target polynucleotide sequence.

[0214] In one implementation, the ligase is essentially devoid of single-strand ligation activity.

[0215] In some embodiments, the kit includes a single-stranded probe oligonucleotide A0, which is capable of forming a first intermediate with a target polynucleotide sequence, said intermediate being at least partially double-stranded;

[0216] (a) Ligase;

[0217] (b) Pyrophosphatase, which is capable of digesting the first intermediate product from the end of A0 in the 3'-5' direction to produce a partially digested chain A1;

[0218] (c) A suitable buffer solution.

[0219] In some implementations, the kit may alternatively also include:

[0220] - Two or more linked chain reaction (LCR) probe oligonucleotides complementary to the adjacent sequence on A1, wherein, upon successful annealing, the 5' phosphate of one LCR probe is directly adjacent to the 3' OH of another LCR probe; and

[0221] - One or more ligases.

[0222] In some implementations, in the presence of A2, two LCR probes will successfully anneal to A2 and be ligated together to form an oligonucleotide molecule. This oligonucleotide molecule then acts as a new target for a second round of covalent ligation, leading to the geometric amplification of the target of interest, in this case, A2. The ligation product, or amplicon, is complementary to A2 and acts as the target in the next amplification cycle. Thus, in the presence of excess LCR probes, exponential amplification of a specific target DNA sequence is achieved through repeated cycles of denaturation, hybridization, and ligation. From this, the presence of A2 is inferred, and therefore the presence of the target polynucleotide sequence is inferred.

[0223] In some implementations, in the presence of A2, two PCR probes will successfully anneal to A2 and be ligated together to form an oligonucleotide molecule, which then acts as a new target for a second round of covalent ligation, resulting in the target of interest, in this case, geometric amplification of A2, and then the target of interest, in this case, is detected by A2.

[0224] In some implementations, the kit may alternatively also include:

[0225] -Link probe oligonucleotide C;

[0226] -Clamping oligonucleotide D;

[0227] C has a 5' phosphate, the 3' end of the intercalation oligonucleotide D is complementary to the 5' end of C, and the 5' end of D is complementary to the 3' end of A1, so that A1 and C can be linked together to form A2.

[0228] In some implementations, the kit may also include:

[0229] - A hairpin oligonucleotide 1 (HO1) containing a fluorophore-quencher pair, wherein HO1 is complementary to A2, and upon annealing with A2, the hairpin structure of HO1 opens and the fluorophore-quencher pair separates; and

[0230] - A hairpin oligonucleotide 2 (HO2) containing a fluorophore-quencher pair, wherein HO2 is complementary to an open HO1, and when annealed with HO1, the hairpin structure of HO2 opens and the fluorophore-quencher pair separates.

[0231] In some implementations, the kit may also include more than one HO1 and HO2.

[0232] In some implementations, the kit may alternatively also include oligonucleotide A, which comprises a substrate arm, a portion of the catalytic core, and a sensor arm;

[0233] -Oligonucleotide B, which includes a substrate arm, a partial catalytic core, and a sensor arm; and

[0234] -Substrates containing fluorophore-quencher pairs;

[0235] The sensor arms of oligonucleotides A and B are complementary to the flanking regions of A2, enabling oligonucleotides A and B to bind in the presence of A2 to form a catalytic, multicomponent nuclease (MNAzyme).

[0236] In some implementations, the kit may alternatively also include a partially double-stranded nucleic acid construct, wherein:

[0237] - A strand contains at least one RNA base, at least one fluorophore, and wherein a region of the strand is complementary to the region of A2, and wherein the strand may be referred to as a "substrate" strand;

[0238] - The other strand contains at least one quencher, and a region of that strand is complementary to a region of A2 that is complementary to the region of the substrate strand, such that in the presence of A2, the partially double-stranded nucleic acid construct becomes substantially more double-stranded.

[0239] In other words, in the presence of A2, some double-stranded nucleic acid constructs have larger double-stranded portions.

[0240] In some implementations, the kit may also include an enzyme for removing at least one RNA base.

[0241] In some implementations, the enzyme is uracil-DNA glycosylase (UDG) and the RNA base is uracil.

[0242] In some implementations, the kit may alternatively also include:

[0243] - An oligonucleotide complementary to the region containing the linker site of A2, the oligonucleotide comprising one or more fluorophores, the one or more fluorophores being arranged such that their fluorescence is quenched by approaching each other or by approaching one or more fluorescence quenchers;

[0244] - Double-stranded DNA-specific digestive enzymes;

[0245] In this process, the labeled oligonucleotides are digested in the presence of A2, causing the fluorophores to separate from each other or from their respective quenchers, and the fluorescence signal, and therefore the presence of A2, becomes detectable.

[0246] In some implementations, the double-stranded specific DNA digesting enzyme is an exonuclease.

[0247] In some implementations, the double-strand-specific DNA digesting enzyme is a polymerase with proofreading activity.

[0248] In some implementations, the fluorophore of the kit may be selected from dyes of the fluorescein family, carboxyrhodamine family, anthocyanin family, rhodamine family, polyhalogenated fluorescein family dyes, hexachlorofluorescein family dyes, coumarin family dyes, oxazine family dyes, thiazine family dyes, squaraine family dyes, and chelated lanthanides.

[0249] In some implementations, the fluorophore of the kit can be selected from any commercially available dye.

[0250] In some implementations, the quencher in the kit may be selected from products marketed under the trade name Black Hole. TM Eclipse TM Dark, Qx1J and Iowa Black TM Those provided.

[0251] In some implementations, the quencher in the kit can be selected from any commercially available quencher.

[0252] In some embodiments, the kit may also include one or more partially double-stranded DNA constructs, each containing one or more fluorophores and one or more quenchers. In some embodiments, when the constructs are partially double-stranded, the one or more fluorophores and one or more quenchers are located close enough to each other that the one or more fluorophores are sufficiently quenched.

[0253] In some implementations, the construct is a DNA strand with its own complementary regions that loop back on itself.

[0254] In some implementations, the construct includes one primer in a primer pair.

[0255] In some implementations, the kit may also include another primer of the primer pair.

[0256] In some embodiments, a portion of the single-stranded segment of the construct hybridizes to A2 and is extended to A2 via DNA polymerase. In some embodiments, another primer in the primer pair then hybridizes to the extended construct. This primer is then extended to the construct, replacing its complementary region. Thus, one or more fluorophores and one or more dyes are sufficiently separated to detect a fluorescent signal indicating the presence of A2 in the reaction mixture.

[0257] In such an implementation, the construct can be called a Sunrise Primer.

[0258] In some implementations, the construct contains two separate DNA strands.

[0259] In some embodiments, a portion of the single-stranded segment of the construct hybridizes to A2 and is extended to A2 via DNA polymerase. In some embodiments, another primer in the primer pair then hybridizes to the extended construct. This primer then extends to the construct in the direction of the double-stranded segment, displacing the shorter strand in the DNA strand, and thus one or more fluorophores and one or more dyes are sufficiently separated to detect a fluorescent signal indicating the presence of A2 in the reaction mixture.

[0260] In such an implementation, the construct can be referred to as a molecular zipper.

[0261] Those skilled in the art will understand that, for both sunrise primers and molecular zippers, one or more fluorophores and one or more quencher pairs may be located at different positions within each respective construct. A key feature is that each pair is located close enough to each other that no fluorescent signal is emitted in the absence of A2, i.e., when no extension and chain substitution occur.

[0262] In one embodiment, the kit also includes a pyrophosphate ion source.

[0263] Suitable sources of pyrophosphate ions are as previously described.

[0264] In some implementations, the kit also includes appropriate positive and negative controls.

[0265] In some implementations, the kit may also include one or more control probes (E0) as previously described.

[0266] In some implementations, the kit may also include one or more blocking oligonucleotides as previously described.

[0267] In some implementations, the kit may also include one or more control probes (E o ) and one or more blocked oligonucleotides.

[0268] In some implementations, the 5' end of A0 may be conferred resistance to digestion by 5'-3' exonucleases, and the kit may also contain 5'-3' exonucleases.

[0269] In some implementations, the kit may also include a linker probe oligonucleotide C.

[0270] In some implementations, the kit may also include splint oligonucleotide D.

[0271] In some implementations, the kit may include both C and D.

[0272] The ligation probe C may contain 3' modifications or internal modifications to protect it from digestion by 3'-5' exonucleases.

[0273] D may contain an oligonucleotide region complementary to the 3' end of A1 and a region complementary to the 5' end of oligonucleotide C or the 5' end of A1.

[0274] In some implementations, D may not be able to extend for A1 due to 3' modification or mismatch between the 3' end of D and the corresponding region of A1 or C.

[0275] In some implementations, the kit may also include dNTPs, polymerase, and a suitable buffer for initial amplification of the target polynucleotide sequence present in the sample.

[0276] In some implementations, the kit may also include a high-fidelity polymerase incorporating dUTP, dUTP, and uracil-DNA N-glycosylation enzyme (UDG).

[0277] In some implementations, the kit may also include phosphatase or phosphorylase.

[0278] In some implementations, the kit may also include pyrophosphatase. The pyrophosphatase may be hot-start.

[0279] In some implementations, the kit may also include a protease.

[0280] In some implementations, the kit may also include one or more oligonucleotide binding dyes or molecular probes.

[0281] In some implementations, the kit may also include more than one A0, each A0 being selective for different target sequences, and each A0 containing a recognition region.

[0282] In some implementations, the kit may also include an enzyme for forming DNA from an RNA template.

[0283] In some implementations, the enzyme is a reverse transcriptase.

[0284] In some implementations, one or more enzymes in the kit may be hot-started.

[0285] In some implementations, one or more enzymes in the kit may be thermostable.

[0286] In some implementations, the kit may also include suitable washing and buffering reagents.

[0287] In some implementations, the amplification enzyme and pyrophosphatase in (e) are the same.

[0288] In some implementations, the amplification enzyme and pyrophosphate hydrolase are the same.

[0289] The kit may also include purification equipment and reagents for isolating and / or purifying a fraction of the polynucleotides following the treatments described herein. Suitable reagents are well known in the art and include gel filtration columns and wash buffers.

[0290] In some implementations, a kit is provided, comprising:

[0291] (a) A single-stranded probe oligonucleotide A0, wherein the single-stranded probe oligonucleotide A0 is capable of forming a first intermediate product with a target polynucleotide sequence, wherein the intermediate product is at least partially double-stranded;

[0292] (b) Ligase;

[0293] (c) Pyrophosphate hydrolase, which is capable of digesting the first intermediate product from the end of A0 in the 3'-5' direction to produce a partially digested chain A1;

[0294] (d) Suitable buffer solution.

[0295] In some implementations, the kit may also include a pyrophosphate ion source.

[0296] In some implementations, the kit may also include appropriate positive and negative controls.

[0297] In some embodiments of the kit, the 5' end of A0 is conferred resistance to digestion by 5'-3' exonucleases, and the kit may also contain 5'-3' exonucleases.

[0298] In some implementations, the kit may also include dNTPs, polymerase, and a suitable buffer for initial amplification of the target polynucleotide sequence present in the sample.

[0299] In some implementations, the kit may also include dUTP high-fidelity polymerase, dUTP, and uracil-DNA N-glycosylation enzyme (UDG).

[0300] In some implementations, the kit may also include a protease.

[0301] In some implementations, the kit may also include a linker probe oligonucleotide C.

[0302] In some implementations, the kit may also include splint oligonucleotide D.

[0303] In some implementations, the kit may also include a linker oligonucleotide C for probes and a splint oligonucleotide D for splints.

[0304] In some implementations of the kit, oligonucleotide C contains 3' modifications or internal modifications to protect it from digestion by 3'-5' exonucleases.

[0305] In some embodiments of the kit, D contains an oligonucleotide region complementary to the 3' end of A1 and a region complementary to the 5' end of oligonucleotide C or the 5' end of A1.

[0306] In some implementations of the kit, D cannot be extended against A1 due to 3' modification or mismatch between the 3' end of D and the corresponding region of A1 or C.

[0307] In some implementations, the kit also includes at least one single-stranded primer oligonucleotide substantially complementary to the A0 portion, an amplification enzyme, and dNTPs.

[0308] In some implementations, the kit also includes one or more oligonucleotide binding dyes or molecular probes.

[0309] In some implementations, the kit also includes more than one A0, each A0 being selective for a different target sequence, and each A0 containing a recognition region.

[0310] In some implementations, the kit also includes:

[0311] - Two or more linked chain reaction (LCR) probe oligonucleotides complementary to the adjacent sequence on A1, wherein, upon successful annealing, the 5' phosphate of one LCR probe is directly adjacent to the 3' OH of another LCR probe; and

[0312] - One or more ligases.

[0313] In some implementations, the kit also includes one or more polymerases.

[0314] In some implementations of the kit, one or more polymerases are the same as pyrophosphate hydrolase.

[0315] In some implementations, the kit also includes:

[0316] -Link probe oligonucleotide C;

[0317] -Clamping oligonucleotide D;

[0318] E has a 5' phosphate, the 3' end of the intercalation oligonucleotide D is complementary to the 5' end of E, and the 5' end of D is complementary to the 3' end of A1, so that A1 and E can be linked together to form A2.

[0319] In some implementations, the kit also includes:

[0320] - A hairpin oligonucleotide 1 (HO1) containing a fluorophore-quencher pair, wherein HO1 is complementary to A2, and upon annealing with A2, the hairpin structure of HO1 opens and the fluorophore-quencher pair separates; and

[0321] - A hairpin oligonucleotide 2 (HO2) containing a fluorophore-quencher pair, wherein HO2 is complementary to an open HO1, and when annealed with HO1, the hairpin structure of HO2 opens and the fluorophore-quencher pair separates.

[0322] In some implementations, the kit also includes more than one HO1 and HO2.

[0323] In some implementations, the kit also includes:

[0324] -Oligonucleotide A, wherein oligonucleotide A comprises a substrate arm, a partial catalytic core and a sensor arm;

[0325] -Oligonucleotide B, wherein oligonucleotide B comprises a substrate arm, a partial catalytic core, and a sensor arm; and

[0326] -Substrates containing fluorophore-quencher pairs;

[0327] The sensor arms of oligonucleotides A and B are complementary to the flanking regions of A2, enabling oligonucleotides A and B to bind in the presence of A2 to form a catalytic, multicomponent nuclease (MNAzyme).

[0328] In some implementations, the kit also includes a partially double-stranded nucleic acid construct, wherein:

[0329] - A strand contains at least one RNA base, at least one fluorophore, and wherein a region of the strand is complementary to the region of A2, and wherein the strand may be referred to as a "substrate" strand;

[0330] - The other strand contains at least one quencher, and a region of that strand is complementary to a region of A2 that is complementary to the region of the substrate strand, such that in the presence of A2, the partially double-stranded nucleic acid construct becomes substantially more double-stranded.

[0331] In some implementations, the kit also includes an enzyme for removing at least one RNA base.

[0332] In some implementations of the kit, the enzyme is uracil-DNA glycosylase (UDG), and the RNA base is uracil.

[0333] In some implementations, the kit also includes:

[0334] - An oligonucleotide complementary to the region containing the linker site of A2, the oligonucleotide containing one or more fluorophores, the fluorophores being arranged such that their fluorescence is quenched by their proximity to each other or by their proximity to one or more fluorescence quenchers;

[0335] - Double-stranded DNA-specific digestive enzymes;

[0336] In this process, the labeled oligonucleotides are digested in the presence of A2, causing the fluorophores to separate from each other or from their respective quenchers, and the fluorescence signal, and therefore the presence of A2, becomes detectable.

[0337] In some implementations of the kit, the double-stranded specific DNA digesting enzyme is an exonuclease.

[0338] In some implementations of the kit, the double-strand-specific DNA digesting enzyme is a polymerase with proofreading activity.

[0339] In some implementations of the kit, the fluorophore is selected from dyes of the fluorescein family, carboxyrhodamine family, anthocyanin family, rhodamine family, polyhalogenated fluorescein family dyes, hexachlorofluorescein family dyes, coumarin family dyes, oxazine family dyes, thiazine family dyes, squaric acid family dyes, and chelated lanthanides.

[0340] In some implementations of the kit, the quencher is selected from products marketed under the trade name Black Hole. TM Eclipse TM Dark, Qx1J and Iowa Black TM Those provided.

[0341] In some implementations, the kit also includes phosphatase or phosphorylase.

[0342] In some implementations, the kit also includes pyrophosphatase.

[0343] In some implementations, the kit also includes an enzyme for forming DNA from an RNA template.

[0344] In some implementations of the kit, the enzyme is a reverse transcriptase.

[0345] In some implementations of the kit, one or more enzymes are hot-started.

[0346] In some implementations of the kit, one or more enzymes are thermostable.

[0347] In some implementations, the kit may also include suitable washing and buffering reagents.

[0348] In one embodiment of the present invention, an apparatus is provided, the apparatus comprising:

[0349] At least one fluid channel between the first region, the second region, and the third region, wherein the first region includes one or more orifices, each orifice comprising:

[0350] dNTP;

[0351] At least one single-stranded primer oligonucleotide;

[0352] Amplifying enzymes used to initially amplify DNA present in the sample; and

[0353] The second region includes one or more holes, each hole comprising:

[0354] A single-stranded probe oligonucleotide A0, which can form a first intermediate product with a target polynucleotide sequence, wherein the intermediate product is at least partially double-stranded;

[0355] Pyrophosphate hydrolase, which digests the first intermediate product from the end of A0 in the 3'-5' direction to produce partially digested chain A1; and

[0356] The third region includes one or more holes, each hole comprising:

[0357] dNTP;

[0358] Buffer solution;

[0359] Amplification enzyme;

[0360] A means for detecting signals originating from A2 or a portion thereof, or more than one copy of A2 or more than one copy of a portion thereof; and

[0361] The pores in the second or third region also include at least one single-stranded primer oligonucleotide substantially complementary to the portion of A0.

[0362] In some implementations, the means for detecting the signal is located in one or more holes in the third region.

[0363] In some implementations, the means for detecting the signal is located in a third area of ​​the device.

[0364] In some implementations, the means for detecting signals is located in an adjacent area of ​​the device.

[0365] In some implementations, the dNTPs in each well of the first region may be dUTP, dGTP, dATP, and dCTP, and each well may also include a high-fidelity polymerase incorporating dUTP and a uracil-DNA N-glycosylation enzyme (UDG).

[0366] In some implementations, the dNTPs in each well of the third region may be dUTP, dGTP, dATP, and dCTP, and each well may also include a high-fidelity polymerase incorporating dUTP and a uracil-DNA N-glycosylation enzyme (UDG).

[0367] In some implementations, each pore in the second region may also include a pyrophosphate ion source.

[0368] In some implementations, the 5' end of A0 is conferred resistance to digestion by 5'-3' exonucleases, and the pores in the second region may also include 5'-3' exonucleases.

[0369] In some implementations, each well in the second or third region may also include a ligase and a ligation probe oligonucleotide C or a splint oligonucleotide D.

[0370] The ligation probe C may contain 3' modifications or internal modifications to protect it from digestion by 3'-5' exonucleases.

[0371] The splice oligonucleotide D may contain an oligonucleotide region complementary to the 3' end of A1 and a region complementary to the 5' end of oligonucleotide C or the 5' end of A1.

[0372] Due to a mismatch between the 3' modification or the corresponding region of A1 or C via the 3' end of D, D may not be able to extend against A1.

[0373] In some implementations, dNTPs can be hot-started, and each well in the second region can also include a phosphatase or a phosphorylase.

[0374] In some implementations, each well in the second region may also include pyrophosphatase.

[0375] In some implementations, pyrophosphatase can be hot-started.

[0376] In some implementations, each well in the third region may also include one or more oligonucleotide binding dyes or molecular probes.

[0377] In some implementations, each pore in the second region may include at least one or more different A0s that are selective to the target sequence including the recognition region.

[0378] In some implementations, the amplification enzyme and pyrophosphate hydrolase in the second region can be the same.

[0379] In some implementations, a fourth region may be present, comprising one or more pores, each pore of which may include a protease, and wherein the fourth region may be located between the first and second regions.

[0380] In some implementations, the second and third regions of the device can be combined, such that the aperture in the second region further includes:

[0381] dNTP;

[0382] Buffer solution;

[0383] amplification enzyme; and

[0384] A means for detecting signals originating from A2 or a portion thereof, or more than one copy of A2 or more than one copy of a portion thereof.

[0385] In some implementations, the means for detecting the signal is located in one or more holes in the second region.

[0386] In some implementations, the means for detecting the signal is located in a second area of ​​the device.

[0387] In some implementations, the means for detecting signals is located in an adjacent area of ​​the device.

[0388] In some implementations, a device is provided, comprising:

[0389] A fluid path between a first region and a second region, wherein the first region includes one or more orifices, and one or more orifices include:

[0390] A single-stranded probe oligonucleotide A0, wherein the single-stranded probe oligonucleotide A0 is capable of forming a first intermediate product with a target polynucleotide sequence, wherein the intermediate product is at least partially double-stranded;

[0391] A pyrophosphate hydrolase capable of digesting the first intermediate from the end of A0 in a 3'-5' direction to produce a partially digested chain A1; and

[0392] One or more ligases, said ligases being able to ligate A1 to produce oligonucleotide A2.

[0393] The second region includes one or more holes.

[0394] In some embodiments, one or more pores in the first region may also include an ion source that drives the pyrophosphate reaction forward.

[0395] In some implementations, the ion is the pyrophosphate ion.

[0396] In some embodiments, the 5' end of A0 is resistant to digestion by 5'-3' exonucleases, and the pores in the first region also include 5'-3' exonucleases.

[0397] In some embodiments, the device may further include a third region comprising one or more orifices connected to the first region via fluid channels, wherein one or more orifices in the third region comprise:

[0398] dNTP;

[0399] Single-stranded primer oligonucleotides; and

[0400] Amplification enzyme.

[0401] In some implementations, the dNTPs in the third region can be dUTP, dGTP, dCTP, and dATP; the amplification enzyme can be a high-fidelity polymerase incorporating dUTP; and one or more pores in the third region can also contain uracil-DNA N-glycosylation enzymes.

[0402] In some embodiments, the device may also include a fourth region located between the first and third regions, the fourth region including one or more pores, one or more of which may include a protease.

[0403] In some implementations, one or more wells in the first or second region may also include a ligase and a ligation probe oligonucleotide C complementary to the region A0.

[0404] In some implementations, one or more wells in the first or second region may also include a ligase and a splint oligonucleotide D complementary to the region of A0.

[0405] In some implementations, one or more wells in the first or second region may also include a ligase, a splint oligonucleotide D, and a ligation probe oligonucleotide C.

[0406] In some implementations, the linker probe oligonucleotide C may include 3' modifications or internal modifications to protect it from 3'-5' exonuclease digestion.

[0407] In some embodiments, D may include an oligonucleotide region complementary to the 3' end of A1 and a region complementary to the 5' end of oligonucleotide C or the 5' end of A1.

[0408] In some implementations, D may not be able to extend for A1 due to 3' modification or mismatch between the 3' end of D and the corresponding region of A1 or C.

[0409] In some implementations, one or more holes in the first region may include at least one or more different A0s, each A0 being selective for a different target sequence and each A0 containing a recognition region.

[0410] In some implementations, the hole in the second region may include:

[0411] dNTP;

[0412] Buffer solution;

[0413] Amplification enzyme;

[0414] A means for detecting signals originating from A2 or a portion thereof, or more than one copy of A2 or more than one copy of a portion thereof.

[0415] In some implementations, the means for detecting the signal is located in one or more holes in the second region.

[0416] In some implementations, the means for detecting the signal is located in a second area of ​​the device.

[0417] In some implementations, the means for detecting signals is located in an adjacent area of ​​the device.

[0418] In some implementations, one or more wells in the second region may also include one or more oligonucleotide binding dyes or molecular probes.

[0419] In some implementations, the amplification enzyme and pyrophosphate hydrolase of the device are the same.

[0420] In some implementations, the holes in the second region also include:

[0421] - Two or more linked chain reaction (LCR) probe oligonucleotides complementary to the adjacent sequence on A1, wherein, upon successful annealing, the 5' phosphate of one LCR probe is directly adjacent to the 3' OH of another LCR probe; and

[0422] - One or more ligases.

[0423] In some implementations, the hole in the second region may include:

[0424] -Link probe oligonucleotide C;

[0425] -Clamping oligonucleotide D;

[0426] C has a 5' phosphate, the 3' end of the splice oligonucleotide D is complementary to the 5' end of C, and the 5' end of D is complementary to the 3' end of A1, so that A1 and C can be linked together to form oligonucleotide A2.

[0427] In some implementations, the hole in the second region may further include:

[0428] - A hairpin oligonucleotide 1 (HO1) containing a fluorophore-quencher pair, wherein HO1 is complementary to A2, and upon annealing with A2, the hairpin structure of HO1 opens and the fluorophore-quencher pair separates; and

[0429] - A hairpin oligonucleotide 2 (HO2) containing a fluorophore-quencher pair, wherein HO2 is complementary to an open HO1, and when annealed with HO1, the hairpin structure of HO2 opens and the fluorophore-quencher pair separates.

[0430] In some implementations, the holes in the second region may also include more than one HO1 and HO2.

[0431] In some implementations, the hole in the second region may further include:

[0432] -Oligonucleotide A, wherein oligonucleotide A comprises a substrate arm, a partial catalytic core and a sensor arm;

[0433] -Oligonucleotide B, wherein oligonucleotide B comprises a substrate arm, a partial catalytic core, and a sensor arm; and

[0434] -Substrates containing fluorophore-quencher pairs;

[0435] The sensor arms of oligonucleotides A and B are complementary to the flanking regions of A2, enabling oligonucleotides A and B to bind in the presence of A2 to form a catalytic, multicomponent nuclease (MNAzyme).

[0436] In some implementations, the pores in the second region may include partially double-stranded nucleic acid constructs, wherein:

[0437] - A strand contains at least one RNA base, at least one fluorophore, and wherein a region of the strand is complementary to the region of A2, and wherein the strand may be referred to as a "substrate" strand;

[0438] - The other strand contains at least one quencher, and a region of that strand is complementary to a region of A2 that is complementary to the region of the substrate strand, such that in the presence of A2, the partially double-stranded nucleic acid construct becomes substantially more double-stranded.

[0439] In some implementations, the pores in the second region may also include an enzyme for removing at least one RNA base.

[0440] In some implementations, the enzyme is uracil-DNA glycosylase (UDG) and the RNA base is uracil.

[0441] In some implementations, one or more holes in the second region may further include:

[0442] Oligonucleotides complementary to the region containing the linker site of A2, the oligonucleotides comprising one or more fluorophores, the one or more fluorophores being arranged such that their fluorescence is quenched by approaching each other or by approaching one or more fluorescence quenchers through them;

[0443] Double-stranded DNA-specific digestive enzymes;

[0444] In this process, the labeled oligonucleotides are digested in the presence of A2, causing the fluorophores to separate from each other or from their respective quenchers, and the fluorescence signal, and therefore the presence of A2, becomes detectable.

[0445] In some implementations, the double-stranded specific DNA digesting enzyme is an exonuclease.

[0446] In some implementations, the double-strand-specific DNA digesting enzyme is a polymerase with proofreading activity.

[0447] In some implementations, the fluorophore is selected from dyes belonging to the fluorescein family, carboxyrhodamine family, anthocyanin family, rhodamine family, polyhalogenated fluorescein family dyes, hexachlorofluorescein family dyes, coumarin family dyes, oxazine family dyes, thiazine family dyes, squaric acid family dyes, and chelated lanthanides.

[0448] In some implementations, the fluorophore of the device can be selected from any commercially available dye.

[0449] In some implementations, the quenching agent for the device is selected from products marketed under the trade name Black Hole. TM Eclipse TM Dark, Qx1J, Iowa Black TM Those provided by ZEN and / or TAO.

[0450] In some implementations, the quenching agent for the device can be selected from any commercially available quenching agent.

[0451] In some embodiments, one or more wells in the second region may further include one or more partially double-stranded DNA constructs, each containing one or more fluorophores and one or more quenchers. In some embodiments, when the constructs are partially double-stranded, the one or more fluorophores and one or more quenchers are located close enough to each other that the one or more fluorophores are sufficiently quenched.

[0452] In some implementations, the construct is a DNA strand having self-complementary regions that return to circularity on itself.

[0453] In some implementations, the construct includes one primer in a primer pair.

[0454] In some implementations, one or more wells in the second region may also include another primer of the primer pair.

[0455] In some embodiments, a portion of the single-stranded segment of the construct hybridizes to A2 and is extended against A2 via DNA polymerase. In some embodiments, another primer in the primer pair then hybridizes to the extended construct, revealing A2. This primer is then extended against the construct, replacing its complementary region. Thus, one or more fluorophores and one or more dyes are sufficiently separated to detect a fluorescent signal indicating the presence of A2.

[0456] In such an implementation, the construct can be called a sunrise primer.

[0457] In some implementations, the construct contains two separate DNA strands.

[0458] In some embodiments, a portion of the single-stranded segment of the construct hybridizes to A2 and is extended to A2 via DNA polymerase. In some embodiments, another primer in the primer pair then hybridizes to the extended construct, revealing A2. This primer then extends to the construct in the direction of the double-stranded segment, displacing the shorter DNA strand, and thus one or more fluorophores and one or more dyes are sufficiently separated to detect a fluorescent signal indicating the presence of A2.

[0459] In such an implementation, the construct can be referred to as a molecular zipper.

[0460] Those skilled in the art will understand that, for both sunrise primers and molecular zippers, one or more fluorophores and one or more quencher pairs may be located at different positions within each respective construct. A key feature is that each pair is located close enough to each other that no fluorescent signal is emitted in the absence of A2, i.e., when no extension and chain substitution occur.

[0461] In some implementations, one or more wells in one or more regions may also include pyrophosphatase.

[0462] In some implementations, one or more pores in one or more regions of the device may also include phosphatases or phosphorylases.

[0463] In some implementations, one or more wells in the first region of the device may also include an enzyme for forming DNA from an RNA template.

[0464] In some implementations, the enzyme is a reverse transcriptase.

[0465] In some implementations, one or more enzymes present in the device are hot-started.

[0466] In some implementations, one or more enzymes present in the device are thermally stable.

[0467] In some implementations, the first and second regions of the device are combined.

[0468] In some implementations, one or more fluid channels are positioned between one or more holes in a region and / or between one or more regions of the device.

[0469] In some implementations, the first region can be fluidly connected to the sample container via a fluid interface.

[0470] In some implementations, heating and / or cooling elements may be present in one or more areas of the device.

[0471] In some implementations, heating and / or cooling may be applied to one or more areas of the device.

[0472] In some implementations, each area of ​​the device may independently contain at least 100 or 200 holes.

[0473] In some implementations, each region of the device may independently contain between approximately 100 and 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 or more holes. The holes may be of any shape, and their positions may be arranged on the substrate in any format or pattern.

[0474] In some embodiments, the porous substrate may be made of a metal (e.g., as a non-limiting example, a gold, platinum, or nickel alloy), ceramic, glass, or other PCR-compatible polymeric material or composite material. The porous substrate may include more than one pore.

[0475] In some implementations, the holes can be formed as blind or through holes within the hole substrate. For example, holes can be created within the hole substrate by laser drilling (e.g., using an excimer laser or a solid-state laser), ultrasonic embossing, thermal embossing, electroforming nickel molds, injection molding, and injection compression molding.

[0476] In some implementations, the volume of a single pore can range from 0.1 nL to 1500 nL. In one implementation, it ranges from 0.5 nL to 50 nL. Each hole can have a diameter of approximately 0.1 nL, 0.2 nL, 0.3 nL, 0.4 nL, 0.5 nL, 0.6 nL, 0.7 nL, 0.8 nL, 0.9 nL, 1 nL, 1.5 nL, 2 nL, 2.5 nL, 3 nL, 3.5 nL, 4 nL, 4.5 nL, 5 nL, 5.5 nL, 6 nL, 6.5 nL, 7 nL, 7.5 nL, 8 nL, 8.5 nL, 9 nL, 9.5 nL, 10 nL, 11 nL, 12 nL, 13 nL, 14 nL, 15 nL, 16 nL, 17 nL, 18 nL, 19 nL, 20 nL, or 25 nL. Volumes of 30nL, 35nL, 40nL, 45nL, 50nL, 55nL, 60nL, 65nL, 70nL, 75nL, 80nL, 85nL, 90nL, 95nL, 100nL, 110nL, 120nL, 130nL, 140nL, 150nL, 160nL, 170nL, 180nL, 190nL, 200nL, 225nL, 250nL, 275nL, 300nL, 325nL, 350nL, 375nL, 400nL, 425nL, 450nL, 475nL, or 500nL.

[0477] In some implementations, the aperture dimension can have any shape, such as circular, elliptical, square, rectangular, oval, hexagonal, octagonal, conical, and other shapes well known to those skilled in the art.

[0478] In some implementations, the hole shape may have a cross-sectional area that varies along the axis. For example, a square hole may gradually shrink from a first size to a second size that is a fraction of the first size.

[0479] In some implementations, the hole dimensions can be square, with the diameter and depth being approximately equal.

[0480] In some implementations, the walls defining the hole may be non-parallel.

[0481] In some implementations, the walls defining the aperture may converge to a single point. The aperture dimension can be derived from the total volume of the aperture substrate.

[0482] In some implementations, the pore depth can range from 25 μm to 1000 μm.

[0483] In one embodiment, the pore may have a depth of 25 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm or 1000 μm.

[0484] In some implementations, the pore diameter can range from about 25 μm to about 500 μm.

[0485] In some implementations, the aperture may have a width of 25 μm, 50 μm, 75 μm, 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 225 μm, 250 μm, 275 μm, 300 μm, 325 μm, 350 μm, 375 μm, 400 μm, 425 μm, 450 μm, 475 μm or 500 μm.

[0486] In some implementations, portions of one or more areas of the device can be modified to promote or prevent fluid adhesion. The surfaces defining the pores can be coated with a hydrophilic material (or modified to be hydrophilic), thereby promoting fluid retention.

[0487] In some embodiments, portions of one or more areas of the device may be coated with a hydrophobic material (or modified to be hydrophobic), thereby preventing fluid from remaining thereon. Those skilled in the art will understand that other surface treatments may be performed such that fluid is preferably retained within the pores rather than on the upper surface, in order to facilitate the drainage of excess fluid.

[0488] In some embodiments, the holes in the hole substrate can be patterned with simple geometric patterns having aligned rows and columns, or with patterns arranged diagonally or in hexagons. In one embodiment, the holes in the hole substrate can be patterned with complex geometric patterns, such as chaotic patterns or isomorphic geometric designs.

[0489] In some implementations, the pores may be geometrically separated from each other and / or have a large depth-to-width ratio to help prevent cross-contamination of reagents.

[0490] In some embodiments, the device may include one or more auxiliary zones that can be used to supply process fluid, such as oil or other chemical solutions, to one or more areas of the device. Such auxiliary zones may be fluidly connected to one or more areas of the device via one or more membranes, valves, and / or pressure-separable substrates (i.e., materials that rupture when subjected to a predetermined amount of pressure from fluid in an adjacent portion of the auxiliary zone or fluid path), such as metal foils or films.

[0491] In some implementations, the fluid passages of the device may include extensive tortuous sections. The tortuous path between the inlet passage of the fluid passage and one or more regions of the device facilitates the control and handling of fluid processing. The tortuous path can help reduce the formation of bubbles, which can interfere with the flow of oil through the fluid passages.

[0492] In some embodiments, the device may further include a gas-permeable membrane that allows gas to escape from pores in one or more regions of the device while preventing fluid passage. The gas-permeable membrane can be adhered to the porous substrate of the device using a gas-permeable adhesive. In one embodiment, the membrane may be made of polydimethylsiloxane (PDMS) and have a thickness ranging from 20 μm to 1000 μm. In some embodiments, the membrane may have a thickness ranging from 100 μm to 200 μm.

[0493] In some embodiments, all or part of the hole substrate may include a conductive metallic portion (e.g., gold) to allow heat to be transferred from the metal to the hole. In one embodiment, the inner surface of the hole may be coated with metal to facilitate heat transfer.

[0494] In some implementations, after the appropriate reagent has filled the orifices in one or more areas of the device, a barrier oil or thermally conductive liquid may be applied to the device to prevent crosstalk.

[0495] In some implementations, the orifices in one or more areas of the device can be shaped to gradually taper from a large diameter to a smaller diameter, similar to a cone. Conical orifices with sloping walls enable the use of non-contact deposition methods for reagents (e.g., inkjet printing). The conical shape also facilitates drying and has been found to prevent bubbles and leaks in the presence of a gas-permeable membrane.

[0496] In some embodiments, pores in one or more regions of the device can be filled by propelling sample fluid (e.g., by pressure) along the fluid channels of the device. As fluid passes through the pores in one or more regions of the device, each pore becomes filled with fluid, which is retained within the pore primarily by surface tension. As previously described, portions of the pore substrate of the device may be coated with a hydrophilic / hydrophobic material as needed to promote complete and uniform filling of the pores as sample fluid passes through.

[0497] In some implementations, the pores in one or more areas of the device can be “capped” with oil after filling. This can then help reduce evaporation as the pore substrate undergoes thermal cycling. In one implementation, after oil coating, an aqueous solution can be filled into one or more areas of the device to improve thermal conductivity.

[0498] In some implementations, a static aqueous solution can be pressurized in one or more areas of the device to prevent the movement of fluid and any bubbles.

[0499] In some implementations, oils such as mineral oils may be used to isolate orifices in one or more areas of the device and to provide thermal conductivity. However, any thermally conductive liquid, such as fluorinated liquids (e.g., 3M FC-40), may be used. Reference to oils in this disclosure should be understood to include applicable alternatives that will be understood by those skilled in the art.

[0500] In some implementations, the device may also include one or more sensor components.

[0501] In some embodiments, one or more sensor assemblies may include charge-coupled device (CCD) / complementary metal-oxide-semiconductor (CMOS) detectors coupled to an optical fiber panel (FOFP). A filter may be stacked on top of the FOPF and positioned against or adjacent to the aperture substrate. In one embodiment, the filter may be directly laminated (bonded) on top of the CCD, with the FOPF placed on top.

[0502] In some implementations, an hydration fluid, such as distilled water, may be heated in one of the first or auxiliary zones, such that one or more zones of the device have a humidity of up to 100%, or at least sufficient humidity to prevent excessive evaporation during thermal cycling.

[0503] In some implementations, after the device is filled, the well substrate can be heated by an external device that is in thermal contact with the device to perform thermal cycling of PCR.

[0504] In some implementations, non-contact heating methods, such as RFID, Curie point, induction heating, or microwave heating, may be employed. These and other non-contact heating methods will be well known to those skilled in the art. During thermal cycling, the chemical reactions of the device can be monitored using the sensor arrangement described previously.

[0505] In some embodiments, reagents deposited in one or more pores in one or more regions of the device are deposited in a predetermined arrangement.

[0506] In some implementations, a method is provided that includes:

[0507] Sample fluid is supplied to the fluid channels of the device, wherein the device includes at least one fluid channel between a first region, a second region, and a third region, wherein the first region, the second region, and the third region independently include one or more orifices;

[0508] The second region is filled with amplification fluid from the first region, such that one or more wells in the second region are covered by the amplification fluid;

[0509] Amplification fluid is drawn from the second region such that one or more wells remain wetted by at least some of the amplification fluid;

[0510] The third region is filled with fluid extracted from the second region, such that one or more orifices in the third region are covered by the fluid; and

[0511] Fluid is drawn from the third chamber such that one or more orifices remain wetted by at least some of the fluid.

[0512] In some implementations of this method, the fluid passage may be valveless.

[0513] In some implementations of this method, the extracted second region can be filled with a hydrophobic material.

[0514] In some implementations of this method, the extracted third region can be filled with a hydrophobic material.

[0515] In some embodiments of the method, a hydrophobic substance can be supplied from an oil chamber that is in fluid communication with the second and third regions.

[0516] In some implementations of this method, the sample fluid can travel along the fluid channel in a meandering manner.

[0517] In some implementations, the method may also include applying heating and cooling cycles to one or more of the first, second, or third regions.

[0518] In view of this disclosure, various other aspects and embodiments of the invention will be apparent to those skilled in the art.

[0519] As used herein, “and / or” is considered to be a specific disclosure of each of two specified features or components with or without the other. For example, “A and / or B” is considered to be a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were listed separately herein.

[0520] Unless the context otherwise indicates, the description and definition of the features listed above are not limited to any particular aspect or embodiment of the invention, and are equally applicable to all aspects and embodiments described.

[0521] Those skilled in the art will further understand that although the invention has been described by way of example with reference to several embodiments, the invention is not limited to the disclosed embodiments, and alternative embodiments may be developed without departing from the scope of the invention as defined in the appended claims.

[0522] Example 1 - Simplified Solution

[0523] For the purposes of this section and the following sections, embodiments of the present invention are illustrated by way of example and are referred to as embodiments 1-5, respectively.

[0524] Figure 1 An overview of the different options is provided.

[0525] The following table provides an overview of the time required to execute each plan:

[0526]

[0527] In one embodiment, TIPP is not present in any of the schemes, methods, kits, and / or devices of the present invention.

[0528] In one embodiment, the 5'-3' exonuclease is not present in any of the schemes, methods, kits and / or devices of the present invention.

[0529] The following table provides an overview of the enzymes that can be used in each protocol:

[0530]

[0531]

[0532] In one implementation, the presence of pyrophosphatase is optional.

[0533] In one implementation, the presence of a 5'-3' exonuclease is optional.

[0534] In one implementation, the presence of UDG is optional.

[0535]

[0536]

[0537] It can be seen that the inventors reduced the total number of enzymes required, thereby lowering the cost and complexity of the method. Surprisingly, the inventors found that adding 5'-3' exonucleases from the pre-amplification step to the pyrosequencing / ligation step of the protocol (as in protocols 3-5) resulted in... Figure 2 The higher fluorescence signal shown indicates the detection of a specific target analyte sequence.

[0538] Example 2: Pyrophosphate hydrolase (PPL)

[0539] The inventors have tested the method of Scheme 3 of the present invention using a series of different PPL enzymes, and the results are shown in Figure 3. Figure 3(A) shows the detection of 1% MAF T790M using Mako, Klenow, and Bsu. Figure 3(B) shows the detection of 0.5% MAF T790M using Bst LF at different PPi concentration ranges.

[0540] The inventors have tested the method of Scheme 4 of the present invention using a series of different PPL enzymes, and the results are shown in Figure 4.

[0541] Example 3: Comparison of Scheme 1 and Scheme 4

[0542] The inventors used schemes 1 and 4 to detect exon 19del_6223 at 0.5%, 0.10%, and 0.05% MAF, which can be seen in... Figure 5 It can be seen that the fluorescence peak is larger when using scheme 4.

[0543] Example 4: Scheme 4 - Sensitivity

[0544] The inventors tested for EGFR exon 20T790M mutations in 0.10%, 0.50%, and 1% MAF according to scheme 4. Figure 6 As shown.

[0545] Example 5: Scheme 4 - Is an exonuclease digestion step required in the RCA process?

[0546] The inventors have demonstrated that the exonuclease digestion step is not necessary during RCA. However, if the exonuclease digestion step is omitted, a detectable signal is detected later in RCA. Figure 7 The detection of EGFR exon 20T790M in 1% MAF was shown in the presence and absence of exonuclease during the RCA step.

[0547] Example 6: Scheme 4 - PPL:RCA mixing ratio

[0548] The inventors investigated the effect of the PPL:RCA mixing ratio on the signal intensity of 0.5% MAF EGFR exon 20T790M detection, and the results showed that... Figure 8 As can be seen, the 1:2 PPL:RCA mixing ratio resulted in the lowest signal strength, but at the earliest time point. Immediately following in time was the 1:4 PPL:RCA mixing ratio, which had a greater signal strength. At the latest time point, the 1:8 PPL:RCA mixing ratio exhibited the highest signal strength.

[0549] Example 7: Scheme 4 - Dye Selection

[0550] The inventors investigated whether the dyes used during RCA could be optimized. Figure 9 The results of comparative experiments using SybrGreenI (50°C and 60°C) and Syto82 (50°C and 60°C) according to Scheme 4 are shown. The Syto82 dye allows RCA to run at a lower temperature of 50°C, while SybrGreenI requires a higher temperature of 60°C. Scheme 5 requires a lower RCA temperature, which eliminates the need for adding proteinase K to the reaction mixture. The amplification enzymes used to prepare at least one single-stranded analyte containing the target polynucleotide region for detection using the method of the present invention require temperatures above 50°C to operate. Using SybrGreenI requires a reaction temperature of 60°C, and therefore proteinase K must be added at some point during the method to inactivate the amplification enzyme before RCA.

[0551] The lower RCA temperature allows the method of this invention to be performed in a plate reader instead of qPCR.

[0552] The response time is faster with Syto82, as shown in Figure 9 This can be seen in the results, and although the total fluorescence of Syto82 is lower—this can be mitigated by using a higher concentration of Syto82 dye.

[0553] Example 8: Scheme 4 - BST LF vs. BST 2.0WS

[0554] The inventors investigated the use of two different enzymes, BST LF and BST 2.0WS, according to scheme 4 to perform RCA to detect the 0.5% MAF EGFR exon 20T790M mutation. This result showed... Figure 10 The results show that the BST 2.0WS response is the fastest. The BST 2.0WS is designed to incorporate dUTP, which contributes to the reaction speed. There is a negligible difference in total signal intensity between the BST LF and BST 2.0WS. According to its description, the BST 2.0WS supplied by New England Biolabs (NEB) is only more stable and active above 45°C.

[0555] Example 9: Effect of PPL enzyme on signal detection

[0556] The inventors investigated the effects of different PPL enzymes on the RCA reaction at different PPL:RCA reaction mixture ratios. The results are shown in Figure 11(A) 1:4 PPL:RCA and Figure 11(B) 1:8 PPL:RCA. Except for BST, all PPL enzymes affected the RCA reaction at a 1:4 PPL:RCA ratio. At a 1:8 PPL:RCA ratio, BST and Klenow had no effect on the RCA reaction.

[0557] Example 10: Pyrophosphate digestion, targeting the specificity of single nucleotide mismatches in linking

[0558] Prepare single-stranded first oligonucleotide 1 (SEQ ID NO:1) having the following nucleotide sequence:

[0559]

[0560] Prepare single-stranded linked oligonucleotide 2 (SEQ ID NO:2) having the following nucleotide sequence:

[0561]

[0562] A, C, G, and T represent nucleotides that carry the relevant characteristic nucleoside bases of DNA.

[0563] / 5Phos / represents the 5' terminal phosphate.

[0564] * Represents a thiophosphate bond

[0565] A group of single-stranded oligonucleotides 3-4 (SEQ ID NO: 3-4) were also prepared, which have the following nucleotide sequences in the 5' to 3' directions:

[0566]

[0567] Oligonucleotide 3 includes a 17-base region complementary to the 17 bases at the 3' end of oligonucleotide 1, and oligonucleotide 4 includes the same region at position 3 with a single nucleotide mismatch. SEQ ID 3 and SEQ ID 4 are portions of the human EGFR gene with / without the C797S mutation, respectively.

[0568] Then, a first reaction mixture is prepared, having a composition corresponding to the composition obtained by the following formulation:

[0569] 0.5 μL 20x buffer, pH 7.0

[0570] 0.25 μL 5x buffer solution, pH 8.0

[0571] 0.25uL 5x HF buffer

[0572] 0.2 μL oligonucleotide 1, 1000 nM

[0573] 0.3 μL oligonucleotide 2, 1000 nM

[0574] 1 μL of oligonucleotide 2 (500 nM) or a mixture of oligonucleotides 2 and 3 (500 nM and 0.5 nM, respectively).

[0575] 0.3U Klenow fragment exo-(NEB)

[0576] 0.01 μL inorganic pyrophosphate, 10 mM

[0577] 0.0132U adenosine triphosphate bisphosphatase (ex. NEB)

[0578] 1U E. coli DNA ligase (ex. NEB)

[0579] Water up to 10ul

[0580] The 20x buffer solution comprises the following mixture:

[0581] 200 μL Tris-acetic acid, 1 M, pH 7.0

[0582] 342.5 μL magnesium acetate aqueous solution, 1M

[0583] 120 μL potassium acetate aqueous solution, 5M

[0584] 50ul Triton X-100 surfactant (10%)

[0585] Water to 1mL

[0586] The 5x buffer solution comprises the following mixture:

[0587] 50 μL Trizma (acetic acid), 1 M, pH 8.0

[0588] 25 μL magnesium acetate aqueous solution, 1M

[0589] 25 μL potassium acetate aqueous solution, 5M

[0590] 50ul Triton X-100 surfactant (10%)

[0591] Water to 1mL

[0592] The mixture was then subjected to pyrophosphate digestion by incubating at 45°C for 15 minutes, followed by cyclization of oligonucleotide 1 via ligation, and the resulting mixture was used for amplification reactions (Example 11).

[0593] Example 11: Amplification of circularized probes

[0594] A pair of single-stranded oligonucleotide primers, 1 (SEQ ID NO 5) and 2 (SEQ ID NO 6), were prepared, having the following nucleotide sequences:

[0595]

[0596] A, C, G, and T represent nucleotides that carry the relevant characteristic nucleoside bases of DNA.

[0597] A second reaction mixture is then prepared, which has a composition corresponding to the composition obtained by the following formulation:

[0598] 3uL 10x Thermopol buffer

[0599] 3.2U BST 2.0WS

[0600] 0.32 μL oligonucleotide 1, 10 μM

[0601] 0.32 μL oligonucleotides, 10 μM

[0602] 1.125uL Syto82, 30uM

[0603] 0.165U inorganic pyrophosphatase

[0604] 1.2 μL dNTP mixture, 10 mM

[0605] 1.25 μL of the reaction mixture in Example 10

[0606] Water up to 11.25uL

[0607] The 10x Thermopol buffer comprises the following mixture:

[0608] 200 μL Tris-HCl pH = 8.8, 1 M

[0609] 100 μL (NH4)2SO4, 1 M

[0610] 100uL KCl, 1M

[0611] 20mM MgSO4, 1M

[0612] 10uL X-100, 10%

[0613] Water to 1mL

[0614] The reaction mixture was then incubated at 50°C for 40 minutes, and the resulting reaction product was analyzed by real-time fluorescence analysis. The results are as follows: Figure 12 As shown in the figure. This analysis shows that when both oligonucleotides 3 and 4 are present, the fluorescence signal appears more quickly in the reaction, indicating that the pyrophosphate digestion and ligation of oligonucleotide 3 have already occurred in the first reaction mixture.

[0615] Example 12: Multicolor detection using Sunrise primers

[0616] 1. Target oligonucleotide dilution solution

[0617] WT oligonucleotide diluent consists of the following components

[0618] 0.5x A7 buffer

[0619] 0.5x Phusion U buffer

[0620] 200 nM WT oligonucleotide (SEQ ID NO:7)

[0621] Total volume: 5uL

[0622] A mixture of T790M and C797S 1% AF mutant oligonucleotides;

[0623] 0.5x A7 buffer

[0624] 0.5x Phusion U buffer

[0625] 100 nM WT oligonucleotide (SEQ ID NO:7)

[0626] 2nM T790M oligonucleotide (SEQ ID NO:8)

[0627] 2nM C797S_2389 oligonucleotide (SEQ ID NO:9)

[0628] Total volume: 5uL

[0629] WT oligonucleotide (SEQ ID NO:7):

[0630]

[0631] T790M oligonucleotide (SEQ ID NO:8):

[0632]

[0633] C797S_2389 oligonucleotide (SEQ ID NO:9):

[0634]

[0635] 1x A7 composition

[0636] Tris-acetic acid pH = 8.0 10mM

[0637] Potassium acetate 25mM

[0638] Magnesium acetate 5mM

[0639] Triton-X 0.01%

[0640] PhusionU buffer

[0641] The PhusionU buffer composition is not publicly available.

[0642] 2.PPL

[0643] A mixture corresponding to the following was prepared:

[0644] 1xBFF1

[0645] 37.5 U / mL Mako DNA polymerase (3'→5'exo-)

[0646] 100 U / mL E. coli ligase

[0647] 1.2 U / mL adenosine triphosphate diphosphatase

[0648] 0.6mM PPi

[0649] 20nM T790M probe

[0650] 20nM C797S_2389 probe

[0651] 30nM T790M splice oligonucleotides

[0652] 30nM C797S_2389 splint oligonucleotide

[0653] 5uL of WT or 1% AF mutant dilution at point 1.

[0654] Total volume 10uL

[0655] The mixture was then incubated at 41°C for 30 minutes.

[0656] 1xBFF1 composition

[0657] Tris-acetic acid pH = 7.0 10mM

[0658] Potassium acetate 30mM

[0659] Magnesium acetate 17.125 mM

[0660] Triton-X 0.01%

[0661] T790M probe (SEQ ID NO:10):

[0662]

[0663] C797S_2389 probe (SEQ ID NO:11):

[0664]

[0665] * represents a thiophosphate bond.

[0666] 3.TIPP

[0667] A mixture corresponding to the following was prepared:

[0668] 1xA7

[0669] 66.6 U / mL TIPP

[0670] 10 μL of the mixture from point 2.

[0671] Total volume: 20uL

[0672] The mixture was then incubated at 25°C for 5 minutes and then at 95°C for 5 minutes.

[0673] 4. Connection

[0674] A mixture corresponding to the following was prepared:

[0675] 1xA7

[0676] 100 U / mL E. coli ligase

[0677] 20 μL of mixture from point 3.

[0678] 10nM T790M splice oligonucleotides

[0679] 10nM C797S_2389 splint oligonucleotide

[0680] Total volume: 30uL

[0681] The mixture was then incubated at 37°C for 10 minutes and at 95°C for 10 minutes.

[0682] T790M splint oligonucleotide (SEQ ID NO:12):

[0683]

[0684] C797S_2389 splint oligonucleotide (SEQ ID NO:13):

[0685]

[0686] 5. Exonuclease treatment

[0687] A mixture corresponding to the following was prepared:

[0688] 1xA7

[0689] 100 U / mL E. coli ligase

[0690] 30uL of the mixture from point 4.

[0691] 625 U / mL exonuclease III

[0692] 62.5 U / mL T5 exonuclease

[0693] Total volume: 40uL

[0694] The mixture was then incubated at 30°C for 5 minutes and then at 95°C for 5 minutes.

[0695] 6. RCA

[0696] Mixtures corresponding to the following were prepared:

[0697] 1x Thermopol buffer (53.2mM Tris-HCl, 26.6mM (NH4)2SO4, 26.6mM KCl, 5.32mM MgSO4, 0.266%) X-100 (pH 8.8)

[0698] 0.2 μM primer mixture 1

[0699] 0.4 μM reverse primer

[0700] 533.3 U / mL BST LF

[0701] 0.4mM dNTP

[0702] 10 μL of the reaction mixture from point 5.

[0703] Total volume 15uL

[0704] Primer mixture 1:

[0705] Cy5 primer (SEQ ID NO:14):

[0706]

[0707] TexasRed primers (SEQ ID NO:15):

[0708]

[0709] Where / BHQ2 / represents Black Hole quencher;

[0710] Reverse primer (SEQ ID NO:16):

[0711]

[0712] The mixture was then incubated at 60°C for 90 minutes. Fluorescence measurements were performed every minute. Cq was obtained based on the automated threshold values ​​provided by the Bio-rad instrument. The results are available on [link to relevant documentation]. Figure 13 .

[0713] Example 13: Multicolor Detection Using Molecular Zippers

[0714] 1. Target oligonucleotide dilution solution

[0715] WT oligonucleotide diluent is made from the following ingredients

[0716] 0.5x A7 buffer

[0717] 0.5x Q5U buffer

[0718] 100 nM WT oligonucleotide (SEQ ID NO:17)

[0719] Total volume: 1.25uL

[0720] G719X_6239, G719X_6252, G719X_6253 0.5% AF mutant oligonucleotide mixture:

[0721] 0.5x A7 buffer

[0722] 0.5x Q5U buffer

[0723] 100 nM WT oligonucleotide (SEQ ID NO:17)

[0724] 0.5 nM G719X_6239 oligonucleotide (SEQ ID NO:18)

[0725] 0.5 nM G719X_6252 oligonucleotide (SEQ ID NO:19)

[0726] 0.5 nM G719X_6253 oligonucleotide (SEQ ID NO:20)

[0727] Total volume: 1.25uL

[0728] WT oligonucleotide (SEQ ID NO:17):

[0729]

[0730] G719X_6239 oligonucleotide (SEQ ID NO:18):

[0731]

[0732] G719X_6252 oligonucleotide (SEQ ID NO:19):

[0733]

[0734] G719X_6253 oligonucleotide (SEQ ID NO:20):

[0735]

[0736] 1xA7 composition

[0737] Tris-acetic acid pH = 8.0 10mM

[0738] Potassium acetate 25mM

[0739] Magnesium acetate 5mM

[0740] Triton-X 0.01%

[0741] Q5U buffer

[0742] The Q5U buffer composition is not publicly available.

[0743] 2. Pyrophosphorylation (PPL) and Ligation

[0744] A mixture corresponding to the following was prepared:

[0745] 1xBFF1

[0746] 10 U / mL Klenow(exo-)

[0747] 100 U / mL E. coli ligase

[0748] 1.2 U / mL adenosine triphosphate diphosphatase

[0749] 100U / mL Lambda exo

[0750] 0.25mM PPi

[0751] 6.6 nM G719X_6239 probe oligonucleotide (SEQ ID NO:21)

[0752] 6.6 nM G719X_6252 probe oligonucleotide (SEQ ID NO:22)

[0753] 6.6 nM G719X_6253 probe oligonucleotide (SEQ ID NO:23)

[0754] 30 nM splice oligonucleotide (SEQ ID NO:24)

[0755] 1.25 μL of the mixture from point 1.

[0756] Total volume 10uL

[0757] The mixture was then incubated at 45°C for 15 minutes.

[0758] 1xBFF1 composition

[0759] Tris-acetic acid pH = 7.0 10mM

[0760] Potassium acetate 30mM

[0761] Magnesium acetate 17.125 mM

[0762] Triton-X 0.01%

[0763] G719X_6239 probe oligonucleotide (SEQ ID NO:21):

[0764]

[0765] G719X_6252 probe oligonucleotide (SEQ ID NO:22):

[0766]

[0767] G719X_6253 probe oligonucleotide (SEQ ID NO:23):

[0768]

[0769] Splint oligonucleotide (SEQ ID NO:24):

[0770]

[0771] * represents a thiophosphate bond.

[0772] 3. Inspection - RCA

[0773] A mixture corresponding to the following was prepared:

[0774] 2.66x Thermopol buffer (53.2mM Tris-HCl, 26.6mM (NH4)2SO4, 26.6mM KCl, 5.32mM MgSO4, 0.266%) X-100 (pH 8.8)

[0775] 0.28 μM dye-primer mixture 1

[0776] 0.56 μM quencher primer 1

[0777] 0.28 μM quencher primer 2

[0778] 0.84 μM reverse primer

[0779] 568.8 U / mL BST 2.0 WarmStart

[0780] 14.67 U / mL TIPP

[0781] 1.06mM dNTP

[0782] 1.25 μL of the reaction mixture from point 2.

[0783] Total volume 11.25uL

[0784] Dye-primer mixture 1 consists of the following:

[0785] Dye primer 1 (SEQ ID NO:25):

[0786]

[0787] Dye primer 2 (SEQ ID NO:26):

[0788]

[0789] Dye primer 3 (SEQ ID NO:27):

[0790]

[0791] Where * represents a thiophosphate bond, / 5Cy5 / represents a Cy5 dye at the 5' end, / 5TEX615 represents a TEX dye at the 5' end, and / 5HEX / represents a Hex dye at the 5' end.

[0792] Quencher primer 1 (SEQ ID NO:28):

[0793]

[0794] Where / 3IAbRQSp / represents 3'Iowa RQ quencher

[0795] Quencher primer 2 (SEQ ID NO:29):

[0796]

[0797] Where / 3IAbkFQ / represents 3'Iowa FQ quencher

[0798] Reverse primer (SEQ ID NO:30):

[0799]

[0800] * represents a thiophosphate bond.

[0801] The mixture was then incubated at 58°C for 150 minutes. Fluorescence measurements were performed every minute. The results are available in [the table / document / etc.]. Figure 14 .

[0802] Example 14: Pyrophospholysis and Target-Specific Binding

[0803] 1. Preparation of oligonucleotide dilution buffer

[0804] Prepare oligonucleotide dilutions in 0.5xA7 and 0.5xQ5 buffers:

[0805] WT oligonucleotide 200nM

[0806] + / - mutant oligonucleotide 500pM

[0807] Total volume 1.25uL

[0808] WT oligonucleotides (SEQ ID NO 31):

[0809]

[0810] Mutant oligonucleotide (SEQ ID NO 32):

[0811]

[0812] 2. Pyrophosphate hydrolysis and ligation

[0813] A PPL mixture consisting of the following components was prepared:

[0814] 1xBFF1

[0815] 10 U / mL Klenow(exo-)

[0816] 100 U / mL E. coli ligase

[0817] 1.2 U / mL adenosine triphosphate diphosphatase

[0818] 100U / mL Lambda exo

[0819] 0.25mM PPi

[0820] 20nM probe A0

[0821] 1.25 μL of oligonucleotides from point 1

[0822] Total volume 10uL

[0823] Probe A0 (SEQ ID NO 33):

[0824]

[0825] Among them, the thiophosphate bond

[0826] 1xBFF1 composition

[0827] Tris-acetic acid pH = 7.0 10mM

[0828] Potassium acetate 30mM

[0829] Magnesium acetate 17.125 mM

[0830] Triton-X 0.01%

[0831] 1xA7 composition

[0832] Tris-acetic acid pH = 8.0 10mM

[0833] Potassium acetate 25mM

[0834] Magnesium acetate 5mM

[0835] Triton-X 0.01%

[0836] Q5 buffer

[0837] Q5 buffer compositions are not publicly available.

[0838] The resulting mixture was incubated at 45°C for 15 minutes.

[0839] 3. Inspection - RCA

[0840] An RCA mixture was prepared, consisting of the following:

[0841] 2.66x Thermopol buffer (53.2mM Tris-HCl, 26.6mM (NH4)2SO4, 26.6mM KCl, 5.32mM MgSO4, 0.266% Triton-X, pH 8.8)

[0842] 0.28 μM primer mixture

[0843] 284.4 U / mL BST 2.0 WarmStart

[0844] 14.67 U / mL TIPP

[0845] 1.06mM dNTP

[0846] Syto82 dye 3uM

[0847] 1.25 μL of reaction from point 2

[0848] Total volume 11.25uL

[0849] Primer mixture:

[0850]

[0851] The resulting mixture was incubated at 50°C for 70 minutes.

[0852] Fluorescence readings are taken every minute. Results can be found in Figure 16 .

[0853] Example 15: Application of further selection of the present invention and implementation scheme

[0854] KRAS testing

[0855] The KRAS gene controls cell proliferation. When it is mutated, this negative signaling is disrupted, and cells can proliferate uncontrollably, often leading to cancer. Single amino acid substitutions, and especially single nucleotide substitutions, are the cause of activating mutations involved in a variety of cancers: lung adenocarcinoma, mucinous adenoma, pancreatic ductal carcinoma, and colorectal cancer. KRAS mutations have been used as prognostic biomarkers for diseases such as lung cancer.

[0856] Driver mutations in KRAS are associated with up to 20% of human cancers, and targeted therapies for these mutations and their associated diseases are currently being developed. The table below lists some of these therapies in a non-limiting manner:

[0857]

[0858] The presence of KRAS mutations has been found to reflect very poor responses to the EGFR inhibitors panitumumab (Vectibix) and cetuximab (Erbitux). Activating mutations in the gene encoding KRAS occur in 30%–50% of colorectal cancers, and studies have shown that patients whose tumors express this mutated form of the KRAS gene do not respond to panitumumab and cetuximab. The presence of the wild-type KRAS gene does not guarantee a response to these drugs; however, studies have shown that cetuximab is significantly effective in patients with metastatic colorectal cancer who have wild-type KRAS tumors. The response rate to the EGFR antagonists erlotinib or gefitinib in KRAS-mutant (wild-type EGFR) lung cancer patients is estimated to be 5% or lower, compared to 60% in patients without KRAS mutations.

[0859] Early detection of the presence of KRAS mutations (activation or overexpression) (a common driver of acquired resistance to cetuximab therapy (anti-EGFR therapy) in colorectal cancer) allows for modifications to treatment (e.g., early initiation of mitogen-activated protein kinase kinase [MEK] inhibitors) to delay or reverse resistance, and therefore the method of the present invention allows for rapid and inexpensive detection of a patient's KRAS status, which is advantageous.

[0860] The non-restrictive list of mutations is: G12D, G12A, G12C, G13D, G12V, G12S, G12R, A59T / E / G, Q61H, Q61K, Q61R / L, K117N, and A146P / T / V.

[0861] The table below shows a further non-restrictive list of mutations:

[0862] Exons Mutation name COSM Number Mutant sequence 2 G12A COSM522 c.35G>C 2 G12C COSM516 c.34G>T 2 G12D COSM521 c.35G>A 2 G12F COSM512 c.34_35delinsTT 2 G12R COSM518 c.34G>C 2 G12S COSM517 c.34G>A 2 G12V COSM520 c.35G>T 2 G12V COSM515 c.35_36delinsTC 2 G13A COSM533 c.38G>C 2 G13C COSM527 c.37G>T 2 G13D COSM532 c.38G>A 2 G13R COSM529 c.37G>C 2 G13S COSM528 c.37G>A 3 Q61E COSM550 c.181C>G 3 Q61H COSM1146992 c.183A>T 3 Q61H COSM554 / COSM1135364 c.183A>C 3 Q61K COSM549 / COSM1159597 c.181C>A 3 Q61L COSM553 c.182A>T 3 Q61R COSM552 c.182A>G

[0863] BRAF detection

[0864] BRAF is a human gene that encodes a protein called B-Raf, which is involved in sending signals within cells that guide cell growth. It has been shown to be mutated in some human cancers. B-Raf is a member of the Raf kinase family of growth signal transduction protein kinases and plays a role in regulating the MAP kinase / ERK signaling pathway, which, among other things, affects cell division.

[0865] Some other inherited BRAF mutations can lead to birth defects.

[0866] More than 30 mutations in the BRAF gene associated with human cancer have been identified. In 90% of cases, thymine is replaced by adenine at nucleotide 1799. This results in the substitution of valine (V) for glutamate (E) at codon 600 of the activation region found in human cancers (now known as V600E). This mutation is widely observed in the following:

[0867] -Colorectal cancer

[0868] -Melanoma

[0869] - Papillary thyroid carcinoma

[0870] Non-small cell lung cancer

[0871] - Ameloblastoma

[0872] The non-restrictive list of other mutations that have been discovered is: R461I, I462S, G463E, G463V, G465A, G465E, G465V, G468A, G468E, N580S, E585K, D593V, F594L, G595R, L596V, T598I, V599D, V599E, V599K, V599R, V600K, and A727V.

[0873] Drugs have been developed to treat cancers driven by BRAF mutations; vemurafenib and dabrafenib have been approved by the FDA for the treatment of advanced melanoma. For metastatic melanoma, vemurafenib treatment has a response rate of 53%, compared to 7%–12% for dacarbazine, previously considered the best chemotherapy drug.

[0874] ERBB2 / HER2 testing

[0875] Human epidermal growth factor receptor 2 (HER2) (also known as CD340 (differentiation cluster 340), proto-oncogene Neu, Erbb2 (rodents), or ERBB2 (humans)) is a protein encoded by the ERBB2 gene. Amplification or overexpression of this oncogene plays a crucial role in the progression of aggressive types of breast cancer. Overexpression of the ERBB2 gene is also known to occur in ovarian cancer, gastric cancer, lung adenocarcinoma, aggressive uterine cancer, and 30% of salivary gland duct carcinomas. Structural alterations leading to receptor-independent ligand-based firing in the absence of overexpression have also been identified.

[0876] There are many approved and developing targeted therapies for this mutation and related diseases. The table below lists some of these therapies in a non-limiting manner:

[0877]

[0878] HER2 testing is routinely performed in breast cancer patients to assess prognosis, monitor response to treatment, and determine the suitability of targeted therapies (such as trastuzumab). Because trastuzumab is expensive and associated with serious side effects (cardiotoxicity), it is important to select only HER2+ patients to receive it, and therefore the method of this invention allows for rapid and inexpensive testing of a patient's HER2 status, which is advantageous.

[0879] In one embodiment, the method of the present invention is used to detect the presence or absence of an ERRB2 exon 20 insertion mutation.

[0880] The table below shows a further non-restrictive list of ERBB2 mutations:

[0881]

[0882] EML4-ALK detection

[0883] EML4-ALK is an aberrant fusion of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene. This gene fusion results in the production of the protein EML4-ALK, which exhibits characteristics that promote and maintain the malignant behavior of cancer cells. EML4-ALK-positive lung cancer is a primary malignant lung tumor in which cells contain this mutation.

[0884] There are many approved and developing targeted therapies for this mutation and related diseases. The table below lists some of these therapies in a non-limiting manner:

[0885]

[0886] EML4-ALK gene fusion accounts for approximately 5% of non-small cell lung cancer (NSCLC), with about 9,000 new cases annually in the United States and about 45,000 cases globally.

[0887] Many EML4-ALK variants exist that require transforming activity, all of which possess the necessary coiled-coil domain in both the N-terminal portion of EML4 and the kinase domain of exon 20 of ALK. The fusion of exon 13 of EML4 with exon 20 of ALK (variant 1: V1) (its detection can be seen in...) Figure 20 The fusion of exon 20 of EML4 with exon 20 of ALK (V2) and the fusion of exon 6 of EML4 with exon 20 of ALK (V3) are some of the more common variants. The clinical significance of these different variants has only recently become clearer.

[0888] V3 has become a biomarker for patients who may have shorter progression-free survival (PFS) after non-tyrosine kinase inhibitor (TKI) therapy (such as chemotherapy and radiotherapy). Further evidence suggests that V3, compared to V1 and V2 of EML4-ALK, is associated with shorter PFS and worse overall survival (OS) in patients receiving first- and second-generation therapy.

[0889] It was also found that V3-positive patients develop resistance to first and second-line treatments by developing resistance mutations, and this resistance may be promoted by incomplete tumor cell suppression due to the higher IC50 of wild-type V3. Detection of unfavorable V3 can be used to select patients requiring more aggressive surveillance and treatment strategies. It has been shown that administering third-generation loratinib to patients with V3 may confer a longer PFS than to patients with V1, and therefore the method of the present invention allows for rapid and inexpensive detection of variants that patients may have, which is advantageous.

[0890] The method of the present invention also allows for the detection of resistance mutations, such as, but not limited to: G1202R, G1269A, E1210K, D1203, S1206C, L1196M, F1174C, I1171T, I1171N / S, V1180L, T1151K, and C1156Y.

[0891] For example, G1202R is a solvent-front mutation that interferes with drug binding and confers high levels of resistance to first- and second-generation ALK inhibitors. Therefore, the method of this invention allows for the identification of patients who may have this mutation and would benefit from third-generation therapy rather than first- or second-generation therapy.

[0892] The table below shows a further non-restrictive list of EML4-ALK mutations:

[0893]

[0894] EGFR testing

[0895] The identification of epidermal growth factor receptor (EGFR) as an oncogene has led to the development of targeted therapies, such as gefitinib, erlotinib, afatinib, brigatinib, and icotinib for lung cancer, and cetuximab for colorectal cancer. However, many patients have developed resistance to these therapies. The two main sources of resistance are the T790M mutation and the MET oncogene.

[0896] EGFR mutations occur in EGFR exons 18-21 and exons 18, 19, and 21, and indicate the suitability for treatment with EGFR-TKIs (tyrosine kinase inhibitors). Mutations in exon 20 (with a few exceptions) indicate that the tumor is EGFR-TKI resistant and not suitable for EGFR-TKI treatment.

[0897] The two most common EGFR mutations are short in-frame deletions in exon 19 and a point mutation at nucleotide 2573 in exon 21 (CTG to CGG), which results in the substitution of leucine for arginine at codon 858 (L858R). These two mutations together account for approximately 90% of all EGFR mutations in non-small cell lung cancer (NSCLC). Screening for these mutations in NSCLC patients can be used to predict which patients will respond to TKIs.

[0898] Therefore, the method of the present invention allows for the identification of patients who may have these mutations and would benefit from initiating TKI treatment. Those skilled in the art will understand that the method of the present invention allows for the identification of a range of EGFR mutations, a non-exhaustive list of which includes: G719X, Ex19Del, S768I, Ex20Ins, and L861Q.

[0899] The table below shows a further non-restrictive list of mutations:

[0900]

[0901]

[0902] ROS1

[0903] ROS1 is a receptor tyrosine kinase (encoded by the gene ROS1) that has a structural similarity to the anaplastic lymphoma kinase (ALK) protein (encoded by the c-ros oncogene).

[0904] The following table shows a list of non-restrictive ROS1 mutations:

[0905]

[0906]

[0907] RET proto-oncogene

[0908] The RET proto-oncogene encodes a receptor tyrosine kinase for a member of the glial cell-derived neurotrophic factor (GDNF) family.

[0909] The following table shows a list of non-restrictive RET mutations:

[0910]

[0911] MET exon 14

[0912] MET exon 14 skipping occurs in NSCLC at a frequency of about 5%, and is visible in both squamous cell carcinoma and adenocarcinoma histology.

[0913] The table below shows a non-restrictive list of MET mutations:

[0914] Exons Mutation name COSM Number Mutant sequence Skip to 14 MET-MET COSM29312 M13_M15

[0915] NTRK proto-oncogene

[0916] NTRK gene fusion results in an abnormal protein called a TRK fusion protein, which may contribute to cancer cell growth. NTRK gene fusions can be present in certain types of cancer, including brain cancer, head and neck cancer, thyroid cancer, soft tissue cancer, lung cancer, and colon cancer. It is also known as neurotrophic tyrosine receptor kinase gene fusion.

[0917] The table below shows a non-restrictive list of NTRK mutations:

[0918]

[0919] Group (panel)

[0920] In one embodiment of the invention, a group comprising more than one probe molecule (A0) is provided, wherein each A0 is complementary to a target mutation. The mutation may be selected from any mutation previously or subsequently described or known. Therefore, those skilled in the art will understand that the scope of the invention includes a group that can be used to detect one or more mutations in any previously or subsequently described or known proto-oncogenes or oncogenes.

[0921] In one embodiment, the group comprises 5-500 individual probe molecules, each complementary to a specific target mutation. In one embodiment, the group comprises 5-400 individual probe molecules, each complementary to a specific target mutation. In one embodiment, the group comprises 5-300 individual probe molecules, each complementary to a specific target mutation. In one embodiment, the group comprises 5-200 individual probe molecules, each complementary to a specific target mutation. In one embodiment, the group comprises 5-100 individual probe molecules, each complementary to a specific target mutation. In one embodiment, the group comprises 5-50 individual probe molecules, each complementary to a specific target mutation.

[0922] In one implementation, there may be more than one probe molecule specific to the same mutation. In another implementation, there may be only one probe molecule specific to each mutation in the group.

[0923] In one embodiment, a group is provided, wherein the group includes more than one probe molecule, wherein one or more probes are complementary to an EGFR mutation, one or more probes are complementary to a KRAS mutation, one or more probes are complementary to an ERBB2 / HER2 mutation, one or more probes are complementary to an EML4-ALK mutation, one or more probes are complementary to a ROS1 mutation, one or more probes are complementary to a RET mutation, and one or more probes are complementary to a MET mutation.

[0924] In one embodiment, a group is provided, wherein the group includes more than one probe molecule, wherein one or more probes are complementary to an EGFR mutation, one or more probes are complementary to a KRAS mutation, one or more probes are complementary to an ERBB2 / HER2 mutation, one or more probes are complementary to an EML4-ALK mutation, one or more probes are complementary to a ROS1 mutation, one or more probes are complementary to a RET mutation, and one or more probes are complementary to a MET mutation.

[0925] In one embodiment, a group of probes selective for one or more EGFR, KRAS, BRAF, ERBB2 / HER2, EML4-ALK, ROS1, RET, and MET mutations is provided.

[0926] In one implementation, a set of probe molecules selective for EGFR mutations is provided.

[0927] In one implementation, a group of probe molecules selective for KRAS mutations is provided.

[0928] In one implementation, a set of probe molecules selective for BRAF mutations is provided.

[0929] In one implementation, a set of probe molecules selective for ERBB2 / HER2 mutations is provided.

[0930] In one embodiment, a group of probe molecules selective for EML4-ALK mutations is provided.

[0931] In one implementation, a group of probe molecules selective for ROS1 mutations is provided.

[0932] In one implementation, a set of probe molecules selective for RET mutations is provided.

[0933] In one implementation, a group of probe molecules selective for NTRK mutations is provided.

[0934] In one implementation, a group of probe molecules selective for ROS1 mutations is provided.

[0935] In one implementation, a set of probe molecules selective for MET exon 14 mutations is provided.

[0936] In one embodiment, a group of more than one probe molecule selective for one or more coding sequences (CDS) is provided.

[0937] In one implementation, a method is provided for detecting one or more mutations using one or more of the previously described groups.

[0938] In one implementation, a method is provided for detecting the presence or absence of one or more mutations using one or more of the previously described groups.

[0939] In one embodiment, a kit is provided comprising a group that may be described previously or subsequently, in combination with one or more reagents that may be described previously or subsequently.

[0940] Those skilled in the art will understand that the implementation of the reagent kit disclosed in A0 includes implementations within its scope, wherein there are groups that include more than one A0.

[0941] In one embodiment, a device is provided, wherein one or more areas of the device comprise one or more groups that may be described previously or subsequently.

[0942] Companion diagnostics

[0943] The method of this invention can be used to detect specific genetic markers in a sample, which can help guide the selection of appropriate therapies. These markers can be tumor-specific mutations or wild-type genomic sequences, and can be detected using tissue, blood, or any other type of patient sample.

[0944] Resistance monitoring

[0945] Repeated testing of patient samples during disease treatment can allow for the early detection of treatment resistance. One example of this application is non-small cell lung cancer (NSCLC), where epidermal growth factor receptor (EGFR) inhibitors (such as gefitinib and erlotinib) are commonly used as first-line therapy. During treatment, tumors may often develop mutations in the EGFR gene (e.g., T790M, C797S), which confer resistance to the drug. Early detection of these mutations can allow patients to switch to alternative therapies (such as Tagrisso).

[0946] Typically, patients being monitored for resistance development may be too ill to undergo repeat tissue biopsies. Repeated tissue biopsies can also be expensive, invasive, and carry associated risks. Blood testing is preferable, but very low copy numbers of the mutation of interest may be present in reasonable blood samples. Therefore, monitoring requires sensitive testing from blood samples using the method of this invention, which is simple and cost-effective to implement and can be performed periodically.

[0947] Recurrence monitoring

[0948] In this application example, patients declared disease-free after treatment may be monitored over time to detect disease recurrence. This needs to be non-invasive and requires sensitive detection of the target sequence from a blood sample. Using the method of this invention, a simple and low-cost approach that can be performed periodically is provided. The targeted sequence can be a common mutation known to be prevalent in the disease of interest, or it can be a set of custom targets designed for a specific patient based on the detection of variants in pre-remission tumor tissue.

[0949] Minimal residual disease (MRD) surveillance

[0950] For some cancers, residual cancer cells remain in the patient's body after treatment, which is a major cause of cancer and leukemia relapse. MRD monitoring and testing play several important roles: determining whether treatment has eradicated the cancer or left any residue, comparing the efficacy of different treatments, monitoring the patient's remission status, detecting leukemia relapse, and selecting the treatment that best meets these needs.

[0951] Screening

[0952] Population screening for early disease detection is a long-term goal, particularly in cancer diagnosis. The challenge is twofold: identifying a set of biomarkers that allow for reliable disease detection with few false negatives, and developing methods with sufficient sensitivity and cost. Compared to PCR-based assays, the method of this invention can handle a larger mutant set, but compared to sequencing-based diagnostics, the method of this invention has a simpler workflow and is less expensive.

[0953] Organ transplant rejection

[0954] When a transplanted organ is rejected by a recipient, DNA from that organ sheds into the recipient's bloodstream. Early detection of this DNA would allow for early detection of rejection. This can be achieved using a customized set of donor-specific biomarkers, or by using a set of variants known to be common in the population (some of which will be present in the donor and some in the recipient). Routine monitoring of organ recipients over time can be achieved through the low-cost and simple workflow of the invention disclosed herein.

[0955] Non-invasive prenatal testing (NIPT)

[0956] It has long been known that fetal DNA is present in the mother's blood, and the NIPT market is now saturated with companies that use sequencing to identify mutations and count the copy number of specific chromosomes to detect fetal abnormalities. The method of the present invention disclosed herein has the ability to detect mutations with very low allele fractions, potentially allowing for earlier detection of fetal DNA. Identifying common mutations in a given population would allow for the development of assays that target mutations that may be present in maternal or fetal DNA, or allow for the detection of abnormalities at an earlier stage of pregnancy.

[0957] In view of this disclosure, various other aspects and embodiments of the invention will be apparent to those skilled in the art.

[0958] As used herein, “and / or” is considered to be a specific disclosure of each of two specified features or components with or without the other. For example, “A and / or B” is considered to be a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were listed separately herein.

[0959] Unless the context otherwise indicates, the description and definition of the features listed above are not limited to any particular aspect or embodiment of the invention, and are equally applicable to all aspects and embodiments described.

[0960] Those skilled in the art will further understand that although the invention has been described by way of example with reference to several embodiments, the invention is not limited to the disclosed embodiments, and alternative embodiments may be constructed without departing from the scope of the invention as defined in the appended claims.

[0961] Those skilled in the art will understand that the reference to "partially digested chain A1" can refer to a single-stranded oligonucleotide formed by the stepwise digestion of A0 in the 3'-5' direction when hybridizing with a target analyte sequence, until the chain dissociates due to lack of complementarity.

[0962] Those skilled in the art will understand that references to "partially double-stranded" nucleic acids can refer to nucleic acids in which one or more portions are double-stranded and one or more portions are single-stranded.

[0963] Those skilled in the art will understand that references to "substantially double-stranded" nucleic acids can refer to nucleic acids in which one or more portions are double-stranded and one or more smaller portions are single-stranded. sequence list <110> Bio-Fidelity Co., Ltd. <120> Simplified polynucleotide sequence detection method <130> P32007WO1 <160> 35 <170> PatentIn version 3.5 <210> 1 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> A at position 1 has 5' phosphate. A, T, G, and T (positions 1-4) are all linked by thiophosphate bonds. <400> 1 atgttcgatg agctttgaca atacttgaag ctcgcagata taggatgttg cgatagtcca 60 ggaggctgc 69 <210> 2 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence <400> 2 tgtcaaagct catcgaacat cctggactat gtctcc 36 <210> 3 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Part of the human EGFR gene <400> 3 tgctgggcat ctgcctcacc tccaccgtgc agctcatcac gcagctcatg cccttcggca 60 gcctcctgga ctatg 75 <210> 4 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> A portion of the human EGFR gene with the C797S mutation <400> 4 tgctgggcat ctgcctcacc tccaccgtgc agctcatcac gcagctcatg cccttcggct 60 gcctcctgga ctatg 75 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence <400> 5 tcgcaacatc ctatatctgc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence <400> 6 tgagctttga caatacttga 20 <210> 7 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> WT oligonucleotides <400> 7 catctgcctc acctccaccg tgcagctcat cacgcagctc atgcccttcg gctgcctcct 60 ggactatgtc cgggaacaca aagacaatat 90 <210> 8 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> T790M oligonucleotide <400> 8 catctgcctc acctccaccg tgcagctcat catgcagctc atgcccttcg gctgcctcct 60 ggactatgtc cgggaacaca aagacaatat 90 <210> 9 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> C797S_2389 oligonucleotide <400> 9 catctgcctc acctccaccg tgcagctcat cacgcagctc atgcccttcg gcagcctcct 60 ggactatgtc cgggaacaca aagacaatat 90 <210> 10 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> T790M probe. 5' Phosphate. Phosphothiophosphate bonds between A, T, G, and T at positions 1-4. <400> 10 atgttcgatg agctttgaca atacttgagc acggcagata taggatgttg cgaagggcat 60 gagctgcatg atgagctg 78 <210> 11 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> C797S_2389 probe. 5' Phosphate. Phosphothiophosphate bonds between A, T, G, and T at positions 1-4. <400> 11 atgttcgatg agctttgaca atacttgaag ctcgcagata taggatgttg cgatagtcca 60 ggaggctgc 69 <210> 12 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> T790M splint oligonucleotides. <400> 12 tgtcaaagct catcgaacat gcccttcgca acatct 36 <210> 13 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> C797S_2389 splint oligonucleotide. <400> 13 tgtcaaagct catcgaacat tcctggacta tcgcat 36 <210> 14 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Cy5 primer. 3' Qusar670 dye, quencher between G at position 50 and A at position 51. <400> 14 acgcctggtt accgagccag gttcgcacat gtaggctcgg taaccaggcg acatcctata 60 tctgccgtgc 70 <210> 15 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> Texas Red primer. 5' Texas Red dye. Quencher between G at position 38 and C at position 39. <400> 15 acgcctggtt acaggttcgc acatgtagta accaggcgca acatcctata tctgcgag 58 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer. <400> 16 atgttcgatg agctttgaca 20 <210> 17 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> WT oligonucleotides. <400> 17 cccaaccaag ctctcttgag gatcttgaag gaaactgaat tcaaaaagat caaagtgctg 60 ggctccggtg cgttcggcac ggtgtataag gtaaggtccc 100 <210> 18 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> G719X_6239 oligonucleotide. <400> 18 cccaaccaag ctctcttgag gatcttgaag gaaactgaat tcaaaaagat caaagtgctg 60 gcctccggtg cgttcggcac ggtgtataag gtaaggtccc 100 <210> 19 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> G719X_6252 oligonucleotide. <400> 19 cccaaccaag ctctcttgag gatcttgaag gaaactgaat tcaaaaagat caaagtgctg 60 agctccggtg cgttcggcac ggtgtataag gtaaggtccc 100 <210> 20 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> G719X_6253 oligonucleotide. <400> 20 cccaaccaag ctctcttgag gatcttgaag gaaactgaat tcaaaaagat caaagtgctg 60 tgctccggtg cgttcggcac ggtgtataag gtaaggtccc 100 <210> twenty one <211> 77 <212> DNA <213> Artificial Sequence <220> <223> G719X_6239 probe oligonucleotide. 5' phosphate. Phosphothiophosphate bonds between A, T, G, and T at positions 1-4. <400> twenty one atgttcgatg agctttgaca atacttgaca tgcgcagata taggatgttg cgaaacgcac 60 cggaggccag cactttg 77 <210> twenty two <211> 77 <212> DNA <213> Artificial Sequence <220> <223> G719X_6252 probe oligonucleotide. 5' phosphate. Phosphothiophosphate bonds between A, T, G, and T at positions 1-4. <400> twenty two atgttcgatg agctttgaca atacttgaca tgccgagtaa tgagagtttc gcaaacgcac 60 cggagctcag cactttg 77 <210> twenty three <211> 77 <212> DNA <213> Artificial Sequence <220> <223> G719X_6253 probe oligonucleotide. 5' phosphate. Phosphothiophosphate bonds between A, T, G, and T at positions 1-4. <400> twenty three atgttcgatg agctttgaca atacttgaca tgcgagcaat taggtagtgt cgtaacgcac 60 cggagcacag cactttg 77 <210> twenty four <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Splint oligonucleotides. <400> twenty four tgtcaaagct catcgaacat ccggtgcgtt cggcaa 36 <210> 25 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Dye primer 1.5' Cy5 dye, thiophosphate bond between A at position 1 and C at position 2. <400> 25 actgaccagc tccatgacaa tcgctgtcgc catgatcgat cgcaacatcc tatatctgc 59 <210> 26 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Dye primer 2.5' TEX dye, thiophosphate bond between A at position 1 and C at position 2. <400> 26 actgaccagc tccatgacaa tcgctgtcgc catgatcgat gcgaaactct cattactcg 59 <210> 27 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> The dye primer 3' is a 5' TEX dye, with a thiophosphate bond between the T at position 1 and the A at position 2. <400> 27 tacgaccgac tcactcctta cagcagtccg cagtatgcta cgacactacc taattgctc 59 <210> 28 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Quencher primer 1.3' Iowa Black RQ quencher. <400> 28 tcgatcatgg cgacagcgat tgtcatggag ctggtcagt 39 <210> 29 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Quencher primer 2.3' Iowa Black FQ quencher. <400> 29 agcatactgc ggactgctgt aaggagtgag tcggtcgta 39 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer. Phosphothiophosphate bonds between T, G, and A at positions 1-3. <400> 30 tgagctttga caatacttga 20 <210> 31 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Wild-type oligonucleotides <400> 31 ctgctgggca tctgcctcac ctccaccgtg cagctcatca cgcagctcat gcccttcggc 60 tgcctcctgg actatgtccg g 21 <210> 32 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Wild-type oligonucleotides <400> 32 ctgctgggca tctgcctcac ctccaccgtg cagctcatca tgcagctcat gcccttcggc 60 tgcctcctgg actatgtccg g 21 <210> 33 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> 5' Phosphoric acid. Thiophosphate bonds between a, g, c, and t at positions 1-4. <400> 33 agctgcatct gagctttgac aatacttgag cacggcagat ataggatgtt gcgaagggca 60 tgagctgcat gatg 14 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> The thiophosphate bonds between t, c, and g at positions 1-3. <400> 34 tcgcaacatc ctatatctgc 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> <400> 35 atgttgcgaa gggcatatgt 20

Claims

1. A reagent kit, the reagent kit comprising: (a) A single-stranded probe oligonucleotide A0, which is capable of forming a first intermediate with a target polynucleotide sequence, the intermediate being at least partially double-stranded, wherein the single-stranded probe oligonucleotide A0 includes a 3' terminal region complementary to the target polynucleotide sequence. (b) Ligase; (c) Pyrophosphate hydrolase, which is capable of digesting the first intermediate product from the end of A0 in the 3'-5' direction to produce a partially digested chain A1; (d) An ion source driving the pyrophosphate reaction, wherein the ion is optionally a pyrophosphate ion; and (e) A suitable buffer solution, The 5' end of A1 is complementary to the region of the target polynucleotide exposed by digestion of A0; or The kit also contains a splint oligonucleotide D, wherein D contains an oligonucleotide region complementary to the 3' end of A1 and a region complementary to the 5' end of A1; or The kit also includes a linker oligonucleotide C and a splice oligonucleotide D, wherein D contains an oligonucleotide region complementary to the 3' end of A1 and a region complementary to the 5' end of oligonucleotide C.

2. The kit according to claim 1 further includes a positive control and a negative control.

3. The kit according to claim 1 or 2, wherein the 5' end of A0 is conferred resistance to digestion by 5'-3' exonuclease, and wherein the kit further comprises a 5'-3' exonuclease.

4. The kit according to any one of claims 1 to 3, wherein the kit further comprises deoxynucleotide triphosphates (dNTPs), polymerase, and buffer for initial amplification of the target polynucleotide sequence present in the sample.

5. The kit according to claim 4, wherein the kit further comprises a high-fidelity polymerase incorporating dUTP, dUTP, and uracil-DNA N-glycosylation enzyme (UDG).

6. The kit according to any one of claims 1 to 5, wherein the kit further comprises a protease.

7. The kit according to any one of claims 1 to 6, wherein the 5' end of A1 is complementary to the region of the target polynucleotide exposed by digestion of A0.

8. The kit according to any one of claims 1 to 6, wherein the kit comprises a splint oligonucleotide D, wherein D comprises an oligonucleotide region complementary to the 3' end of A1 and a region complementary to the 5' end of A1.

9. The kit according to any one of claims 1 to 6, wherein the kit comprises a linker oligonucleotide C and a splice oligonucleotide D, wherein D comprises an oligonucleotide region complementary to the 3' end of A1 and a region complementary to the 5' end of oligonucleotide C.

10. The kit of claim 9, wherein the ligation probe oligonucleotide C comprises a 3' modification that protects it from digestion by 3'-5' exonucleases, and the kit further comprises 3'-5' exonucleases.

11. The kit of claim 9, wherein the ligation probe oligonucleotide C comprises an internal modification that protects it from digestion by 3'-5' exonucleases, and the kit further comprises 3'-5' exonucleases.

12. The kit according to claim 8 or 9, wherein D cannot be extended against A1 due to 3' modification or mismatch between the 3' end of D and the corresponding region of A1 or C.

13. The kit according to any one of claims 1 to 12, wherein the kit further comprises at least one single-stranded primer oligonucleotide complementary to a portion of A0, an amplification enzyme, dNTPs, and one or more oligonucleotide binding dyes or molecular probes.

14. The kit according to any one of claims 1 to 13, wherein the kit further comprises more than one A0, each A0 being selective for different target sequences and each A0 containing a recognition region.

15. The kit according to any one of claims 1 to 12, wherein the kit further comprises: - Two or more linker chain reaction (LCR) probe oligonucleotides complementary to adjacent sequences on A1, wherein, upon successful annealing, the 5' phosphate of one LCR probe is directly adjacent to the 3' OH of the other LCR probe; and - One or more ligases.

16. The kit according to claim 13, wherein the amplification enzyme is the same as the pyrophosphate hydrolase.

17. The kit according to any one of claims 1 to 12, wherein the kit further comprises... -Link probe oligonucleotide C; -Clamping oligonucleotide D; C has a 5' phosphate, the 3' end of the intercalation oligonucleotide D is complementary to the 5' end of C, and the 5' end of D is complementary to the 3' end of A1, so that A1 and C can be linked together to form A2.

18. The kit of claim 17, wherein the kit further comprises: - A hairpin oligonucleotide 1 (HO1) comprising a fluorophore-quencher pair, wherein HO1 is complementary to A2, and upon annealing with A2, the hairpin structure of HO1 opens and the fluorophore-quencher pair dissociates; and - A hairpin oligonucleotide 2 (HO2) comprising a fluorophore-quencher pair, wherein HO2 is complementary to an open HO1, and the hairpin structure of HO2 opens and the fluorophore-quencher pair separates when annealed with HO1.

19. The kit according to claim 18, wherein the kit further comprises more than one HO1 and HO2.

20. The kit according to any one of claims 1 to 12, wherein the kit further comprises: -Oligonucleotide A, wherein oligonucleotide A comprises a substrate arm, a partial catalytic core and a sensor arm; -Oligonucleotide B, wherein oligonucleotide B comprises a substrate arm, a partial catalytic core and a sensor arm; and -Substrates containing fluorophore-quencher pairs; The sensor arms of oligonucleotides A and B are complementary to the flanking regions of A2, enabling oligonucleotides A and B to combine in the presence of A2 to form a catalytic multicomponent nuclease (MNAzyme).

21. The kit according to any one of claims 1 to 12, wherein the kit further comprises a partially double-stranded nucleic acid construct, wherein: - A strand contains at least one RNA base, at least one fluorophore, and wherein a region of the strand is complementary to the region of A2, and wherein the strand is a "substrate" strand; and - Another chain contains at least one quencher, and a region of this chain is complementary to a region of A2 that is complementary to the region of the substrate chain, such that, in the presence of A2, the partially double-stranded nucleic acid construct has a larger double-stranded portion.

22. The kit of claim 21, wherein the kit further comprises an enzyme for removing the at least one RNA base.

23. The kit according to any one of claims 1 to 12, wherein the kit further comprises: - An oligonucleotide complementary to the region containing the linker site of A2, the oligonucleotide comprising one or more fluorophores arranged such that their fluorescence is quenched by their proximity to each other or by their proximity to one or more fluorescence quenchers; - Double-stranded DNA-specific digestive enzymes; In the presence of A2, the labeled oligonucleotides are digested, causing the fluorophores to separate from each other or from their respective quenchers, and the fluorescence signal, and therefore the presence of A2, is detectable.

24. The kit according to any one of claims 1 to 23, wherein the kit further comprises phosphatase.

25. The kit according to any one of claims 1 to 23, wherein the kit further comprises phosphohydrolase.

26. The kit according to any one of claims 1 to 25, wherein the kit further comprises pyrophosphatase.

27. The kit according to any one of claims 1 to 26, wherein the kit further comprises an enzyme for forming DNA from an RNA template.

28. An apparatus, the apparatus comprising: A fluid channel between a first region and a second region, wherein the first region includes one or more orifices, and one or more orifices include: A single-stranded probe oligonucleotide A0, which is capable of forming a first intermediate with a target polynucleotide sequence, the intermediate being at least partially double-stranded, wherein the single-stranded probe oligonucleotide A0 includes a 3' terminal region complementary to the target polynucleotide sequence. Pyrophosphate hydrolase, which is capable of digesting the first intermediate product from the end of A0 in a 3'-5' direction to produce a partially digested chain A1; an ion source, which drives the pyrophosphate hydrolysis reaction forward, wherein the ion is optionally a pyrophosphate ion; and One or more ligases capable of ligating A1 to produce oligonucleotide A2, wherein the 5' end of A1 is complementary to the region of the target polynucleotide exposed by digestion of A0; or The second region includes one or more pores, wherein one or more pores in the first or second region include a splint oligonucleotide D, wherein D comprises an oligonucleotide region complementary to the 3' end of A1 and a region complementary to the 5' end of A1; or One or more wells in the first or second region include a connecting probe oligonucleotide C and a splice oligonucleotide D, wherein D contains an oligonucleotide region complementary to the 3' end of A1 and a region complementary to the 5' end of oligonucleotide C.

29. The device according to claim 28, wherein, The 5' end of A0 is resistant to digestion by 5'-3' exonucleases, and the pores in the first region further include 5'-3' exonucleases.

30. The device according to any one of claims 28 or 29, wherein the device further comprises a third region, the third region including one or more orifices connected to the first region via fluid channels, and wherein the one or more orifices of the third region comprise: dNTP; At least one single-stranded primer oligonucleotide; and Amplification enzyme.

31. The device according to claim 30, wherein: The dNTPs in the third region are dUTP, dGTP, dCTP, and dATP; The amplification enzyme is a high-fidelity polymerase incorporating dUTP; and One or more pores in the third region also include uracil-DNA N-glycosylation enzyme.

32. The device of claim 30 or claim 31, wherein the device further comprises a fourth region located between the first region and the third region, the fourth region comprising one or more pores, wherein the one or more pores comprise a protease.

33. The device according to any one of claims 28 to 32, wherein one or more pores of the first region or the second region comprise a ligase, and the 5' end of A1 is complementary to the region of the target polynucleotide exposed by digestion of A0.

34. The device according to any one of claims 28 to 32, wherein one or more pores of the first region or the second region comprise a ligase and a splint oligonucleotide D complementary to the region of A0, wherein D comprises an oligonucleotide region complementary to the 3' end of A1 and a region complementary to the 5' end of A1.

35. The device according to any one of claims 28 to 32, wherein one or more pores in the first region or the second region comprise a ligase, a splint oligonucleotide D, and a ligation probe oligonucleotide C, wherein D comprises an oligonucleotide region complementary to the 3' end of A1 and a region complementary to the 5' end of oligonucleotide C.

36. The device of claim 35, wherein the ligation probe oligonucleotide C comprises a 3' modification that protects it from digestion by 3'-5' exonucleases.

37. The device of claim 35, wherein the ligation probe oligonucleotide C contains an internal modification that protects it from digestion by 3'-5' exonucleases.

38. The device according to any one of claims 28 to 37, wherein one or more holes in the first region comprise at least one or more different A0s, each A0 being selective for a different target sequence.

39. The device of claim 38, wherein the hole in the second region comprises: dNTP; Buffer solution; Amplification enzyme; One or more oligonucleotides bind to dyes or molecular probes; and A means for detecting signals originating from A1 or a portion thereof, or more than one copy of A1 or more than one copy of a portion thereof.

40. The device according to any one of claims 28 to 37, wherein the hole in the second region further comprises: - Two or more linked chain reaction (LCR) probe oligonucleotides complementary to the adjacent sequence on A1, wherein, upon successful annealing of the probes, the 5' phosphate of one LCR probe is directly adjacent to the 3' OH of the other LCR probe; and - One or more ligases.

41. The device according to any one of claims 28 to 37, wherein the hole in the second region comprises: -Link probe oligonucleotide C; -Clamping oligonucleotide D; C has a 5' phosphate, the 3' end of the splice oligonucleotide D is complementary to the 5' end of C, and the 5' end of D is complementary to the 3' end of A1, so that A1 and C can be linked together to form oligonucleotide A2.

42. The device of claim 41, wherein the hole in the second region further comprises: - A hairpin oligonucleotide 1 (HO1) comprising a fluorophore-quencher pair, wherein HO1 is complementary to A2 and, upon annealing with A2, the hairpin structure of HO1 opens and the fluorophore-quencher pair dissociates; and - A hairpin oligonucleotide 2 (HO2) comprising a fluorophore-quencher pair, wherein HO2 is complementary to an open HO1, and the hairpin structure of HO2 opens and the fluorophore-quencher pair separates when annealed with HO1.

43. The device according to any one of claims 28 to 37, wherein the hole in the second region further comprises: -Oligonucleotide A, wherein oligonucleotide A comprises a substrate arm, a partial catalytic core and a sensor arm; -Oligonucleotide B, wherein oligonucleotide B comprises a substrate arm, a partial catalytic core and a sensor arm; and -Substrates containing fluorophore-quencher pairs; The sensor arms of oligonucleotides A and B are complementary to the flanking regions of A2, such that oligonucleotides A and B bind in the presence of A2 to form a catalytic multicomponent nuclease (MNAzyme).

44. The device according to any one of claims 28 to 37, wherein the pore in the second region further comprises a partially double-stranded nucleic acid construct, wherein: - A strand contains at least one RNA base, at least one fluorophore, and wherein a region of the strand is complementary to the region of A2, and wherein the strand may be referred to as a "substrate" strand; - The other chain contains at least one quencher, and a region of this chain is complementary to a region of A2 that is complementary to the region of the substrate chain, such that, in the presence of A2, the partially double-stranded nucleic acid construct becomes more double-stranded; and The pores in the second region also include an enzyme for removing the at least one RNA base.

45. The device according to any one of claims 28 to 37, wherein one or more holes in the second region further include: Oligonucleotides complementary to the region containing the linker site of A2, wherein the oligonucleotides contain one or more fluorophores, wherein the one or more fluorophores are arranged such that their fluorescence is quenched by approaching each other or by approaching one or more fluorescence quenchers. Double-stranded DNA-specific digestive enzymes; In the presence of A2, the labeled oligonucleotides are digested, causing the fluorophores to separate from each other or from their respective quenchers, and the fluorescence signal, and thus the presence of A2, becomes detectable.

46. ​​The device according to any one of claims 28 to 45, wherein one or more pores in one or more regions further comprise pyrophosphatase.

47. The device according to any one of claims 28 to 46, wherein one or more pores in one or more regions further comprise phosphatase.

48. The device according to any one of claims 28 to 46, wherein one or more pores in one or more regions further comprise a phosphorylase.

49. The device according to any one of claims 28 to 48, wherein one or more pores in the first region further include an enzyme for forming DNA from an RNA template.

50. The device according to any one of claims 28 to 49, wherein the first region and the second region are merged.

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