Simplified polynucleotide sequence detection method

Abstract

Provided herein are methods for improved polynucleotide detection.

Description

[0001] The present invention relates to a simplified polynucleotide sequence detection method suitable for detecting the presence of a large number of diagnostic markers, including those used to identify cancer, infectious diseases and transplant organ rejection. It is also useful for companion diagnostic testing, in which a panel of markers must be identified reliably and at low cost.

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

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

[0004] Another problem is that quantification of the target is difficult because the PCR reaction cycles exponentially; small changes in reaction efficiency have a large effect on the amount of detectable material generated. Therefore, even with appropriate controls and proper calibration, quantification is usually limited to a precision of about 3-fold.

[0005] Finally, mutations in the target region studied by PCR amplification methods may have unwanted side effects. For example, there are instances where FDA-approved tests have to be revoked because the target organism mutated in the genetic region targeted by the test primers, resulting in a large number of false negatives. In contrast, 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 efficacy 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 mismatches, which is generally an inefficient and not highly specific reaction. In addition, as disclosed in Wang et al., because cleavage is used at mismatches, off-target hybridization of probes and similar sequences in samples can lead to false positive results. It is also impossible to distinguish two different genetic variants at the same or adjacent positions, because they will all lead to cleavage and amplification of the same probe. Therefore, the teachings of Wang et al. will lead to low sensitivity and low specificity of the reaction scheme. In contrast, the technical effect of the method disclosed in the present invention provides a fast and efficient method, which has high specificity for dsDNA and can be effectively blocked by mismatches. In addition, the method of the present invention is extremely specific to targeted genetic variants and can distinguish between different variants at the same or adjacent positions.

[0007] US2009 / 239283 A1 (Liu et al.) teaches the use of non-extendable 3' ends removed by pyrophosphorolysis, making it necessary to genetically engineer a custom polymerase capable of removing the 3' blocking modification. In contrast, the present invention utilizes the natural pyrophosphorolysis activity inherent to existing polymerases and does not use a 3' blocking modification. The method disclosed in Liu et al. also relies on removing only the terminal base from a portion of the probe to achieve subsequent amplification, and is limited to this embodiment by using a 3' blocking modification. In contrast, the method disclosed in the present invention implements an embodiment in which more than one base needs to be gradually removed from the probe to initiate the reaction, making it substantially more robust to transient off-target annealing of background DNA or other probes, which may result in unnecessary removal of the terminal base. Summary of the invention

[0008] We have now developed a new simplified method, which is based on the experience of using the pyrophosphorolysis method used in our early patent (PCT / GB2019 / 052017) to overcome many of these limitations. To this end, it utilizes the double-stranded specificity of pyrophosphorolysis (a reaction that can not be effectively carried out on a single-stranded oligonucleotide substrate or a double-stranded substrate containing a blocking group or nucleotide mismatch). The new method runs faster, has lower complexity and lower cost than the method disclosed in PCT / GB2019 / 052017. Therefore, according to the present invention, a method for detecting a target polynucleotide sequence in a given nucleic acid analyte is provided, the method comprising the following steps:

[0009] (a) introducing one or more nucleic acid analytes into a first reaction mixture comprising:

[0010] i. single-stranded probe oligonucleotide A0;

[0011] ii. pyrophosphatase; and

[0012] iii. Ligase

[0013] wherein A0 is pyrophosphorolyzed in the 3'-5' direction from the 3' terminus to produce at least partially digested chain A1 and A1 undergoes ligation to form A2;

[0014] (b) detecting a signal derived from a 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 inferring the presence or absence of the polynucleotide target sequence in the analyte.

[0015] The analytes to which the method of the present invention can be applied are those nucleic acids including the target polynucleotide sequence being sought, such as naturally occurring or synthetic DNA or RNA molecules. In one embodiment, the analyte is generally present in an aqueous solution containing the analyte and other biological materials, and in one embodiment, the analyte will be present together with other background nucleic acid molecules, which are not of concern for the purpose of the test. 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, before the step (a) of the method is performed, 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 substances. 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 cracked so as to release the analyte by destroying any cells present. 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. Sequence Listing <110> Bio-Fidelity 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> The A at position 1 has a 5' phosphate. A, T, G and T (positions 1-4) are all linked by phosphorothioate 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> Part 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 oligonucleotide <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. Phosphorothioate 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. Phosphorothioate 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 oligonucleotide. <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. Phosphorothioate 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. Phosphorothioate 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. Phosphorothioate 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, phosphorothioate 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, phosphorothioate 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> Dye Primer 3. 5' TEX dye, phosphorothioate bond between T at position 1 and 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. Phosphorothioate 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 oligonucleotide <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 oligonucleotide <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' phosphate. Phosphorothioate bonds between a, g, c, 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> Phosphorothioate 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 BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Protocol for a simplified method of polynucleotide sequence detection.

[0018] Figure 2 : A graph comparing the fluorescence levels detected (representing the presence of a specific target analyte sequence) when the 5'-3' exonuclease digestion step occurs during the pre-amplification step and when it moves to the pyrophosphorolysis / ligation step of the protocol (as in Protocols 3-5). In this example, the 5'-3' exonuclease is Lambda.

[0019] Figure 3: The inventors have tested the method of Scheme 3 of the present invention using a range of different PPL enzymes. 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. Even without extended optimization, all four enzymes performed very well.

[0020] FIG. 4 : Detection results of 1% MAF T790M using four different pyrophosphorolytic (PPL) enzymes Mako, Klenow, Bsu and Bst LF using the method of Scheme 4 of the present invention.

[0021] Figure 5 : Graph showing the fluorescence levels (representing the presence of a specific target analyte sequence) detected in the presence of 0.5%, 0.10% and 0.05% MAF exon 19del_6223 using methods according to Protocol 1 and Protocol 4.

[0022] Figure 6 : The inventors detected EGFR exon 20 T790M at 0.10%, 0.50% and 1% MAF according to Scheme 4.

[0023] Figure 7 : Detection of EGFR exon 20 mutation T790M at 1% MAF with and without exonuclease in the RCA step.

[0024] Figure 8 The inventors studied 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 ratio of the PPL:RCA mix resulted in the lowest signal intensity, but at the earliest time point. This was followed in time by a 1:4 PPL:RCA mix with a greater signal intensity. At the latest time point of the reaction, the greatest signal intensity was observed for the 1:8 PPL:RCA mix.

[0025] Fig. 9 : shows the results of comparative experiments performed according to Scheme 4 using SybrGreen I (50°C and 60°C) and Syto82 (50°C and 60°C).

[0026] Fig.10 : The inventors investigated RCA according to Scheme 4 using two different enzymes, BST LF and BST 2.0WS.

[0027] Figure 11: The inventors studied the effect of different PPL enzymes on the RCA reaction at different PPL:RCA reaction mixture ratios. The results can be seen in Figure 11 (A) 1:4 PPL:RCA and Figure 11 (B) 1:8 PPL:RCA. Except for BST LF, all PPL enzymes affect the RCA reaction at a 1:4 PPL:RCA ratio. At a 1:8 PPL:RCA ratio, all enzymes except BST LF and Klenow affect the RCA reaction.

[0028] Fig.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 pyrophosphorolysis and ligation of oligonucleotide 3 have occurred in the first reaction mixture.

[0029] Fig.13 : Detection of mutations of T790M and C797S_2389 at 1% allele fraction in the same reaction.

[0030] Fig.14 : Three mutations (0.5% allele fraction) were detected simultaneously in one well: G719X_6239, G719X_6252, G719X_6253.

[0031] Fig.15 : Schematic diagram of the circularization of A1 to form A2 against the analyte target sequence. A0 is digested stepwise from the 3' end of A0 in the 3'-5' direction relative to the target to form partially digested strand A1, which is shown as steps (A) and (B). This stepwise digestion reveals a target region complementary to the 5' end of A0 / A1, and the 5' end of A1 then hybridizes to this region, which is shown in step (C). A1 is then ligated together to form circularized A2, step (D).

[0032] Fig.16 : The fluorescence measurement results of Example 14 show the results from an embodiment in which A0 undergoes pyrophosphorolysis to form A1, which is then cyclized to form A2 directed to the target sequence.

[0033] Fig.17 : A single-stranded probe oligonucleotide A0 is annealed to a target polynucleotide sequence to produce a first intermediate product that is at least partially double-stranded and in which the 3' end of A0 forms a double-stranded complex with the target polynucleotide sequence. In this simplified embodiment of the invention, there are two A0 molecules and one target polynucleotide sequence to illustrate how A0 that is not annealed to the target does not participate in the additional 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' chemical blocking group, a common priming sequence, and a barcode region.

[0034] The partially double-stranded first intermediate is pyrophosphorolyzed in the 3'-5' direction from the 3' end of A0 in the presence of pyrophosphorylase to produce partially digested strand A1, analyte, and undigested A0 molecules that have not annealed to the target.

[0035] Fig.18 : A1 is annealed to a single-stranded trigger oligonucleotide B, and the A1 strand is extended in the 5'-3' direction against B to produce oligonucleotide A2. In this illustrative example, trigger oligonucleotide B has a 5' chemical block. Any undigested A0 anneals to trigger oligonucleotide B, but it cannot be extended in the 5'-3' direction against B to produce a sequence that is a target for the subsequent part of the method. In this example, A2 is primed with at least one single-stranded primer oligonucleotide, and more than one copy of A2 or a region of A2 is produced.

[0036] Fig.19 : A1 is annealed to the splint oligonucleotide D and then cyclized by ligating its 3' and 5' ends. The now cyclized A2 is primed with at least one single-stranded primer oligonucleotide and more than one copy of A2 or a region of A2 is produced. In this illustrative example, the splint oligonucleotide D cannot be extended against A1 due to a 3'-modification (chemical modification in this example) or by a nucleotide mismatch between the 3' end of D and the corresponding region of A2.

[0037] Fig. 20 : The 3' region of the splint oligonucleotide D anneals to the 3' region of A1, while the 5' region of the splint oligonucleotide D anneals to the 5' region of the ligation probe C. Thus, the second intermediate product A2 consists of A1, C, and optionally an intermediate region formed by extending A1 in the 5'-3' direction to join with the 5' end of C. In this illustrative example, the ligation probe C has a 3' chemical blocking group so that a 3'-5' exonuclease can be used to digest any unligated A1.

[0038] A2 is primed with at least one single-stranded primer oligonucleotide, and more than one copy of A2 or a region of A2 is produced.

[0039] Description of the implementation plan

[0040] In one aspect of the present invention, a method for detecting a target polynucleotide sequence in a given nucleic acid analyte present in a sample is provided, the method comprising the steps of:

[0041] (a) introducing one or more nucleic acid analytes into a first reaction mixture, the first reaction mixture comprising:

[0042] i. single-stranded probe oligonucleotide A0;

[0043] ii. pyrophosphatase; and

[0044] iii. Ligase

[0045] wherein A0 is pyrophosphorolyzed in the 3'-5' direction from the 3' terminus to produce at least partially digested chain A1 and A1 undergoes ligation to form A2;

[0046] (b) detecting a signal derived from a 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 inferring the presence or absence of the polynucleotide target sequence in the analyte.

[0047] In some embodiments, the first reaction mixture further comprises a source of pyrophosphate ions.

[0048] In some embodiments, the 3' end of A0 is fully complementary to the target polynucleotide sequence.

[0049] In some embodiments, the ligase substantially lacks single-stranded ligation activity.

[0050] In some embodiments, the first reaction mixture further comprises at least one single-stranded primer oligonucleotide substantially complementary to a portion of AO, and deoxyribonucleotide triphosphates (dNTPs).

[0051] In some embodiments, the first reaction mixture further comprises an amplification enzyme.

[0052] 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.

[0053] In some embodiments, the second reaction mixture further comprises an amplification enzyme.

[0054] In some embodiments, one or more nucleic acid analytes can be introduced into the first reaction mixture and the second reaction mixture simultaneously.

[0055] In some embodiments, one or more nucleic acid analytes can be introduced sequentially to the first reaction mixture and the second reaction mixture.

[0056] In some embodiments, the dNTPs are hot start dNTPs.

[0057] Hot-start dNTPs are dNTPs modified at the 3' end with a thermolabile protecting group. The presence of this modification prevents nucleotide incorporation by DNA polymerase until the nucleotide protecting group is removed using a heat activation step.

[0058] In some embodiments, during step (a), the analyte anneals to the single-stranded probe oligonucleotide A0 to produce a first intermediate product that is at least partially double-stranded and in which the 3' end of A0 forms a double-stranded complex with the analyte target sequence.

[0059] In some embodiments, during step (a), the first intermediate is pyrophosphorolyzed in the 3'-5' direction from the 3' end of A0 to produce partially digested strand A1 and the analyte.

[0060] In some embodiments, the first reaction mixture further comprises a ligation probe oligonucleotide C, and the partially digested strand A1 is ligated at the 3' end to the 5' end of C to produce oligonucleotide A2.

[0061] In some embodiments, partially digested chain A1 is circularized by ligation of its 3' and 5' ends to produce oligonucleotide A2.

[0062] In some embodiments, the linking of A1 occurs:

[0063] during step (a); or

[0064] during step (b); or

[0065] Between steps (a) and (b).

[0066] In some embodiments, the first reaction mixture further comprises a 5'-3' exonuclease, and the 5' end of A0 is rendered resistant to digestion by the 5'-3' exonuclease.

[0067] In some embodiments, after amplification of a given nucleic acid analyte and prior to addition to the first reaction mixture (step (a)), the sample is also treated with a protease.

[0068] In some embodiments, the first reaction mixture further comprises a phosphatase or a phosphohydrolase.

[0069] In some embodiments, prior to step (b) or during step (b), the product of the previous step is treated with pyrophosphatase.

[0070] In some embodiments, prior to step (b) or during step (b), the product of the previous step is treated with an exonuclease.

[0071] In some embodiments, oligonucleotide C further includes a 3' modification or an internal modification that protects it from 3'-5' exonuclease digestion.

[0072] In some embodiments, oligonucleotide C further comprises a 5' modification that protects it from 5'-3' exonuclease digestion.

[0073] In some embodiments, the first reaction mixture or the second reaction mixture further comprises a splint oligonucleotide D.

[0074] 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.

[0075] In some embodiments, D cannot be extended with respect to A1 due to a 3' modification or by a mismatch between the 3' end of D and the corresponding region of A1.

[0076] In some embodiments, the enzyme that performs the pyrophosphorolysis of A0 to form partially digested chain A1 also amplifies A2.

[0077] In some embodiments, detection is achieved using one or more oligonucleotide fluorescent conjugated dyes or molecular probes.

[0078] In some 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.

[0079] In some embodiments, more than one probe A0 is used, each probe A0 is selective for a different target sequence, and each probe A0 comprises a recognition region, and is further characterized in that the amplicon derived from A2 comprises this recognition region, and thus the target sequence present in the analyte is inferred by detecting the recognition region.

[0080] In some embodiments, detection of the recognition region is performed using molecular probes or by sequencing.

[0081] In some embodiments, the final step of the method further comprises the following steps:

[0082] i. labeling the product of step (b) using one or more oligonucleotide fluorescent binding dyes or molecular probes;

[0083] ii. measuring the fluorescence signal of the product;

[0084] iii. exposing the product to a set of denaturing conditions; and

[0085] The polynucleotide target sequence in the analyte is identified by monitoring the change in the fluorescent signal of the product during exposure to denaturing conditions.

[0086] In some embodiments, one or more nucleic acid analytes are split into more than one reaction volume, each volume having one or more probe oligonucleotides A0 introduced to detect a different target sequence.

[0087] In some embodiments, the different probes A0 contain a common priming site, allowing the use of a single primer or set of primers to amplify the region of A2.

[0088] In some embodiments, a method for detecting a target polynucleotide sequence in a given nucleic acid analyte present in a sample is provided, the method comprising the steps of:

[0089] (a) amplifying a given nucleic acid analyte present in a sample;

[0090] (b) introducing the product of step (a) into a first reaction mixture comprising:

[0091] i. single-stranded probe oligonucleotide A0;

[0092] ii. pyrophosphatase; and

[0093] iii. Ligase

[0094] wherein A0 is pyrophosphorolyzed in the 3'-5' direction from the 3' terminus to produce at least partially digested chain A1 and A1 undergoes ligation to form A2;

[0095] (c) detecting a signal derived from a 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 inferring the presence or absence of the polynucleotide target sequence in the analyte.

[0096] According to the present invention, a method for detecting a target polynucleotide sequence in a given nucleic acid analyte is provided. The analyte to which the various methods of the present invention can be applied can be prepared from the above-mentioned biological sample by a series of preliminary steps, which are intended to amplify the analyte and separate it from the background genomic DNA that is usually present in significant excess. The method is generally applicable to the production of single-stranded target analytes, and therefore the method is also useful in addition to being integrated with the method of the first aspect of the present invention or also including a part of the method. Therefore, a method for preparing at least one single-stranded analyte of a nucleic acid comprising a target polynucleotide region is provided, characterized in that the following steps are provided: (1) an amplicon of the analyte is produced by amplifying a biological sample comprising the analyte and optionally background genomic DNA through amplification cycles. In a preferred embodiment, amplification is performed using a polymerase chain reaction (PCR) in the presence of a polymerase, a nucleoside triphosphate and at least one corresponding primer pair, wherein one primer comprises 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.

[0097] In a preferred embodiment, steps (1) to (4) are performed before step (a) of the method of the first aspect of the invention to produce an integrated method for detecting a target sequence derived from a biological sample. In another embodiment, the biological sample has been subjected to cell lysis before performing step (1).

[0098] In some embodiments of step (1), the nucleoside triphosphate is a mixture of four deoxynucleoside triphosphates that are unique to naturally occurring DNA. 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 amplicons from previous assays. In yet another embodiment, a high-fidelity polymerase is used in step (1), such as commercially available polymerase Or one of those polymerases sold by Q5. In yet another embodiment, the polymerase can be KAPA HiFi Uracil+ DNA polymerase.

[0099] High-fidelity DNA polymerases have several safeguards to prevent making and propagating errors when copying DNA. During polymerization, these enzymes have a distinct binding preference between correct and incorrect nucleoside triphosphates. If an incorrect nucleotide does bind to the polymerase active site, incorporation is slowed due to the suboptimal structure of the active site complex. This lag time increases the chance that the incorrect nucleotide will dissociate before the polymerase progresses, allowing the process to restart with the correct nucleoside triphosphate. Proofreading DNA polymerases have an additional line of defense if an incorrect nucleotide is inserted. The disturbance caused by the mismatched base is detected, and the polymerase moves the 3' end of the growing DNA chain to the proofreading 3'→5' exonuclease domain. There, the incorrect nucleotide is removed by the 3'→5' exonuclease activity, and the chain is then moved back to the polymerase domain, where polymerization can continue.

[0100] In some embodiments, the nucleoside triphosphate is a mixture of synthetic or modified deoxynucleoside triphosphates.

[0101] In some embodiments, the nucleoside triphosphates are a mixture of the four deoxynucleoside triphosphates and synthetic or modified deoxynucleotide triphosphates.

[0102] In some embodiments, 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 the analyte. Therefore, the need for quantification of the analyte before subsequent steps is avoided. In another embodiment of step (1) (which has the advantage that step (2) is not required), amplification is performed in the presence of a primer pair, wherein one of the two primers is present in excess of the other, resulting in the production of a single-stranded amplicon once one primer is fully utilized.

[0103] In a preferred embodiment of step (2), the 5' primer is blocked by an exonuclease blocking group selected from the group consisting of a phosphorothioate bond, an inverted base, a DNA spacer, and other oligonucleotide modifications known in the art. In another embodiment, the other primer in the pair of primers has a phosphate group at its 5' end.

[0104] 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, 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 point after step (2), the reaction medium is treated with a phosphatase or phosphohydrolase to remove any residual nucleoside triphosphates that may be present.

[0105] In some embodiments, the target polynucleotide sequence in the analyte will be a gene or chromosomal region in the DNA or RNA of a cancerous tumor cell and is 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 will be useful for monitoring and / or treating disease recurrence. Patients who are declared disease-free after treatment may be monitored over time to detect recurrence of the disease. This needs to be done non-invasively and requires sensitive detection of the target sequence from a blood sample. Likewise, for some cancers, there are still residual cancer cells 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 detection of recurrence of the disease or failure of the current treatment and the need to switch to an alternative.

[0106] In some embodiments, detection of the target polynucleotide sequence will allow repeated testing of patient samples during disease treatment to allow early detection of the therapeutic resistance produced. For example, epidermal growth factor receptor (EGFR) inhibitors, such as gefitinib, erlotinib, are commonly used as first-line treatments for non-small cell lung cancer (NSCLC). During treatment, tumors will typically produce mutations (e.g., T790M, C797S) in the EGFR gene, which produces resistance to treatment. Early detection of these mutations allows patients to turn to alternative therapies.

[0107] In some embodiments, the target polynucleotide sequence in the analyte will be a gene or chromosome region in DNA or RNA of fetal origin and is characterized by the presence of one or more mutations; for example, in the form of one or more single nucleotide polymorphisms (SNPs). Therefore, compared with other available detection technologies, the present invention can be used to detect mutations with very low allele fractions at an earlier stage of pregnancy.

[0108] In another embodiment, the target polynucleotide sequence may be a gene or genomic region derived from an otherwise healthy individual, but the genetic information obtained may aid in generating valuable companion diagnostic information in one or more defined groups within the human population that allows medical or therapeutic conclusions to be drawn.

[0109] In yet another embodiment, the target polynucleotide sequence can be specific to an infectious disease, or specific to resistance of an infectious disease 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 a therapy.

[0110] In some embodiments, the target polynucleotide sequence can be unique to the donor DNA. When the transplanted organ is rejected by the patient, the DNA from the organ falls off and enters the patient's bloodstream. Early detection of the DNA will allow early detection of rejection. This can be achieved using a customized group of donor-specific markers, or by using a group of variants known to be common in a population, some of which will be present in the donor, and some in the recipient. Therefore, routine monitoring of organ recipients can be performed over time by the method for claiming protection.

[0111] In yet another embodiment, different versions of the method using different combinations of probes (see below) are used in parallel, so that more than one target sequence of an analyte can be screened simultaneously; for example, a cancer source, a cancer indicator, or more than one infectious source. In this method, the amplification products obtained by the parallel application of the method are contacted with a detection group including one or more oligonucleotide binding dyes or sequence-specific molecular probes such as molecular beacons, hairpin probes, etc. Therefore, in another aspect of the present invention, there is provided the use of at least one probe and optionally one connecting oligonucleotide in combination with one or more chemical and biological probes selective for a target polynucleotide sequence, or the use of at least one probe and optionally one connecting oligonucleotide in combination with the use of sequencing to identify the amplified probe region.

[0112] In some embodiments, the single-stranded probe oligonucleotide A0 comprises a priming region and a 3' end complementary to the target polynucleotide sequence to be detected. In this way, a first intermediate product that is at least partially double-stranded is produced. In one embodiment, this step is carried out in the presence of an excess of A0 and in an aqueous medium containing the analyte and any other nucleic acid molecules.

[0113] During step (a), the double-stranded region of the first intermediate product is pyrophosphorolyzed from the 3' end of its A0 chain in the 3'-5' direction. Therefore, the A0 chain is gradually digested to produce a partially digested chain; hereinafter referred to as A1. When the probe oligonucleotide mistakenly hybridizes with a non-target sequence, the pyrophosphorolysis reaction will stop at any mismatch, preventing the subsequent steps of the method from continuing. In another embodiment, this digestion continues until A1 lacks sufficient complementarity to form a stable duplex with the analyte or the target region therein. At this point, the individual chains are then separated by melting, thereby producing A1 in single-stranded form. Under typical pyrophosphorolysis conditions, this separation occurs when there are 6 to 20 complementary nucleotides between the analyte and A0.

[0114] In another embodiment, digestion continues until A1 lacks sufficient complementarity with the analyte or target region therein to allow pyrophosphorylase binding or pyrophosphorylation reaction to proceed. This typically occurs when 6 to 20 complementary nucleotides remain between the analyte and the probe. In some embodiments, this occurs when 6 to 40 complementary nucleotides remain.

[0115] In another embodiment in which a splint oligonucleotide D (see below) having complementarity to both the 5' and 3' ends of A1 is used, digestion is continued until the length of complementarity between A1 and the target is reduced to a point where it is energetically favorable for oligonucleotide D to displace the analyte molecule from A1. This typically occurs when the region of complementarity between A1 and the analyte molecule is similar to or shorter than the length of the region of complementarity between oligonucleotide D and the 3' end of A1, but may also occur when the complementarity between A1 and the analyte molecule is longer than the region of complementarity between oligonucleotide D and the 3' end of A1, in which case oligonucleotide D may have hybridized to the 5' end of A1 because of the favorability of intramolecular hybridization.

[0116] In another embodiment, the attachment of A1 is performed using the analyte molecule as a splint (see Fig.16 ), digestion continues until the 5' end of A1 is able to hybridize with the analyte molecule, so that the 3' and 5' ends of A1 are adjacent and separated only by a nick, at which they are ligated together by the ligase and digestion is no longer possible.

[0117] Suitably, the pyrophosphorolysis is carried out in a reaction medium at a temperature in the range of 20°C to 90°C in the presence of at least one polymerase exhibiting pyrophosphorolysis activity and a source of pyrophosphate ions. Further information on pyrophosphorolysis reactions applied to polynucleotide digestion can be found in, for example, J. Biol. Chem. 244 (1969) pp. 3019-3028 or in our earlier patent applications.

[0118] In some embodiments, the pyrophosphorolysis step is driven by the presence of an excess of a polypyrophosphate source, suitable sources including those compounds containing 3 or more phosphorus atoms.

[0119] In some embodiments, the first reaction mixture comprises an excess of a focusing phosphoric acid source.

[0120] In some embodiments, the pyrophosphorolysis step is driven by the presence of an excessive modified pyrophosphate source. Suitable modified pyrophosphates include those that replace bridging oxygens with other atoms or groups, or pyrophosphates (or focused phosphoric acid) replaced with replacement or modifying groups on other oxygens. Those skilled in the art will appreciate that there are many such examples of pyrophosphates that are suitable for modification of the present invention, and their non-limiting selections are:

[0121]

[0122] In some embodiments, the first reaction mixture comprises an excess of a modified focusing phosphate source.

[0123] In a preferred embodiment, the pyrophosphate ion source is PNP, PCP or tripolyphosphate (PPPi).

[0124] Additionally, but not limited to, examples of pyrophosphate ion sources used in the pyrophosphorolysis step (b) can be found in WO2014 / 165210 and WO00 / 49180.

[0125] In some embodiments, the excess modified pyrophosphate source can be represented as YH, where Y corresponds to the general formula (XO)2P(=B)-(ZP(=B)(OX)) n -, wherein 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 groups are independently selected from -H, -Na, -K, alkyl, alkenyl or heterocyclic groups, provided that when Z and B both correspond to -O-, and when n is 1, at least one X group is not H.

[0126] In some embodiments, Y corresponds to the general formula (XO)2P(=B)-(ZP(=B)(OX)) n -, wherein 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.

[0127] In alternative embodiments, 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 the X groups are all -Na or -K and Z is -NH- or -CH2-.

[0128] In another embodiment, Y corresponds to the formula (HO)2P(=B)-OP(=B)(OH)-, wherein each B group is independently O or S, and at least one is S.

[0129] Specific examples of preferred embodiments of Y include those of the 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) X1 and X2 are both methyl; or (c) X1 and X2 are both ethyl; or (d) X1 is methyl and X2 is ethyl, or vice versa.

[0130] In some embodiments, when a molecular probe is used for detection, the probe oligonucleotide A0 is configured to include an oligonucleotide recognition region at the 5' side of the region complementary to the target sequence, and the molecular probe used is designed to anneal to the recognition region. In one embodiment, only the 3' region of A0 can anneal to the target; that is, any other region lacks sufficient complementarity with the analyte so that a stable duplex exists at a temperature for performing a pyrophosphorolysis step. Here and throughout, the term "sufficient complementarity" refers to a region of complementarity that is more than 10 nucleotides long with respect to a given region having complementarity with a given region on the analyte.

[0131] In another aspect of the method of the present invention, an alternative embodiment is provided in which the phosphorolysis step of any of the foregoing embodiments is replaced by an exonuclease digestion step using a double-stranded specific exonuclease. It will be appreciated by those skilled in the art 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, and the like.

[0132] In some embodiments of the invention, wherein the exonuclease digestion step utilizes a double-stranded specific 5'-3' exonuclease, the 5' end of A0 is complementary to the target analyte, and the common priming sequence and blocking group are located on the 3' side of the region complementary to the target. In another embodiment, when a molecular probe is to be used for detection, 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 the recognition region.

[0133] 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 leaving intact A0 and any material comprising the partially digested strand A1. Suitably, this resistance to exonucleolysis is achieved as described elsewhere in this application.

[0134] In a preferred embodiment of the present invention, the 5' end of A0 or the internal site of the 5' side of the initiation zone is endowed with resistance to exonucleolytic digestion. In this way, and after or simultaneously with the pyrophosphorolysis step, the exonuclease with 5'-3' exonucleolytic activity can be optionally added to the reaction medium to digest any other nucleic acid molecules present, so that A0 and any material comprising the partially digested chain A1 are complete at the same time. Suitably, this resistance to exonucleolytic digestion is realized by introducing one or more blocking groups at the desired site of oligonucleotide A0. In one embodiment, these blocking groups can be selected from other backbone modifications commonly used in the art, C3 spacers, phosphate groups, modified bases, etc., such as thiophosphate bonds (phosphorothioate linkages) and other backbone modifications, C3 spacers, phosphate groups (phosphate group), modified bases, etc.

[0135] In some embodiments, the identification region will comprise or be embedded in a barcoding region having a unique sequence and suitable for indirect identification using sequence-specific molecular probes applied to amplified component A2, or direct identification by sequencing of these components. Examples of molecular probes that can be used include, but are not limited to, molecular beacons, Probe, Probes, etc.

[0136] In some embodiments, the A2 chain or its desired region is subjected to amplification, thereby producing more than one copy, typically millions of copies. This is achieved by initiating the region of A2 and any amplicon subsequently derived from A2 with a single-stranded primer oligonucleotide, which is provided, for example, in the form of a forward / reverse or sense / antisense pair, which can anneal to the complementary region on the region of A2 and any amplicon subsequently derived from A2. The chain initiated 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; when A2 is cyclized, the last one is applicable. By any of these methods, many amplicon copies in the region of A2 and its sequence complement in some cases can be quickly produced. The exact method for executing any of these amplification methods is well known to those of ordinary skill, and the exact conditions and temperature patterns adopted are easily available in the general literature read by the reader. Specifically, in the case of a polymerase chain reaction (PCR), the method generally includes extending a primer oligonucleotide against the A2 strand in the 5'-3' direction using a polymerase and a source of multiple single nucleotide triphosphates until a complementary strand is produced; dehybridizing the resulting double-stranded product to regenerate the A2 strand and the complementary strand; reinitiating the A2 strand and any amplicons thereof, and then repeating these extension / dehybridization / repriming steps multiple times to establish the concentration of the A2 amplicon to a level at which it can be reliably detected.

[0137] In alternative embodiments, the first reaction mixture or the second reaction mixture further comprises components for a hybridization chain reaction (HCR).

[0138] In some embodiments, the first reaction mixture further comprises a ligation probe oligonucleotide C having a 5' phosphate, a splint oligonucleotide D complementary to the 3' end of A1 and the 5' end of C, and the partially digested chain A1 is ligated to the 5' end of C at the 3' end to produce oligonucleotide A2. In some embodiments, the second reaction mixture further comprises a hairpin oligonucleotide 1 (HO1) and a hairpin oligonucleotide 2 (HO2), each of which comprises a fluorophore and a quencher, such that when each oligonucleotide is held in a hairpin configuration, the fluorophore and the quencher are in contact with each other. HO1 is designed to allow A2 to anneal to it, opening the "hairpin" structure and separating the fluorophore from the quencher. The now "opened" HO1 is now able to anneal to HO2, opening the "hairpin" structure and separating the fluorophore from the quencher.

[0139] In some embodiments, there are more than one hairpin oligonucleotide, so that the presence of one A2 can cause a chain reaction of opening of the hairpin oligonucleotide, resulting in the generation of a detectable fluorescent signal. This method is referred to in the literature as hybridization chain reaction (HCR).

[0140] In some embodiments, the fluorophore of the fluorophore-quencher pair is selected from, but not limited to, dyes of the fluorescein family, the carboxyrhodamine family, the cyanine family, and the rhodamine family. Other dye families that can be used include, for example, dyes of the polyhalofluorescein family, dyes of the hexachlorofluorescein family, dyes of the coumarin family, dyes of the oxazine family, dyes of the thiazine family, dyes of the squaraine family, chelated lanthanide dyes, dyes of the trade name AlexaFluor J available from Molecular Probes, dyes of the trade name Atto available from ATTO-TEC (Siegen, Germany), and dyes of the trade name Bodipy J available from Invitrogen (Carlsbad, Calif.). Dyes of the fluorescein family include, for example, 6-carboxyfluorescein (FAM), 2',4',1,4-tetrachlorofluorescein (TET), 2',4',5',7',1,4-hexachlorofluorescein (HEX), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyrhodamine (JOE), 2'-chloro-5'-fluoro-7',8'-fused phenyl-1,4-dichloro-6-carboxyfluorescein (NED), 2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), 6-carboxy-X-rhodamine (ROX), and 2',4',5',7'-tetrachloro-5-carboxy-fluorescein (ZOE). Dyes of the carboxyrhodamine family include tetramethyl-6-carboxyrhodamine (TAMRA), tetraacetone-6-carboxyrhodamine (ROX), Texas Red, R110, and R6G. Dyes of the cyanine family include Cy2, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7. Fluorophores are readily available commercially, for example, from Perkin-Elmer (Foster City, Calif.), Molecular Probes, Inc. (Eugene, Oreg.), and Amersham GE Healthcare (Piscataway, NJ).

[0141] In some embodiments, the quencher of the fluorophore-quencher pair can be a fluorescent quencher or a non-fluorescent quencher. Fluorescent quenchers include, but are not limited to, TAMRA, ROX, DABCYL, DABSYL, cyanine dyes including nitrothiazole blue (NTB), anthraquinone, malachite green, nitrothiazole, and nitroimidazole compounds. Exemplary non-fluorescent quenchers that dissipate energy absorbed from the fluorophore include fluorophores available from Biosearch Technologies, Inc. (Novato, Calif.) under the trade name Black Hole TMThose available are from Epoch Biosciences (Bothell, Wash.) under the trade name Eclipse TM Dark, those available from Anaspec, Inc. (San Jose, Calif.) under the trade name Qx1J, those available from Integrated DNA Technologies (Coralville, Iowa) under the trade name ZEN and TAO, and those available from Integrated DNA Technologies (Coralville, Iowa) under the trade name Iowa Black TM Those that are available.

[0142] In some embodiments, the fluorophore of the fluorophore-quencher pair can be fluorescein, Lucifer Yellow, B-phycoerythrin, 9-acridine isothiocyanate, Lucifer Yellow VS, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, 1-pyrenebutyric acid succinimidyl ester, and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives.

[0143] In some embodiments, the fluorophore of the fluorophore-quencher pair can be LC-Red 640, LC-Red 705, Cy5, Cy5.5, lissamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosin isothiocyanate, fluorescein, diethylenetriamine pentaacetate, or other lanthanide ion chelates (e.g., europium or terbium).

[0144] In some embodiments, the present invention uses a double-quenched fluorescently labeled oligonucleotide. The inclusion of a second internal quencher shortens the distance between the dye and the quencher and provides greater overall dye quenching in conjunction with the first quencher, reducing background and increasing signal detection. The second quencher and the first quencher can be any quencher previously described.

[0145] In some embodiments, the first reaction mixture further comprises a ligation probe oligonucleotide C having a 5' phosphate, a splint oligonucleotide D complementary to the 3' end of A1 and the 5' end of C, and the partially digested chain A1 is ligated at the 3' end to the 5' end of C to produce oligonucleotide A2.

[0146] In some embodiments, the 5' and 3' ends of A1 are linked together to form a circularized A2.

[0147] In some embodiments, A1 is circularized to form A2 for ligation probe oligonucleotide C.

[0148] In some embodiments, A1 is circularized to form A2 directed to a target sequence.

[0149] In some embodiments, A1 is cyclized to form A2 as previously or subsequently described.

[0150] In some embodiments, A2 is formed from partially digested chain A1 as previously or subsequently described.

[0151] In some embodiments, the second reaction mixture further comprises:

[0152] - Oligonucleotide A, comprising a substrate arm, part of the catalytic core and a sensor arm;

[0153] - oligonucleotide B, comprising a substrate arm, a portion of a catalytic core and a sensor arm; and

[0154] - a substrate comprising a fluorophore-quencher pair;

[0155] The sensor arms of oligonucleotides A and B are complementary to the flanking regions of A2, such that oligonucleotides A and B bind to form a catalytic multi-component nucleic acid enzyme (MNAzyme) in the presence of A2. In this embodiment, the MNAzyme forms and cleaves a substrate comprising a fluorophore-quencher pair only in the presence of A2, thereby generating a detectable fluorescent signal.

[0156] In some embodiments, the fluorophore-quencher pair can be as previously described. In some embodiments, the first reaction mixture and the second reaction mixture are combined.

[0157] In some embodiments, the first reaction mixture and the second reaction mixture are combined such that pyrophosphorolysis, ligation, and generation of a detectable fluorescent signal occur without the addition of other reagents.

[0158] In alternative embodiments, the second reaction mixture further comprises one or more DNAzymes.

[0159] In some embodiments, the second reaction mixture further comprises a partially double-stranded nucleic acid construct, wherein:

[0160] - one strand comprises at least one RNA base, at least one fluorophore, and wherein a region of this strand is complementary to a region of A2, and wherein this strand may be referred to as the "substrate" strand;

[0161] - the other strand comprises at least one quencher, and wherein a region of this strand is complementary to a region of A2 adjacent to a region of A2 that is complementary to the substrate strand, such that in the presence of A2, the partially double-stranded nucleic acid construct becomes substantially more double-stranded;

[0162] Wherein the substrate strand of the double stranded nucleic acid construct is cleaved at an RNA base in the process of becoming substantially more double stranded, fluorescence is generated because at least one quencher of the "other" strand is no longer in sufficient proximity to at least one fluorophore of the substrate strand.

[0163] In other words, in the presence of A2, the partially double-stranded nucleic acid construct has a double-stranded portion that is larger in size.

[0164] In some embodiments, the fluorophore-quencher pair can be as previously described.

[0165] In some embodiments, additional reagents are present in the second reaction mixture, such as suitable buffers and / or ions.

[0166] In some embodiments, the second reaction mixture further comprises Mg 2+ ion.

[0167] In some embodiments, the second reaction mixture further comprises Zn 2+ ion.

[0168] In some embodiments, the second reaction mixture further comprises X 2+ ions, where X is a metal.

[0169] In some embodiments, the second reaction mixture further comprises one or more X 2+ ions, where X is a metal.

[0170] In alternative embodiments, the first reaction mixture further comprises reagents for a ligase chain reaction (LCR).

[0171] In such embodiments, the first reaction mixture further comprises:

[0172] - one or more ligases; and

[0173] - Two or more LCR probe oligonucleotides complementary to adjacent sequences on A2, wherein the 5' phosphate of one LCR probe is directly adjacent to the 3' OH of another LCR probe when the probes are successfully annealed to A2.

[0174] In some embodiments, two LCR probes will successfully anneal to A2 and be linked together to form an oligonucleotide molecule in the presence of A2, which then serves as a new target for the second round of covalent attachment, resulting in a geometric amplification of the target of interest, in this case A2. The product or amplicon connected is complementary to A2 and serves as a target in the next amplification cycle. Therefore, in the presence of an excessive amount of LCR probes, the exponential amplification of a specific target DNA sequence is achieved by repeated cycles of denaturation, hybridization and connection. The presence of A2 is thus inferred, and the presence of the target polynucleotide sequence is therefore inferred.

[0175] In some embodiments, in the presence of A2, two PCR probes will successfully anneal to A2 and be ligated together to form one oligonucleotide molecule, which then serves as a new target for a second round of covalent ligation, resulting in geometric amplification of the target of interest, in this case A2, and then the target of interest, in this case A2, is detected.

[0176] In some embodiments, ligated oligonucleotide molecules are detected in real time using intercalating dyes or molecular probes.

[0177] In some embodiments, gel electrophoresis is used to detect ligated oligonucleotide molecules.

[0178] Those skilled in the art will recognize that there are many techniques that allow for detection of ligated oligonucleotide molecules.

[0179] In some embodiments, the dNTPs are hot start dNTPs.

[0180] In some embodiments, one or more ligases are thermostable.

[0181] In some embodiments, one or more ligases are naturally occurring.

[0182] In another embodiment, one or more ligases are engineered.

[0183] In some embodiments, the one or more ligases are selected from any ligase previously or subsequently disclosed.

[0184] In some embodiments, one or more polymerases are thermostable.

[0185] In some embodiments, the one or more polymerases are selected from any polymerase previously or subsequently disclosed.

[0186] In some embodiments, one or more polymerases are naturally occurring.

[0187] In some embodiments, one or more polymerases are engineered.

[0188] In some embodiments, one or more polymerases are the same as the polymerase used for pyrophosphorolysis.

[0189] In some embodiments, one or more enzymes of the invention are hot-start enzymes.

[0190] In some embodiments, one or more enzymes of the invention are thermostable.

[0191] In an alternative embodiment, the product of step (a) is introduced into a second reaction mixture prior to step (b), the second reaction mixture also comprising reagents for a ligase chain reaction (LCR).

[0192] In this embodiment, the second reaction mixture comprises

[0193] - one or more ligases;

[0194] - optionally one or more polymerases;

[0195] - two or more LCR probe oligonucleotides complementary to adjacent sequences on A2, wherein the 5' phosphate of one LCR probe is directly adjacent to the 3' OH of another LCR probe when the probes are successfully annealed to A2;

[0196] - optionally one or more primers for amplifying successfully ligated LCR probes; and

[0197] -Optionally dNTPs.

[0198] In some embodiments, two LCR probes will successfully anneal to A2 and be linked together to form an oligonucleotide molecule in the presence of A2, which then serves as a new target for the second round of covalent attachment, resulting in a geometric amplification of the target of interest, in this case A2. The connected product or amplicon is complementary to A2 and serves as a target in the next amplification cycle. Therefore, in the presence of excessive LCR probes, the exponential amplification of a specific target DNA sequence is achieved by repeated cycles of denaturation, hybridization and connection. The presence of A2 is thus inferred, and the presence of the target polynucleotide sequence is therefore inferred.

[0199] In some embodiments, in the presence of A2, two PCR probes will successfully anneal to A2 and be ligated together to form one oligonucleotide molecule, which then serves as a new target for a second round of covalent ligation, resulting in geometric amplification of the target of interest, in this case A2, and then the target of interest, in this case A2, is detected.

[0200] In some embodiments, ligated oligonucleotide molecules are detected in real time using intercalating dyes or molecular probes.

[0201] In some embodiments, gel electrophoresis is used to detect ligated oligonucleotide molecules.

[0202] Those skilled in the art will recognize that there are many techniques that allow for detection of ligated oligonucleotide molecules.

[0203] In some embodiments, the dNTPs are hot start dNTPs.

[0204] In some embodiments, one or more ligases are thermostable.

[0205] In some embodiments, one or more ligases are naturally occurring.

[0206] In another embodiment, one or more ligases are engineered.

[0207] In some embodiments, the one or more ligases are selected from any ligase previously or subsequently disclosed.

[0208] In some embodiments, one or more polymerases are thermostable.

[0209] In some embodiments, the one or more polymerases are selected from any polymerase previously or subsequently disclosed.

[0210] In some embodiments, one or more polymerases are naturally occurring.

[0211] In another embodiment, one or more polymerases are engineered.

[0212] In some embodiments, one or more polymerases are the same as the polymerase used for pyrophosphorolysis.

[0213] In some embodiments, the second reaction mixture comprises a pyrophosphatase in addition to one or more polymerases.

[0214] In some embodiments, one or more enzymes of the invention are hot-start enzymes.

[0215] In some embodiments, one or more enzymes of the invention are thermostable.

[0216] In an alternative embodiment, the first reaction mixture further comprises:

[0217] - an oligonucleotide complementary to the region of A2 comprising the attachment site, said oligonucleotide comprising one or more fluorophores arranged such that their fluorescence is quenched by their proximity to each other or by one or more fluorescence quenchers;

[0218] -Double-strand specific DNA digesting enzyme;

[0219] Therein, the labeled oligonucleotide is digested in the presence of A2, so that the fluorophores are separated from each other or from their corresponding quenchers, and the fluorescent signal, and thus the presence of A2, is detectable.

[0220] In some embodiments, the fluorophore and quencher can be as previously described.

[0221] In some embodiments, the double-stranded specific DNA digestion enzyme is an exonuclease. In another embodiment, it is a polymerase with proofreading activity. In another embodiment, the second reaction mixture comprises a mixture of one or more of: an exonuclease or a polymerase with proofreading activity.

[0222] In some embodiments, the double-strand-specific DNA digesting enzyme is a hot-start enzyme.

[0223] In some embodiments, the double-strand-specific DNA digesting enzyme has reduced activity at the temperature at which the pyrophosphorolysis reaction of the method occurs.

[0224] In some embodiments, the double-strand-specific DNA digesting enzyme is inactive at the temperature at which the pyrophosphorolysis reaction of the method occurs.

[0225] In an alternative embodiment, the second reaction mixture further comprises:

[0226] - an oligonucleotide complementary to the region of A2 comprising the attachment site, said oligonucleotide comprising one or more fluorophores arranged such that their fluorescence is quenched by their proximity to each other or by one or more fluorescence quenchers;

[0227] -Double-strand specific DNA digesting enzyme;

[0228] Therein, the labeled oligonucleotide is digested in the presence of A2, so that the fluorophores are separated from each other or from their corresponding quenchers, and the fluorescent signal, and thus the presence of A2, is detectable.

[0229] In some embodiments, the fluorophore and quencher can be as previously described.

[0230] In some embodiments, the double-stranded specific DNA digestion enzyme is an exonuclease. In another embodiment, it is a polymerase with proofreading activity. In another embodiment, the second reaction mixture comprises a mixture of one or more of: an exonuclease or a polymerase with proofreading activity.

[0231] In some embodiments, the double-strand-specific DNA digesting enzyme is a hot-start enzyme.

[0232] In some embodiments, the double-strand-specific DNA digesting enzyme has reduced activity at the temperature at which the pyrophosphorolysis reaction of the method occurs.

[0233] In some embodiments, the double-strand-specific DNA digesting enzyme is inactive at the temperature at which the pyrophosphorolysis reaction of the method occurs.

[0234] In some embodiments, the first reaction mixture or the second reaction mixture further comprises one or more partially double-stranded DNA constructs, wherein each construct comprises one or more fluorophores and one or more quenchers. In some embodiments, when the construct is partially double-stranded, the one or more fluorophores and the one or more quenchers are located in a position close enough to each other so that sufficient quenching of the one or more fluorophores occurs.

[0235] In some embodiments, the construct is a DNA strand having a self-complementary region looped back on itself.

[0236] In some embodiments, the construct includes one primer of a primer pair.

[0237] In some embodiments, the first reaction mixture or the second reaction mixture further comprises another primer of the primer pair.

[0238] In some embodiments, the portion of the single-stranded segment of the construct is hybridized with A2 and extended by a DNA polymerase for A2. In some embodiments, another primer of the primer pair is then hybridized to the extended construct. The primer is then extended for the construct, displacing the self-complementary region. Therefore, one or more fluorophores and one or more dyes are fully separated to detect fluorescent signals, indicating the presence of A2 in the reaction mixture.

[0239] In such an embodiment, the construct may be referred to as a Sunrise Primer.

[0240] In some embodiments, the construct comprises two separate DNA strands.

[0241] In some embodiments, a portion of the single-stranded segment of the construct hybridizes with A2 and is extended by a DNA polymerase for A2. In some embodiments, the other primer of the primer pair then hybridizes to the extended construct. Then, the primer extends for the construct in the direction of the double-stranded segment, displacing the shorter chain in the DNA chain, and thus one or more fluorophores and one or more dyes are fully separated to detect a fluorescent signal indicating the presence of A2 in the reaction mixture.

[0242] In such embodiments, the construct may be referred to as a Molecular Zipper.

[0243] Those skilled in the art will appreciate that for both sunrise primers and molecular zippers, the one or more fluorophores and one or more quencher pairs may be located at different positions within each respective construct. The 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 extension and strand displacement do not occur.

[0244] In some embodiments, prior to step (a) of the method, any RNA template present in the sample is used to form the corresponding DNA sequence.

[0245] In some embodiments, this is accomplished by using a reverse transcriptase and appropriate nucleotides.

[0246] In some embodiments, formation of DNA from any RNA template present occurs simultaneously with any pre-amplification by PCR of nucleic acids present in the sample.

[0247] In some embodiments, the formation of DNA from any RNA template present occurs in separate steps from any pre-amplification of nucleic acids present in the sample by PCR.

[0248] In some embodiments of any of the previously or subsequently described methods, the RNA present in the sample is not converted to a corresponding DNA sequence. In such embodiments, A0 is subjected to pyrophosphorolysis against the RNA sequence to form a partially digested chain A1, and the method is then performed as previously or subsequently described.

[0249] In some embodiments of any of the previously or subsequently described methods, one or more reaction mixtures can be combined.

[0250] In some embodiments, the first reaction mixture further comprises a ligation probe oligonucleotide C, and the partially digested strand A1 is ligated at the 3' end to the 5' end of C, while in another embodiment, A1 is circularized by ligating its 3' and 5' ends;

[0251] In each case oligonucleotide A2 was generated.

[0252] In one embodiment, the attachment of A1 occurs:

[0253] during step (a); or

[0254] during step (b); or

[0255] Between steps (a) and (b).

[0256] In one embodiment, A1 is optionally extended in the 5'-3' direction prior to ligation.

[0257] In some embodiments, such optional extension and ligation are performed to the target oligonucleotide, while in another embodiment, they are performed by adding another splint 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 to A1 due to a 3' end modification or by a nucleotide mismatch between the 3' end of D and the corresponding region of A1.

[0258] In some embodiments, the ligation probe C has a 5' region that is complementary to at least a portion of the 5' terminal region of the splint oligonucleotide D or to the target oligonucleotide. By this method, a second intermediate product is formed, in which the A2 chain includes A1, C and optionally an intermediate region formed by extending A1 in the 5'-3' direction to connect with the 5' end of C. In such an embodiment, the primers used in step (c) (see below) are selected to amplify at least one region of A2, including the site where ligation of A1 to C occurs. In this embodiment, we found that it is advantageous to include a 3' blocking group on C so that a 3'-5' nuclease can be used to digest any unligated A1 before amplification. Suitable polymerases that can be used for pre-ligation extension include, but are not limited to, HemoKlenTaq, Mako, and Stoffel fragments.

[0259] In some embodiments, the first reaction mixture further comprises a phosphatase or phosphohydrolase to remove by hydrolysis the nucleoside triphosphates produced by the pyrophosphorolysis reaction, thereby ensuring that the pyrophosphorolysis reaction can proceed and is not out-competed with the forward polymerization reaction.

[0260] In some embodiments, prior to or during step (b), the product of the previous step is treated with a pyrophosphatase to hydrolyze the pyrophosphate ions, preventing further pyrophosphorolysis from occurring and favoring the forward polymerization reaction.

[0261] In some embodiments, prior to or during step (b), the product of the previous step is treated with an exonuclease.

[0262] In some embodiments, the enzyme that performs the pyrophosphorolysis of A0 to form partially digested chain A1 also amplifies A2. One skilled in the art will recognize that there are many such enzymes.

[0263] Amplicon is detected, and the information obtained is used to infer whether the polynucleotide target sequence exists in the original analyte and / or the property associated with it. For example, in this way, the target sequence unique to cancerous tumor cells can be detected with reference to the specific SNP being sought. In another embodiment, the target sequence unique to a virus or bacterial genome (including its new mutation) can be detected. The method for many detection amplicon or recognition zone can be used, including for example oligonucleotide binding dyes, sequence-specific molecular probes such as fluorescently labeled molecular beacons or hairpin probes. Alternatively, the direct sequencing of the A2 amplicon can be carried out using one of the direct sequencing methods adopted or reported in this area. When using oligonucleotide binding dyes, fluorescently labeled beacons or probes, it is convenient to detect amplicon using a source (laser, LED, lamp, etc.) and a photodetector that includes stimulating electromagnetic radiation, and the photodetector is arranged to detect the emitted fluorescence and generate a signal including a data stream therefrom, and the data stream can be analyzed by a microprocessor or a computer using a specially designed algorithm.

[0264] 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 caused by the generation of the amplicon of A2 is used to infer the concentration of the target sequence in the analyte. In one embodiment, the last step of the method further comprises the following steps:

[0265] i. labeling the product of step (b) using one or more oligonucleotide fluorescent binding dyes or molecular probes;

[0266] ii. measuring the fluorescence signal of the product;

[0267] iii. exposing the product to a set of denaturing conditions; and

[0268] The polynucleotide target sequence in the analyte is identified by monitoring the change in the fluorescent signal of the product during exposure to denaturing conditions.

[0269] In some embodiments, more than one probe A0 is used, each probe A0 is selective for a different target sequence, and each probe A0 comprises a recognition region, and is also characterized in that the amplicon of A2 comprises this recognition region, and thus the target sequence present in the analyte is inferred by detecting the recognition region. In such embodiments, detection of the recognition region is performed using a molecular probe or by sequencing.

[0270] In some embodiments, one or more nucleic acid analytes are split into more than one reaction volume, each volume having one or more probe oligonucleotides A0 introduced to detect a different target sequence.

[0271] In some embodiments, where different probes AO are used, the different probes AO contain one or more common priming sites, allowing amplification using a single primer or set of primers.

[0272] In another aspect of the invention, a method for identifying a target polynucleotide sequence in a given nucleic acid analyte is provided, characterized in that the steps of any previous embodiment of the invention, wherein more than one copy of A2 or the region of A2 is labeled using one or more oligonucleotide fluorescent binding dyes or molecular probes. These more than one copy of the fluorescent signal is measured, and more than one copy is exposed to a set of denaturing conditions. The target polynucleotide sequence is identified by monitoring the changes in the fluorescent signal of more than one copy during exposure to denaturing conditions.

[0273] In some embodiments, denaturing conditions can be provided by changing the temperature, for example, raising the temperature to a point where the double strands begin to dissociate. Additionally or alternatively, denaturing conditions can also be provided by changing the pH to make the conditions acidic or alkaline, or by adding additives or reagents such as strong acids or bases, concentrated inorganic salts, or organic solvents such as alcohols.

[0274] In another aspect of the present invention, there is provided the use of the above method to screen mammalian subjects, especially human patients, for the presence of infectious diseases, cancer or to generate companion diagnostic information.

[0275] In another aspect of the present invention, a control probe for the above method is provided. Embodiments of the present invention include those that illustrate the presence of one or more specific target sequences by generating a fluorescent signal. In such an embodiment, there may inevitably be a signal level generated by non-target DNA present in the sample. For a given sample, the background signal is later than the start time of the "real" signal, but such a start may be different between samples. Therefore, accurately detecting the presence of one or more than one target sequence at low concentrations depends on knowing what signal is expected in the absence of the target sequence. For artificial samples (contrived sample), references are available, but for true "blind" samples from patients, this is not the case. The control probe (E0) is used to determine the expected background signal characteristics of each assay probe. The control probe targets sequences that are not expected to be present in the sample, and the signal generated from the probe can then be used to infer the expected rate of signal generation from the sample in the absence of the target sequence.

[0276] Thus, there is provided a method for detecting a target polynucleotide sequence in a given nucleic acid analyte according to any of the aforementioned methods, characterized by the following steps:

[0277] a. using a separate aliquot of the sample or in the same aliquot and using a second detection channel, repeating the steps of the method subsequently or simultaneously using a second single-stranded probe oligonucleotide E0 having a 3' terminal region that is at least partially mismatched with the target sequence;

[0278] b. inferring the background signal expected to be generated from A0 in the absence of any target analyte in the sample; and

[0279] c. Inferring the presence or absence of the polynucleotide target sequence in the analyte by comparing the expected background signal inferred in (a) with the actual signal observed in the presence of the target analyte.

[0280] In some embodiments, the control probe (E0) and A0 are added to different parts of the sample, while in another embodiment, E0 and A0 are added to the same part 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, correction of the background signal can include subtracting the signal observed from E0 from the signal observed from A0, or calibrating the signal observed from A0 by using a calibration curve of the relative signals generated by A0 and E0 under different conditions.

[0281] In some embodiments, one EO can be used to calibrate all assay probes that may be produced.

[0282] In some embodiments, a separate E0 can be used to calibrate each amplicon of the sample DNA generated in the initial amplification step. Each amplicon can contain more than one mutation / target sequence of interest, but a single E0 is sufficient to calibrate all assay probes for a single amplicon.

[0283] In another embodiment, a separate E0 can be used for each target sequence. For example, if a C>T mutation is targeted, then an E0 can be designed that targets a C>G mutation at the same site that is not known to be present in patients. The signal curves generated by the E0 under various conditions can be evaluated in calibration reactions, and these data are used to infer the expected signal from the assay probe targeting the C>T variant when the variant is not present.

[0284] exist Figures 17 to 20 Some embodiments of the method of the present invention can be seen in .

[0285] exist Fig.17In the present invention, a single-stranded probe oligonucleotide A0 is annealed to a target polynucleotide sequence to produce a first intermediate product that is at least partially double-stranded and in which the 3' end of A0 forms a double-stranded complex with the target polynucleotide sequence. In this simplified embodiment of the present invention, there are two A0 molecules and one target polynucleotide sequence to illustrate how A0 that is not annealed to the target does not participate in the additional 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' chemical blocking group, a common priming sequence, and a barcode region.

[0286] The partially double-stranded first intermediate undergoes pyrophosphorolysis in the 3'-5' direction from the 3' end of A0 in the presence of pyrophosphorolysis enzyme to produce partially digested strand A1, analyte, and undigested A0 molecules that have not annealed to the target.

[0287] exist Fig.18 In the example, A1 is annealed to a single-stranded trigger oligonucleotide B, and the A1 strand is extended in the 5'-3' direction against B to produce oligonucleotide A2. In this illustrative example, trigger oligonucleotide B has a 5' chemical block. Any undigested A0 anneals to trigger oligonucleotide B, however it cannot be extended in the 5'-3' direction against B to produce a sequence that is a target for the subsequent part of the method. In this example, A2 is primed with at least one single-stranded primer oligonucleotide, and more than one copy of A2 or a region of A2 is produced.

[0288] exist Fig.19 In the example of the present invention, A1 is annealed to the splint oligonucleotide D and then cyclized by ligating its 3' and 5' ends. The now cyclized A2 is primed with at least one single-stranded primer oligonucleotide and more than one copy of A2 or a region of A2 is produced. In this illustrative example, the splint oligonucleotide D cannot be extended against A1 due to a 3'-modification (chemical modification in this example) or by a nucleotide mismatch between the 3' end of D and the corresponding region of A2.

[0289] exist Fig. 20 In the embodiment, the 3' region of the splint oligonucleotide D anneals to the 3' region of A1, while the 5' region of the splint oligonucleotide D anneals to the 5' region of the ligation probe C. Thus, a second intermediate product A2 is formed, which includes A1, C, and optionally an intermediate region formed by A1 extending in the 5'-3' direction to connect to the 5' end of C. In this illustrative example, the ligation probe C has a 3' chemical blocking group so that a 3'-5' exonuclease can be used to digest any unligated A1.

[0290] A2 is primed with at least one single-stranded primer oligonucleotide, and more than one copy of A2 or a region of A2 is produced.

[0291] The specificity of the inventive method 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 to promote AO to anneal only to the target polynucleotide sequence rather than the wild-type. Alternatively or additionally, blocking oligonucleotides can be used to improve the specificity of polymerase chain reaction (PCR) to prevent the amplification of any wild-type sequence present. Commonly used techniques are to design a kind of oligonucleotides, which anneal between PCR primers and can not be replaced or digested by PCR polymerase. Oligonucleotides are designed to anneal with non-target (usually healthy) sequences, and mismatch (usually differing by a single base) with target (mutated) sequences. This mismatch causes different melting temperatures for two sequences, and oligonucleotides are designed to keep annealing with non-target sequences at the PCR extension temperature, while dissociating from the target sequence.

[0292] The blocking oligonucleotide may typically have modifications to prevent its digestion by the exonuclease activity of the PCR polymerase, or to increase the difference in melting temperature between the target and non-target sequences.

[0293] Incorporation of locked nucleic acid (LNA) or other modifications that alter the melting temperature in blocking oligonucleotides can significantly increase the difference in melting temperatures between oligonucleotides for target and non-target sequences.

[0294] Therefore, embodiments of the present invention are provided in which blocking oligonucleotides are used. Blocking oligonucleotides must tolerate pyrophosphorolysis (PPL) reactions to ensure that they are not digested or replaced. This can be achieved in a variety of different ways, for example by mispairing at the 3' end or by modifications such as phosphorothioate bonds or spacers.

[0295] In such an embodiment or aspect of the invention using a blocking oligonucleotide, the method for detecting a target polynucleotide sequence in a given nucleic acid analyte is characterized in that the single-stranded blocking oligonucleotide is annealed to at least a subset of non-target polynucleotide sequences before or during the same step of annealing the analyte target sequence to the single-stranded probe oligonucleotide A0 to produce a first intermediate product that is at least partially double-stranded and in which the 3' end of A0 forms a double-stranded complex with the analyte target sequence.

[0296] In some embodiments, the blocking oligonucleotide becomes tolerant to pyrophosphorolysis by mispairing at its 3' end. In another embodiment, the blocking oligonucleotide becomes tolerant by the presence of a 3'-blocking group. In another embodiment, the blocking oligonucleotide becomes tolerant by the presence of a spacer or other internal modifications. In another embodiment, the blocking oligonucleotide includes both modifications that increase the melting temperature or modified nucleotide bases and becomes tolerant to pyrophosphorolysis.

[0297] "Phosphatase" mentioned herein refers to any enzyme or its functional fragment having the ability to remove nucleoside triphosphates produced by the method of the present invention by hydrolysis. This includes any enzyme or its functional fragment having the ability to split phosphate monoesters into phosphate ions and alcohols.

[0298] The "pyrophosphatase" mentioned herein refers to any enzyme or a functional fragment thereof that has the ability to catalyze the conversion of one pyrophosphate ion into two phosphate ions.

[0299] This also includes inorganic pyrophosphatases and inorganic bisphosphatases. A non-limiting example is thermostable inorganic pyrophosphatase (TIPP).

[0300] In some embodiments, modifications of any previously described or subsequently described embodiments are provided, wherein the use of pyrophosphatase is optional.

[0301] In one embodiment, A1 is circularized against the analyte target sequence. In this embodiment, the region of the target revealed by stepwise digestion of A0 from the 3' end of A0 in the 3'-5' direction to form A1 is complementary to the 5' end of A0 / A1. In this embodiment, a ligase can be used to connect the 3' end and the 5' end of A1 to form the circularized oligonucleotide A2. This is useful, for example, Fig.15 In one embodiment, the 5' end of A0 / A1 is complementary to the target in a region of 5-50 nucleotides in length. In one embodiment, it is 5-25 nucleotides in length. In one embodiment, it is 5-20 nucleotides in length. In one embodiment, it is 5-15 nucleotides in length. In one embodiment, it is 5-12 nucleotides in length. In one embodiment, it is 5-10 nucleotides in length.

[0302] In an embodiment, a method is provided for diagnosing or monitoring cancer in a patient, comprising:

[0303] - Extracting DNA or RNA from plasma or serum of a patient's blood;

[0304] - introducing one or more DNA or RNA analytes into a first reaction mixture comprising:

[0305] i. single-stranded probe oligonucleotide A0;

[0306] ii. pyrophosphatase; and

[0307] iii. Ligase;

[0308] wherein the analyte anneals with the single-stranded probe oligonucleotide A0 to produce a first intermediate product which is at least partially double-stranded and wherein the 3′ end of A0 forms a double-stranded complex and A0 is pyrophosphorolyzed in the 3′-5′ direction from the 3′ end to produce an at least partially digested strand A1 and A1 undergoes ligation to form A2;

[0309] - detecting a signal derived from a 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 inferring the presence or absence of cancer in the patient therefrom.

[0310] In an embodiment, a method for monitoring cancer in a patient is provided, comprising:

[0311] - Extracting DNA or RNA from plasma or serum of a patient's blood;

[0312] - introducing one or more DNA or RNA analytes into a first reaction mixture comprising:

[0313] i. single-stranded probe oligonucleotide A0;

[0314] ii. pyrophosphatase; and

[0315] iii. Ligase;

[0316] wherein the analyte anneals with the single-stranded probe oligonucleotide A0 to produce a first intermediate product which is at least partially double-stranded and wherein the 3′ end of A0 forms a double-stranded complex and A0 is pyrophosphorolyzed in the 3′-5′ direction from the 3′ end to produce an at least partially digested strand A1 and A1 undergoes ligation to form A2;

[0317] - detecting a signal derived from a 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 inferring the progression or remission of the patient's cancer therefrom.

[0318] In an embodiment, a method is provided for monitoring a patient's response to one or more cancer treatments, comprising:

[0319] - Extracting DNA or RNA from plasma or serum of blood from patients receiving one or more cancer treatments;

[0320] - introducing one or more DNA or RNA analytes into a first reaction mixture comprising:

[0321] i. single-stranded probe oligonucleotide A0;

[0322] ii. pyrophosphatase; and

[0323] iii. Ligase;

[0324] wherein the analyte anneals with the single-stranded probe oligonucleotide A0 to produce a first intermediate product which is at least partially double-stranded and wherein the 3′ end of A0 forms a double-stranded complex and A0 is pyrophosphorolyzed in the 3′-5′ direction from the 3′ end to produce an at least partially digested strand A1 and A1 undergoes ligation to form A2;

[0325] - detecting a signal derived from a 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 inferring the patient's response to one or more cancer treatments therefrom.

[0326] In an embodiment, a method for screening a patient is provided, comprising:

[0327] - Extracting DNA or RNA from plasma or serum of a patient's blood;

[0328] - introducing one or more DNA or RNA analytes into a first reaction mixture comprising:

[0329] i. single-stranded probe oligonucleotide A0;

[0330] ii. pyrophosphatase; and

[0331] iii. Ligase;

[0332] wherein the analyte anneals with the single-stranded probe oligonucleotide A0 to produce a first intermediate product which is at least partially double-stranded and wherein the 3′ end of A0 forms a double-stranded complex and A0 is pyrophosphorolyzed in the 3′-5′ direction from the 3′ end to produce an at least partially digested strand A1 and A1 undergoes ligation to form A2;

[0333] - detecting a signal derived from a 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 inferring the presence or absence of one or more cancers in the patient.

[0334] In an embodiment, a method for non-invasive prenatal testing (NIPT) is provided, comprising:

[0335] - Extraction of DNA or RNA from plasma or serum of maternal blood of pregnant patients;

[0336] - introducing one or more DNA or RNA analytes into a first reaction mixture comprising:

[0337] i. single-stranded probe oligonucleotide A0;

[0338] ii. pyrophosphatase; and

[0339] iii. Ligase;

[0340] wherein the analyte anneals to the single-stranded probe oligonucleotide A0 to produce a first intermediate product that is at least partially double-stranded and wherein the 3′ end of A0 forms a double-stranded complex and A0 is pyrophosphorolyzed in the 3′-5′ direction from the 3′ end to produce an at least partially digested strand A1 and A1 undergoes ligation to form A2;

[0341] - detecting a signal derived from a 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 inferring the presence or absence of one or more fetal abnormalities therefrom.

[0342] In an embodiment, a method for screening a patient is provided, comprising:

[0343] - Extracting DNA or RNA from plasma or serum of a patient's blood;

[0344] - introducing one or more DNA or RNA analytes into a first reaction mixture comprising:

[0345] i. single-stranded probe oligonucleotide A0;

[0346] ii. pyrophosphatase; and

[0347] iii. Ligase;

[0348] wherein the analyte anneals with the single-stranded probe oligonucleotide A0 to produce a first intermediate product which is at least partially double-stranded and wherein the 3′ end of A0 forms a double-stranded complex and A0 is pyrophosphorolyzed in the 3′-5′ direction from the 3′ end to produce an at least partially digested strand A1 and A1 undergoes ligation to form A2;

[0349] - detecting a signal derived from a 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 inferring the presence or absence of one or more cancers in the patient.

[0350] In an embodiment, a method for monitoring a transplant recipient patient is provided, comprising:

[0351] - Extracting DNA or RNA from plasma or serum of the transplant recipient patient's blood;

[0352] - introducing one or more DNA or RNA analytes into a first reaction mixture comprising:

[0353] i. single-stranded probe oligonucleotide A0;

[0354] ii. pyrophosphatase; and

[0355] iii. Ligase;

[0356] wherein the analyte anneals to the single-stranded probe oligonucleotide A0 to produce a first intermediate product that is at least partially double-stranded and wherein the 3′ end of A0 forms a double-stranded complex and A0 is pyrophosphorolyzed in the 3′-5′ direction from the 3′ end to produce an at least partially digested strand A1 and A1 undergoes ligation to form A2;

[0357] - detecting a signal derived 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 inferring the presence or absence of DNA or RNA from the transplanted organ.

[0358] 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:

[0359] (a) a single-stranded probe oligonucleotide A0, which is capable of forming a first intermediate product with a target polynucleotide sequence, wherein the intermediate product is at least partially double-stranded;

[0360] (b) ligase;

[0361] (c) a pyrophosphorylase capable of digesting the first intermediate from the end of A0 in the 3'-5' direction to produce a partially digested chain A1;

[0362] (d) at least one single-stranded primer oligonucleotide that is substantially complementary to a portion of A0;

[0363] (e) an amplification enzyme; and

[0364] (f) A suitable buffer.

[0365] In one embodiment, the kit further comprises a source of pyrophosphate ions.

[0366] Suitable sources of pyrophosphate ions are described previously.

[0367] In some embodiments, the kits also include appropriate positive and negative controls.

[0368] In some embodiments, the kit may also include one or more control probes (E0) as previously described.

[0369] In some embodiments, the kit may also include one or more blocking oligonucleotides as previously described.

[0370] In some embodiments, the kit may also include one or more control probes (E o ) and one or more blocking oligonucleotides.

[0371] In some embodiments, the 5' end of A0 is rendered resistant to 5'-3' exonuclease digestion, and the kit may further include a 5'-3' exonuclease.

[0372] In some embodiments, the kit may further include a ligation probe oligonucleotide C.

[0373] In some embodiments, the kit may further include a splint oligonucleotide D.

[0374] In some embodiments, a kit can include both C and D.

[0375] Ligation probe C may contain a 3' modification or an internal modification that protects it from 3'-5' exonuclease digestion.

[0376] D may comprise 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.

[0377] In some embodiments, D may not be able to extend toward A1 due to a 3' modification or by a mismatch between the 3' end of D and the corresponding region of A1 or C.

[0378] In some embodiments, the kit may also include dNTPs, a polymerase, and a suitable buffer for initial amplification of target polynucleotide sequences present in the sample.

[0379] In some embodiments, the kit may further include a high-fidelity polymerase that incorporates dUTP, dUTP, and uracil-DNA N-glycosylase (UDG).

[0380] In some embodiments, the kit may further include a phosphatase or a phosphohydrolase.

[0381] In some embodiments, the kit may further include a pyrophosphatase. The pyrophosphatase may be hot-start.

[0382] In some embodiments, the kit may further include a protease.

[0383] In some embodiments, the kits may also include one or more oligonucleotide binding dyes or molecular probes.

[0384] In some embodiments, the kit may also include more than one AO, each AO being selective for a different target sequence, and each AO comprising a recognition region.

[0385] In some embodiments, the amplification enzyme and the pyrophosphatase of (e) are the same.

[0386] The kit can also include purification equipment and reagents for separating and / or purifying a portion of polynucleotides after treatment as described herein. Suitable reagents are well known in the art and include gel filtration columns and washing buffer. Other suitable reagents include magnetic beads and silica columns.

[0387] In one embodiment of the present invention, there is provided a device comprising:

[0388] at least one fluid passage between the first region, the second region, and the third region, wherein the first region comprises one or more apertures, wherein each aperture comprises:

[0389] dNTPs;

[0390] at least one single-stranded primer oligonucleotide;

[0391] an amplification enzyme for initially amplifying the DNA present in the sample; and

[0392] wherein the second region comprises one or more apertures, wherein each aperture comprises:

[0393] A single-stranded probe oligonucleotide A0, which is capable of forming a first intermediate product with the target polynucleotide sequence, wherein the intermediate product is at least partially double-stranded;

[0394] a pyrophosphorolyase capable of digesting the first intermediate in the 3'-5' direction from the end of A0 to produce a partially digested chain A1; and

[0395] wherein the third region comprises one or more apertures, wherein each aperture comprises:

[0396] dNTPs;

[0397] Buffer;

[0398] Amplification enzymes;

[0399] means for detecting a signal originating from A2 or a portion thereof, or more than one copy of A2 or more than one copy of a portion thereof; and

[0400] The wells of the second region or the wells of the third region further comprise at least one single-stranded primer oligonucleotide substantially complementary to a portion of A0.

[0401] In some embodiments, the means for detecting a signal is located within one or more wells of the third region.

[0402] In some embodiments, the means for detecting a signal is located within a third region of the device.

[0403] In some embodiments, the means for detecting a signal is located within an adjacent area of ​​the device.

[0404] In some embodiments, the dNTP of each well of the first region may be dUTP, dGTP, dATP, and dCTP, and each well may further include a high-fidelity polymerase and uracil-DNA N-glycosylase (UDG) that incorporates dUTP.

[0405] In some embodiments, the dNTP of each well of the third region may be dUTP, dGTP, dATP, and dCTP, and each well may further include a high-fidelity polymerase and uracil-DNA N-glycosylase (UDG) that incorporates dUTP.

[0406] In some embodiments, each well of the second region may further include a source of pyrophosphate ions.

[0407] In some embodiments, the 5' end of A0 is rendered resistant to digestion by a 5'-3' exonuclease, and the pore of the second region may further comprise a 5'-3' exonuclease.

[0408] In some embodiments, each well of the second region or the third region may further include a ligase and a ligation probe oligonucleotide C or a splint oligonucleotide D.

[0409] Ligation probe C may contain a 3' modification or an internal modification that protects it from 3'-5' exonuclease digestion.

[0410] The splint oligonucleotide D may comprise 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.

[0411] D may not be able to extend toward A1 due to 3' modifications or by mismatches between the 3' end of D and the corresponding regions of A1 or C.

[0412] In some embodiments, the dNTPs can be hot start, and each well of the second region can also include a phosphatase or phosphohydrolase.

[0413] In some embodiments, each well of the second region can also include pyrophosphatase.

[0414] In some embodiments, each well of the third region may further include pyrophosphatase.

[0415] In some embodiments, the pyrophosphatase can be hot-start.

[0416] In some embodiments, each well of the third region may also include one or more oligonucleotide binding dyes or molecular probes.

[0417] In some embodiments, each well of the second region can include at least one or more different AOs that are selective for a target sequence that includes the recognition region.

[0418] In some embodiments, the amplification enzyme and the pyrophosphatase in the second region can be the same.

[0419] In some embodiments, there may be a fourth region comprising one or more wells, wherein each well may comprise a protease, and wherein the fourth region may be located between the first region and the second region.

[0420] In some embodiments, the second region and the third region of the device may be combined such that the apertures in the second region further comprise:

[0421] dNTPs;

[0422] Buffer;

[0423] an amplification enzyme; and

[0424] Means for detecting a signal originating from A2 or a portion thereof, or more than one copy of A2 or more than one copy of a portion thereof.

[0425] In some embodiments, the means for detecting a signal is located within one or more wells of the second region.

[0426] In some embodiments, the means for detecting a signal is located within the second region of the device.

[0427] In some embodiments, the means for detecting a signal is located within an adjacent area of ​​the device.

[0428] In some embodiments, the first region can be fluidically connected to the sample container via a fluid interface.

[0429] In some embodiments, heating and / or cooling elements may be present in one or more regions of the device.

[0430] In some embodiments, heating and / or cooling may be applied to one or more regions of the device.

[0431] In some embodiments, each region of the device may independently contain at least 100 or 200 wells.

[0432] In some embodiments, each region of the device can independently contain between about 100 and 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 or more holes. The holes can be of any shape, and their locations can be arranged in any format or pattern on the substrate.

[0433] In some embodiments, the well substrate may be composed of metal (eg, as non-limiting examples, gold, platinum, or nickel alloys), ceramic, glass, or other PCR compatible polymeric materials or composite materials.The well substrate includes more than one well.

[0434] In some embodiments, the hole can be formed as a blind hole or a through hole in the hole substrate. For example, the hole can be produced in the hole substrate by laser drilling (e.g., excimer laser or solid state laser), ultrasonic embossing, hot embossing lithography, electroforming nickel mold, injection molding and injection molding.

[0435] In some embodiments, the volume of a single pore may be in the range of 0.1 nl to 1500 nl. In one embodiment, 0.5 nL to 50 nL. Each well can have about 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, 25 nL. , 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.

[0436] In some embodiments, the hole dimensions can have any shape, for example, circular, elliptical, square, rectangular, oval, hexagonal, octagonal, conical, and other shapes known to those skilled in the art.

[0437] In some embodiments, the hole shape can have a cross-sectional area that varies along an axis. For example, a square hole can taper from a first size to a second size that is a fraction of the first size.

[0438] In some embodiments, the well dimensions may be square, with approximately equal diameter and depth.

[0439] In some embodiments, the walls defining the apertures may be non-parallel.

[0440] In some embodiments, the walls defining the wells may converge to a point.Well dimensions may be derived from the total volume of the well substrate.

[0441] In some embodiments, the pore depth may be in the range of 25 μm to 1000 μm.

[0442] In one embodiment, the holes may have a depth of 25μm, 50μm, 75μm, 100μm, 150μm, 200μm, 250μm, 300μm, 350μm, 400μm, 450μm, 500μm, 550μm, 600μm, 650μm, 700μm, 750μm, 800μm, 850μm, 900μm, 950μm, or 1000μm.

[0443] In some embodiments, the pore diameter may be in the range of about 25 μm to about 500 μm.

[0444] In some embodiments, the holes 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.

[0445] In some embodiments, portions of one or more regions of the device may be modified to promote or prevent fluid adhesion.Surfaces defining pores may be coated with a hydrophilic material (or modified to be hydrophilic) and thereby promote retention of fluids.

[0446] In some embodiments, portions of one or more regions of the device may be coated with a hydrophobic material (or modified to be hydrophobic) and thereby prevent fluid from being retained thereon. Those skilled in the art will appreciate that other surface treatments may be performed so that fluid is preferably retained within the pores rather than on the upper surface in order to facilitate drainage of excess fluid.

[0447] In some embodiments, the holes of the well substrate can be patterned to have a simple geometric pattern of aligned rows and columns, or a pattern arranged in a diagonal or hexagonal pattern. In one embodiment, the holes of the well substrate can be patterned to have a complex geometric pattern, such as a chaotic pattern or an iso-geometric design pattern.

[0448] In some embodiments, the wells can be geometrically separated from one another and / or have a large depth to width ratio to help prevent cross contamination of reagents.

[0449] In some embodiments, the device may include one or more auxiliary regions that can be used to provide a process fluid, such as an oil or other chemical solution, to one or more regions of the device. Such auxiliary regions may be fluidly connected to one or more regions of the device through one or more membranes, valves, and / or pressure-separable substrates (i.e., materials that rupture when subjected to a predetermined amount of pressure from a fluid in an auxiliary region or adjacent portion of a fluid path) such as a metal foil or film.

[0450] In some embodiments, the fluid passage of the device may include extensive tortuosity. A tortuous path between an inlet passage of the fluid passage and one or more regions of the device helps control and process fluid processing. A tortuous path can help reduce the formation of bubbles that would interfere with the flow of oil through the fluid passage.

[0451] In some embodiments, the device may also include a gas permeable membrane that allows gas to be discharged from the holes of one or more regions of the device without allowing fluid to pass through. The gas permeable membrane may be adhered to the hole substrate of the device by a gas permeable adhesive. In one embodiment, the membrane may be made of polydimethylsiloxane (PDMS) and have a thickness in the range of 20 μm-1000 μm. In some embodiments, the membrane may have a thickness in the range of 100 μm-200 μm.

[0452] In some embodiments, all or part of the well substrate may contain a conductive metal portion (eg, gold) to enable heat transfer from the metal to the well. In one embodiment, the inner surface of the well may be coated with a metal to enable heat transfer.

[0453] In some embodiments, after appropriate reagents have filled the wells of one or more regions of the device, an isolation oil or thermally conductive liquid may be applied to the device to prevent crosstalk.

[0454] In some embodiments, the pores of one or more regions of the device can be shaped to taper from a large diameter to a smaller diameter, similar to a cone. Conical pores with sloping walls enable the use of non-contact deposition methods (e.g., ink jetting) of reagents. The cone shape also aids in drying and has been found to prevent bubbles and leaks when a gas permeable membrane is present.

[0455] In some embodiments, the pores of one or more regions of the device can be filled by advancing a sample fluid (e.g., by pressure) along the fluid pathway of the device. As the fluid passes through the pores of one or more regions of the device, each pore becomes filled with the fluid, which is retained within the pore primarily by surface tension. As previously described, portions of the pore substrate of the device can be coated with a hydrophilic / hydrophobic substance as desired to promote complete and uniform filling of the pores as the sample fluid passes through.

[0456] In some embodiments, the wells of one or more regions of the device can be "capped" with oil after filling. This can then help reduce evaporation when the well substrate undergoes thermal cycling. In one embodiment, after oil capping, an aqueous solution can fill one or more regions of the device to improve thermal conductivity.

[0457] In some embodiments, a stationary aqueous solution may be pressurized in one or more regions of the device to prevent movement of the fluid and any air bubbles.

[0458] In some embodiments, oils such as mineral oil can be used to isolate the holes in one or more areas of the device and provide thermal conductivity. However, any thermally conductive liquid, such as a fluorinated liquid (e.g., 3M FC-40) can be used. Reference to oil in this disclosure should be understood to include applicable alternatives that will be understood by those skilled in the art.

[0459] In some embodiments, the device may also include one or more sensor components.

[0460] In some embodiments, one or more sensor components may include a charge coupled device (CCD) / complementary metal oxide semiconductor (CMOS) detector coupled to a fiber optic faceplate (FOFP). An optical filter may be layered on top of the FOPF and placed against or adjacent to the well substrate. In one embodiment, the optical filter may be layered (bonded) directly on top of the CCD, with the FOPF placed on top.

[0461] In some embodiments, a hydrating fluid, such as distilled water, may be heated within one of the first or secondary regions so that one or more regions of the device have up to 100% humidity, or at least sufficient humidity to prevent excessive evaporation during thermal cycling.

[0462] In some embodiments, after filling of the device is complete, the well substrate can be heated by an external device in thermal contact with the device to perform thermal cycling of PCR.

[0463] In some embodiments, non-contact heating methods such as RFID, Curie point, induction heating or microwave heating may be used. 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 may be monitored by the sensor arrangements described previously.

[0464] In some embodiments, reagents deposited in one or more wells in one or more regions of a device are deposited in a predetermined arrangement.

[0465] In some embodiments, a method is provided, comprising:

[0466] providing a sample fluid to a fluid channel of a device, wherein the device comprises 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 comprise one or more wells;

[0467] filling the second region with the amplification fluid from the first region such that one or more pores of the second region are covered by the amplification fluid;

[0468] withdrawing amplification fluid from the second region such that one or more pores remain wetted with at least some of the amplification fluid;

[0469] filling the third region with the fluid drawn from the second region such that one or more pores of the third region are covered by the fluid; and

[0470] Fluid is withdrawn from the third chamber such that the one or more pores remain wetted with at least some of the fluid.

[0471] In some embodiments of the method, the fluid channel can be valveless.

[0472] In some embodiments of the method, the extracted second region may be filled with a hydrophobic substance.

[0473] In some embodiments of the method, the extracted third region may be filled with a hydrophobic substance.

[0474] In some embodiments of the method, the hydrophobic substance may be supplied from an oil chamber in fluid communication with the second region and the third region.

[0475] In some embodiments of the method, the sample fluid can travel along the fluid channel in a serpentine manner.

[0476] In some embodiments, the method may further include applying a heating and cooling cycle to one or more of the first region, the second region, or the third region.

[0477] One skilled in the art will understand that when the term "infer" is used, for example, "inferring the presence or absence of a particular sequence" means determining the presence or absence of a particular feature based on the presence or absence of A2, or a copy of A2, or a region of A2, or a copy of a region of A2.

[0478] Various other aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0479] As used herein, "and / or" is considered to be a specific disclosure of each of the 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.

[0480] Unless the context indicates otherwise, the descriptions and definitions of the features listed above are not limited to any particular aspect or embodiment of the invention, and apply equally to all aspects and embodiments described.

[0481] It will be further appreciated by those skilled in the art that, while the invention has been described by way of examples with reference to several embodiments, the invention is not limited to the disclosed embodiments and that alternative embodiments may be constructed without departing from the scope of the invention as defined in the appended claims.

[0482] Example 1 - Simplified Protocol

[0483] For purposes of this and the following sections, embodiments of the present invention are illustrated and referred to as Schemes 1-5, respectively.

[0484] Figure 1 An overview of the different options is provided.

[0485] The following table gives an overview of the time required to execute each scenario:

[0486]

[0487] In one embodiment, TIPP is not present in any of the regimens, methods, kits and / or devices of the invention.

[0488] In one embodiment, a 5'-3' exonuclease is not present in any of the protocols, methods, kits and / or devices of the invention.

[0489] The following table shows an overview of the enzymes used in each protocol:

[0490]

[0491] In one embodiment, the presence of pyrophosphatase is optional.

[0492] In one embodiment, the presence of a 5'-3' exonuclease is optional.

[0493] In one embodiment, the presence of UDG is optional.

[0494]

[0495]

[0496] As can be seen, the inventors have reduced the total number of enzymes required, thereby reducing the cost and complexity of the method. Surprisingly, the inventors have found that moving the addition of the 5'-3' exonuclease from the pre-amplification step to the pyrophosphorolysis / ligation step of the protocol (as in Schemes 3-5) results in Figure 2 Higher fluorescence signals (representing detection of specific target analyte sequences) are shown.

[0497] Example 2: Pyrophosphorolysis (PPL) enzyme

[0498] The inventors have tested the method of Scheme 3 of the present invention using a range of different PPL enzymes, and the results can be seen 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.

[0499] The inventors have tested the method of Scheme 4 of the present invention using a series of different PPL enzymes, the results of which can be seen in FIG4 .

[0500] Example 3: Scheme 1 vs. Scheme 4

[0501] The inventors detected exon 19del_6223 at 0.5%, 0.10% and 0.05% MAF using protocol 1 and protocol 4, which can be seen in Figure 5 It can be seen that the fluorescence peak is larger when protocol 4 is used.

[0502] Example 4: Scheme 4-Sensitivity

[0503] The inventors detected the EGFR exon 20T790M mutation at 0.10%, 0.50% and 1% MAF according to Scheme 4. Figure 6 shown.

[0504] Example 5: Scheme 4 - Is an exonuclease digestion step required during the RCA process?

[0505] The inventors have shown that an 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 Shown is the detection of EGFR exon 20 T790M at 1% MAF in the presence and absence of exonuclease in the RCA step.

[0506] Example 6: Scheme 4 - PPL:RCA Mixing Ratio

[0507] The inventors studied the effect of the PPL:RCA mixing ratio on the signal intensity of 0.5% MAF EGFR exon 20T790M detection, and the results are shown in Figure 8 As can be seen, the 1:2 PPL:RCA mix ratio resulted in the lowest signal intensity, but at the earliest time point. This was followed in time by the 1:4 PPL:RCA mix with greater signal intensity. The greatest signal intensity was observed for the 1:8 PPL:RCA mix at the latest time point of the reaction.

[0508] Example 7: Scheme 4 - Dye Selection

[0509] The inventors investigated whether the dyes used during RCA could be optimized. Fig. 9 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 be run at a lower temperature of 50°C, whereas SybrGreenI requires a higher temperature of 60°C. Scheme 5 requires a lower RCA temperature, which eliminates the addition of proteinase K to the reaction mixture. The amplification enzyme used to prepare at least one single-stranded analyte of nucleic acid comprising a target polynucleotide region for detection using the method of the present invention requires a temperature greater than 50°C to work. The use of 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 prior to RCA.

[0510] Lower RCA temperatures may allow the methods of the invention to be performed in a plate reader rather than qPCR.

[0511] Using Syto82, the response is faster, as in Fig. 9 , and despite the lower total fluorescence of Syto82 - this can be alleviated by using higher concentrations of Syto82 dye.

[0512] Example 8: Scheme 4 - BST LF vs. BST 2.0WS

[0513] The inventors studied the use of two different enzymes, BST LF and BST 2.0WS, for RCA to detect the 0.5% MAF EGFR exon 20 T790M mutation according to protocol 4. This result was shown in Fig.10 , from which it can be seen that the reaction using BST 2.0WS is the fastest. BST 2.0WS is designed to incorporate dUTP, which helps the speed of the reaction. There is a negligible difference between the overall signal intensity achieved by BST LF and BST 2.0WS. According to its description, BST 2.0WS provided by New England Biolabs (NEB) is more stable and active only above 45°C.

[0514] Example 9: Effect of PPL enzyme on signal detection

[0515] The inventors studied the effects of different PPL enzymes on the RCA reaction at different PPL:RCA reaction mixture ratios. The results can be seen 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 ratio of 1:4 PPL:RCA. At a ratio of 1:8 PPL:RCA, BST and Klenow had no effect on the RCA reaction.

[0516] Example 10: Pyrophosphorolysis, ligation specificity for single nucleotide mismatches

[0517] A single-stranded first oligonucleotide 1 (SEQ ID NO: 1) was prepared, which had the following nucleotide sequence:

[0518]

[0519] A single-stranded linker oligonucleotide 2 (SEQ ID NO: 2) was prepared, which had the following nucleotide sequence:

[0520]

[0521] A, C, G and T represent nucleotides that carry the relevant characteristic nucleobases of DNA.

[0522] / 5Phos / represents the 5' terminal phosphate

[0523] * represents phosphorothioate bond

[0524] A set of single-stranded oligonucleotides 3-4 (SEQ ID No: 3-4) was also prepared, which had the following nucleotide sequences in the 5' to 3' direction: 3

[0526] 4

[0528]

[0529] 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 with a single nucleotide mismatch at position 3. SEQ ID 3 and SEQ ID 4 are portions of human EGFR gene with / without C797S mutation, respectively.

[0530] A first reaction mixture is then prepared having a composition corresponding to the composition obtained by the following formula:

[0531] 0.5ul 20x buffer pH 7.0

[0532] 0.25uL 5x buffer pH 8.0

[0533] 0.25uL 5x HF buffer

[0534] 0.2ul oligonucleotide 1, 1000nM

[0535] 0.3uL Oligonucleotide 2, 1000nM

[0536] 1ul oligonucleotide 2 (500nM) or a mixture of oligonucleotides 2 and 3 (500nM and 0.5nM, respectively),

[0537] 0.3U Klenow fragment exo-(NEB)

[0538] 0.01ul inorganic pyrophosphate, 10mM

[0539] 0.0132U apyrase (ex.NEB)

[0540] 1U E. coli DNA ligase (ex.NEB)

[0541] Water to 10ul

[0542] The 20x buffer contains the following mixture:

[0543] 200uL Tris-acetic acid, 1M, pH 7.0

[0544] 342.5ul magnesium acetate aqueous solution, 1M

[0545] 120ul potassium acetate aqueous solution, 5M

[0546] 50ul Triton X-100 surfactant (10%)

[0547] Water to 1 mL

[0548] The 5x buffer contains the following mixture:

[0549] 50ul Trizma Acetic Acid, 1M, pH 8.0

[0550] 25ul magnesium acetate aqueous solution, 1M

[0551] 25ul potassium acetate aqueous solution, 5M

[0552] 50ul Triton X-100 surfactant (10%)

[0553] Water to 1 mL

[0554] Pyrophosphorolysis was then performed by incubating the mixture at 45°C for 15 minutes, followed by circularization of oligonucleotide 1 via ligation, and the resulting product mixture was used in an amplification reaction (Example 11).

[0555] Example 11: Amplification of Circularized Probes

[0556] A pair of single-stranded oligonucleotide primers 1 (SEQ ID NO: 5) and 2 (SEQ ID NO: 6) were prepared, which had the following nucleotide sequences:

[0557] 1:

[0558] 2:

[0559] A, C, G and T represent nucleotides that carry the relevant characteristic nucleobases of DNA.

[0560] A second reaction mixture was then prepared having a composition corresponding to that obtained by the following formulation:

[0561] 3uL 10x Thermopol Buffer

[0562] 3.2U BST 2.0WS

[0563] 0.32uL Oligonucleotide 1, 10uM

[0564] 0.32uL Oligonucleotide 2, 10uM

[0565] 1.125uL Syto82, 30uM

[0566] 0.165U inorganic pyrophosphatase

[0567] 1.2uL dNTP mix, 10mM

[0568] 1.25uL of the reaction mixture in Example 10

[0569] Water to 11.25uL

[0570] The 10x Thermopol buffer contains the following mixture:

[0571] 200uL Tris-HCl pH=8.8, 1M

[0572] 100uL(NH4)2SO4, 1M

[0573] 100uL KCl, 1M

[0574] 20mM MgSO4, 1M

[0575] 10uL 10%

[0576] Water to 1 mL

[0577] The reaction mixture was then incubated at 50°C for 40 minutes, and the resulting reaction products were then analyzed by real-time fluorescence. Fig.12 From this analysis, it can be seen that when both oligonucleotides 3 and 4 are present, the fluorescent signal appears faster in the reaction, indicating that pyrophosphorolysis and ligation of oligonucleotide 3 has already occurred in the first reaction mixture.

[0578] Example 12: Multicolor Detection Using Sunrise Primers

[0579] 1. Target oligonucleotide dilution

[0580] WT oligonucleotide dilution solution consists of the following components;

[0581] 0.5x A7 buffer

[0582] 0.5x Phusion U buffer

[0583] 200 nM WT oligonucleotide (SEQ ID NO:7)

[0584] Total volume: 5uL

[0585] T790M and C797S 1% AF mutant oligonucleotide mixture;

[0586] 0.5x A7 buffer

[0587] 0.5x Phusion U buffer

[0588] 100 nM WT oligonucleotide (SEQ ID NO:7)

[0589] 2nM T790M oligonucleotide (SEQ ID NO:8)

[0590] 2nM C797S_2389 oligonucleotide (SEQ ID NO:9)

[0591] Total volume: 5uL

[0592] WT oligonucleotide (SEQ ID NO:7):

[0593]

[0594] T790M oligonucleotide (SEQ ID NO:8):

[0595]

[0596] C797S_2389 oligonucleotide (SEQ ID NO:9):

[0597]

[0598] 1x A7 composition

[0599] Tris-acetate pH = 8.0 10 mM

[0600] Potassium acetate 25mM

[0601] Magnesium acetate 5mM

[0602] Triton-X 0.01%

[0603] PhusionU Buffer

[0604] The PhusionU buffer composition is not publicly available.

[0605] 2.PPL

[0606] A mixture corresponding to the following was prepared:

[0607] 1xBFF1

[0608] 37.5U / mL Mako DNA polymerase (3'→5'exo-)

[0609] 100U / mL E. coli ligase

[0610] 1.2 U / mL apyrase

[0611] 0.6mM PPi

[0612] 20nM T790M probe

[0613] 20nM C797S_2389 probe

[0614] 30nM T790M splint oligonucleotide

[0615] 30nM C797S_2389 splint oligonucleotide

[0616] 5uL of WT or 1% AF mutant dilution from point 1.

[0617] Total volume 10uL

[0618] This mixture was then incubated at 41°C for 30 minutes.

[0619] 1xBFF1 composition

[0620] Tris-acetate pH = 7.0 10 mM

[0621] Potassium acetate 30 mM

[0622] Magnesium acetate 17.125mM

[0623] Triton-X 0.01%

[0624] T790M probe (SEQ ID NO: 10):

[0625]

[0626] C797S_2389 probe (SEQ ID NO: 11):

[0627]

[0628] Where * represents a phosphorothioate bond

[0629] 3. TIPP

[0630] A mixture corresponding to the following was prepared:

[0631] 1xA7

[0632] 66.6U / mL TIPP

[0633] 10uL of the mixture from point 2.

[0634] Total volume: 20uL

[0635] This mixture was then incubated at 25°C for 5 min and 95°C for 5 min.

[0636] 4. Connect

[0637] A mixture corresponding to the following was prepared:

[0638] 1xA7

[0639] 100U / mL E. coli ligase 20uL of the mixture from point 3.

[0640] 10nM T790M splint oligonucleotide

[0641] 10nM C797S_2389 splint oligonucleotide

[0642] Total volume: 30uL

[0643] This mixture was then incubated at 37°C for 10 min and 95°C for 10 min.

[0644] T790M splint oligonucleotide (SEQ ID NO: 12):

[0645]

[0646] C797S_2389 splint oligonucleotide (SEQ ID NO: 13):

[0647]

[0648] 5. Exonuclease Treatment

[0649] A mixture corresponding to the following was prepared:

[0650] 1xA7

[0651] 100U / mL E. coli ligase

[0652] 30uL of the mixture from point 4.

[0653] 625U / mL Exonuclease III

[0654] 62.5U / mL T5 exonuclease

[0655] Total volume: 40uL

[0656] This mixture was then incubated at 30°C for 5 min and 95°C for 5 min.

[0657] 6. RCA

[0658] A mixture corresponding to the following was prepared:

[0659] 1x Thermopol buffer (53.2mM Tris-HCl, 26.6mM (NH4)2SO4, 26.6mM KCl, 5.32mM MgSO4, 0.266% pH 8.8)

[0660] 0.2uM Primer Mix 1

[0661] 0.4uM reverse primer

[0662] 533.3U / mL BST LF

[0663] 0.4 mM dNTP

[0664] 10uL of the reaction mixture from point 5.

[0665] Total volume 15uL

[0666] Primer Mix 1:

[0667] Cy5 primer (SEQ ID NO: 14):

[0668]

[0669] TexasRed Primer (SEQ ID NO: 15):

[0670]

[0671] Where / BHQ2 / represents Black Hole quencher;

[0672] Reverse primer (SEQ ID NO:16):

[0673]

[0674] The mixture was then incubated at 60°C for 90 minutes. Fluorescence measurements were taken every 1 minute. The Cq was obtained based on the automatic threshold given by the Bio-rad machine. The results can be seen in Fig.13 .

[0675] Example 13: Multicolor Detection Using Molecular Zippers

[0676] 1. Target oligonucleotide dilution

[0677] WT oligonucleotide dilution solution is made from the following ingredients

[0678] 0.5x A7 buffer

[0679] 0.5x Q5U buffer

[0680] 100 nM WT oligonucleotide (SEQ ID NO: 17)

[0681] Total volume: 1.25uL

[0682] G719X_6239, G719X_6252, G719X_6253 0.5% AF mutant oligonucleotide mix:

[0683] 0.5x A7 buffer

[0684] 0.5x Q5U buffer

[0685] 100 nM WT oligonucleotide (SEQ ID NO: 17)

[0686] 0.5 nM G719X_6239 oligonucleotide (SEQ ID NO: 18)

[0687] 0.5 nM G719X_6252 oligonucleotide (SEQ ID NO: 19)

[0688] 0.5 nM G719X_6253 oligonucleotide (SEQ ID NO: 20)

[0689] Total volume: 1.25uL

[0690] WT oligonucleotide (SEQ ID NO: 17):

[0691]

[0692] G719X_6239 oligonucleotide (SEQ ID NO: 18):

[0693]

[0694] G719X_6252 oligonucleotide (SEQ ID NO: 19):

[0695]

[0696] G719X_6253 oligonucleotide (SEQ ID NO:20):

[0697]

[0698] 1xA7 Composition

[0699] Tris-acetate pH = 8.0 10 mM

[0700] Potassium acetate 25mM

[0701] Magnesium acetate 5mM

[0702] Triton-X 0.01%

[0703] Q5U Buffer

[0704] The Q5U buffer composition is not publicly available.

[0705] 2. Pyrophosphorolysis (PPL) and ligation

[0706] A mixture corresponding to the following was prepared:

[0707] 1xBFF1

[0708] 10U / mL Klenow(exo-)

[0709] 100U / mL E. coli ligase

[0710] 1.2 U / mL apyrase

[0711] 100U / mL Lambda exo

[0712] 0.25mM PPi

[0713] 6.6nM G719X_6239 probe oligonucleotide (SEQ ID NO:21)

[0714] 6.6nM G719X_6252 probe oligonucleotide (SEQ ID NO:22)

[0715] 6.6nM G719X_6253 probe oligonucleotide (SEQ ID NO: 23)

[0716] 30 nM splint oligonucleotide (SEQ ID NO: 24)

[0717] 1.25uL of the mixture from point 1.

[0718] Total volume 10uL

[0719] This mixture was then incubated at 45°C for 15 minutes.

[0720] 1xBFF1 composition

[0721] Tris Acetate pH = 7.0 10 mM

[0722] Potassium acetate 30 mM

[0723] Magnesium acetate 17.125mM

[0724] Triton-X 0.01%

[0725] G719X_6239 probe oligonucleotide (SEQ ID NO:21):

[0726]

[0727] G719X_6252 probe oligonucleotide (SEQ ID NO:22):

[0728]

[0729] G719X_6253 probe oligonucleotide (SEQ ID NO:23):

[0730]

[0731] Splint oligonucleotide (SEQ ID NO:24):

[0732]

[0733] Where * represents a phosphorothioate bond

[0734] 3. Detection - RCA

[0735] A mixture corresponding to the following was prepared:

[0736] 2.66x Thermopol buffer (53.2 mM Tris-HCl, 26.6 mM (NH4)2SO4, 26.6 mM KCl, 5.32 mM MgSO4, 0.266% pH 8.8)

[0737] 0.28uM Dye Primer Mix 1

[0738] 0.56uM Quencher Primer 1

[0739] 0.28uM Quencher Primer 2

[0740] 0.84uM reverse primer

[0741] 568.8U / mL BST 2.0WarmStart

[0742] 14.67U / mL TIPP

[0743] 1.06mM dNTP

[0744] 1.25uL of reaction mixture from point 2.

[0745] Total volume 11.25uL

[0746] Dye Primer Mix 1 consisted of:

[0747] Dye Primer 1 (SEQ ID NO: 25):

[0748]

[0749] Dye Primer 2 (SEQ ID NO: 26):

[0750]

[0751] Dye Primer 3 (SEQ ID NO: 27):

[0752]

[0753] Where * represents a phosphorothioate bond, / 5Cy5 / represents a Cy5 dye on the 5' end, / 5TEX615 represents a TEX dye on the 5' end, and / 5HEX / represents a Hex dye on the 5' end

[0754] Quencher Primer 1 (SEQ ID NO: 28):

[0755]

[0756] Where / 3IAbRQSp / represents 3'Iowa RQ Quencher

[0757] Quencher Primer 2 (SEQ ID NO: 29):

[0758]

[0759] Where / 3IAbkFQ / stands for 3'Iowa FQ Quencher

[0760] Reverse primer (SEQ ID NO:30):

[0761]

[0762] Where * represents a phosphorothioate bond

[0763] The mixture was then incubated at 58°C for 150 minutes. Fluorescence measurements were taken every 1 minute. The results can be seen in Fig.14 .

[0764] Example 14: Pyrophosphorolysis and ligation to target

[0765] 1. Preparation of Oligonucleotide Dilution Solution

[0766] Prepare oligonucleotide dilutions in 0.5xA7 and 0.5xQ5 buffer:

[0767] WT oligonucleotide 200nM

[0768] + / - mutant oligonucleotide 500pM

[0769] Total volume 1.25uL

[0770] WT oligonucleotide (SEQ ID NO 31):

[0771]

[0772] Mutant oligonucleotide (SEQ ID NO 32):

[0773]

[0774] 2. Pyrophosphorolysis and Ligation

[0775] A PPL mixture consisting of the following was prepared:

[0776] 1xBFF1

[0777] 10U / mL Klenow(exo-)

[0778] 100U / mL E. coli ligase

[0779] 1.2 U / mL apyrase

[0780] 100U / mL Lambda exo

[0781] 0.25mM PPi

[0782] 20 nM probe A0

[0783] 1.25uL oligonucleotide from point 1

[0784] Total volume 10uL

[0785] Probe A0 (SEQ ID NO 33):

[0786]

[0787] *Phosphorothioate bond

[0788] 1xBFF1 composition

[0789] Tris Acetate pH = 7.0 10 mM

[0790] Potassium acetate 30 mM

[0791] Magnesium acetate 17.125mM

[0792] Triton-X 0.01%

[0793] 1xA7 Composition

[0794] Tris-acetate pH = 8.0 10 mM

[0795] Potassium acetate 25mM

[0796] Magnesium acetate 5mM

[0797] Triton-X 0.01%

[0798] Q5 Buffer

[0799] The Q5 buffer composition is not publicly available.

[0800] The resulting mixture was incubated at 45°C for 15 minutes.

[0801] 3. Detection - RCA

[0802] An RCA mixture consisting of the following was prepared:

[0803] 2.66x Thermopol buffer (53.2 mM Tris-HCl, 26.6 mM (NH4)2SO4, 26.6 mM KCl, 5.32 mM MgSO4, 0.266% Triton-X, pH 8.8)

[0804] 0.28uM Primer Mix

[0805] 284.4U / mL BST 2.0WarmStart

[0806] 14.67U / mL TIPP

[0807] 1.06mM dNTP

[0808] Syto82 dye 3uM

[0809] 1.25uL from the reaction at point 2

[0810] Total volume 11.25uL

[0811] Primer Mix:

[0812] Fwd (SEQ ID NO 34):

[0813] Rev (SEQ ID NO 35):

[0814] The resulting mixture was incubated at 50°C for 70 minutes.

[0815] Fluorescence readings were taken every 1 minute. Results can be seen in Fig.16 .

[0816] Example 15: Further selected applications of the present invention and embodiments

[0817] KRAS testing

[0818] The KRAS gene controls cell proliferation, and when it mutates, this negative signaling is disrupted and the cells are able to continue to proliferate, often developing into cancer. Single amino acid substitutions, and especially single nucleotide substitutions, are responsible for 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 example, in lung cancer.

[0819] Driver mutations in KRAS are associated with up to 20% of human cancers, and targeted therapies are currently being developed for this mutation and its associated diseases. A non-limiting list of some of these therapies is provided in the table below:

[0820]

[0821] The presence of KRAS mutations has been found to reflect very poor responses to the EGFR inhibitors panitumumab (Vectibix) and cetuximab (Erbitux). Activating mutations of 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 a wild-type KRAS gene does not guarantee that a patient will respond to these drugs, however, studies have shown that cetuximab has significant efficacy in metastatic colorectal cancer patients with wild-type KRAS tumors. The response rate for lung cancer patients who are KRAS mutation-positive (wild-type EGFR) to the EGFR antagonists erlotinib or gefitinib is estimated to be 5% or less, compared to a 60% response rate for patients who do not have a KRAS mutation.

[0822] Early detection of the emergence of KRAS mutations (activation or overexpression), a common driver of acquired resistance to cetuximab therapy (anti-EGFR therapy) in colorectal cancer, allows modification of treatment (e.g., early initiation of mitogen-activated protein kinase kinase [MEK] inhibitors) to delay or reverse resistance, and it is therefore advantageous that the methods of the present invention allow rapid and inexpensive detection of a patient's KRAS status.

[0823] A non-limiting list of mutations is: G12D, G12A, G12C, G13D, G12V, G12S, G12R, A59T / E / G, Q61H, Q61K, Q61R / L, K117N, and A146P / T / V.

[0824] The following table shows a further non-limiting list of mutations:

[0825] Exon Mutation Name COSM Number Mutation 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

[0826] BRAF testing

[0827] BRAF is a human gene that encodes a protein called B-Raf, which is involved in sending signals within cells that are involved in directing 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 transducing protein kinases and plays a role in regulating the MAP kinase / ERKs signaling pathway, which, among other things, affects cell division.

[0828] Certain other inherited BRAF mutations cause birth defects.

[0829] More than 30 mutations of the BRAF gene have been identified that are associated with human cancer. In 90% of cases, thymine is replaced by adenine at nucleotide 1799. This results in a substitution of valine (V) by glutamic acid (E) at codon 600 of the activation segment found in human cancers (now called V600E). This mutation is widely observed in:

[0830] -Colorectal cancer

[0831] -Melanoma

[0832] - Papillary thyroid cancer

[0833] - Non-small cell lung cancer

[0834] -Ameloblastoma

[0835] A non-limiting list of other mutations that have been discovered are: R461I, I462S, G463E, G463V, G465A, G465E, G465V, G468A, G468E, N580S, E585K, D593V, F594L, G595R, L596V, T598I, V599D, V599E, V599K, V599R, V600K, and A727V.

[0836] Drugs have been developed to treat cancers driven by BRAF mutations; vemurafenib and dabrafenib are FDA-approved for the treatment of advanced melanoma. For metastatic melanoma, vemurafenib treatment has a 53% response rate, compared to 7%-12% for the previous best chemotherapy drug, dacarbazine.

[0837] ERBB2 / HER2 testing

[0838] Human epidermal growth factor receptor 2 (HER2), also known as CD340 (cluster of differentiation 340), proto-oncogene Neu, Erbb2 (rodent) or ERBB2 (human) is a protein encoded by the ERBB2 gene. Amplification or overexpression of this oncogene plays an important 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, invasive uterine cancer and 30% of salivary duct carcinomas. Structural alterations that lead to ligand-independent firing of the receptor in the absence of overexpression have also been identified.

[0839] There are many targeted therapies approved and in development for this mutation and its associated disease, a non-limiting list of some of these therapies is listed in the table below:

[0840]

[0841] HER2 testing is routinely performed in breast cancer patients to assess prognosis, monitor response to treatment, and determine the suitability of targeted therapies (trastuzumab, etc.). Since trastuzumab is expensive and associated with severe side effects (cardiotoxicity), it is important to select only HER2+ patients to receive it, and therefore it is advantageous that the method of the present invention allows rapid and inexpensive detection of a patient's HER2 status.

[0842] In one embodiment, the methods of the invention are used to detect the presence or absence of an ERRB2 exon 20 insertion mutation.

[0843] The following table shows a further non-limiting list of ERBB2 mutations:

[0844]

[0845] EML4-ALK testing

[0846] EML4-ALK is an abnormal gene 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 has been shown to promote and maintain the malignant behavior of cancer cells. EML4-ALK-positive lung cancers are primary malignant lung tumors whose cells contain this mutation.

[0847] There are many targeted therapies approved and in development for this mutation and its associated disease, a non-limiting list of some of these therapies is listed in the table below:

[0848]

[0849] The EML4-ALK gene fusion causes approximately 5% of non-small cell lung cancer (NSCLC), with approximately 9,000 new cases each year in the United States and approximately 45,000 cases worldwide.

[0850] There are many EML4-ALK variants that are required for transforming activity, all of which have the essential coiled-coil domain in the N-terminal portion of EML4 and the kinase domain of ALK exon 20. A fusion of exon 13 of EML4 to exon 20 of ALK (Variant 1: V1) (detected in Fig. 20 ), fusion of exon 20 of EML4 to exon 20 of ALK (V2), and fusion of exon 6 of EML4 to exon 20 of ALK (V3) are some of the more common variants. The clinical significance of these different variants has only recently become clearer.

[0851] V3 has emerged as a marker for selecting patients who may have shorter progression-free survival (PFS) after non-tyrosine kinase inhibitor (TKI) treatments such as chemotherapy and radiotherapy. There is further evidence that V3 is associated with shorter PFS and worse overall survival (OS) in patients receiving first- and second-generation lines of treatment compared with V1 and V2 of EML4-ALK.

[0852] It was also found that V3-positive patients developed resistance to both the first and second lines of treatment by developing resistance mutations, and that this resistance may be facilitated by the higher IC50 of wild-type V3, which results in incomplete tumor cell inhibition. Detection of an unfavorable V3 may be used to select patients who require a more aggressive monitoring and treatment strategy. It was shown that administration of the third generation lorlatinib to patients with V3 may confer longer PFS than patients with V1, and therefore it is advantageous that the methods of the present invention allow for rapid and inexpensive detection of variants that patients may have.

[0853] The methods of the invention also allow 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.

[0854] For example, G1202R is a solvent-front mutation that interferes with drug binding and confers high-level resistance to first and second generation ALK inhibitors. It is therefore advantageous that the methods of the invention allow identification of those patients who may have such a mutation and benefit from treatment beginning with a third generation therapy rather than a first or second generation therapy.

[0855] The following table shows a further non-limiting list of EML4-ALK mutations:

[0856]

[0857] EGFR testing

[0858] The identification of the 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 colon cancer. However, many people develop resistance to these therapies. The two main sources of resistance are the T790M mutation and the MET oncogene.

[0859] EGFR mutations occur in EGFR exons 18-21 and exons 18, 19, and 21 and indicate suitability for treatment with EGFR-TKI (tyrosine kinase inhibitors). Mutations in exon 20 (except for a few mutations) indicate that the tumor is EGFR-TKI-resistant and is not suitable for treatment with EGFR-TKI.

[0860] The two most common EGFR mutations are short in-frame deletions in exon 19 and a point mutation (CTG to CGG) at nucleotide 2573 in exon 21, which results in a substitution of leucine for arginine at codon 858 (L858R). Together, these two mutations account for ~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.

[0861] Therefore, it is advantageous that the methods of the invention allow identification of those patients who may have these mutations and benefit from initiation of treatment with TKIs. It will be appreciated by those skilled in the art that the methods of the invention allow identification of a range of EGFR mutations, a non-exhaustive list of such mutations being: G719X, Ex19Del, S768I, Ex20Ins and L861Q.

[0862] The following table shows a further non-limiting list of mutations:

[0863]

[0864]

[0865] ROS1

[0866] ROS1 is a receptor tyrosine kinase (encoded by the gene ROS1) that has structural similarity to the anaplastic lymphoma kinase (ALK) protein (which is encoded by the c-ros oncogene).

[0867] The following table shows a non-limiting list of ROS1 mutations:

[0868]

[0869]

[0870] RET proto-oncogene

[0871] The RET proto-oncogene encodes a receptor tyrosine kinase that is a member of the glial cell line-derived neurotrophic factor (GDNF) family.

[0872] The following table shows a non-limiting list of RET mutations:

[0873]

[0874] MET exon 14

[0875] MET exon 14 skipping occurs at a frequency of approximately 5% in NSCLC and is seen in both squamous cell and adenocarcinoma histologies.

[0876] The following table shows a non-limiting list of MET mutations:

[0877] Exon Mutation Name COSM Number Mutation sequence Skip 14 MET-MET COSM29312 M13_M15

[0878] NTRK proto-oncogene

[0879] NTRK gene fusions result in abnormal proteins called TRK fusion proteins, which may cause cancer cell growth. NTRK gene fusions may be present in certain types of cancer, including brain cancer, head and neck cancer, thyroid cancer, soft tissue cancer, lung cancer, and colon cancer. Also known as neurotrophic tyrosine receptor kinase gene fusions.

[0880] The following table shows a non-limiting list of NTRK mutations:

[0881]

[0882] Panel

[0883] In one embodiment of the invention, a group comprising more than one probe molecule (A0) is provided, wherein each A0 is complementary to the target mutation. The mutation may be selected from any mutation previously or subsequently described or known. Therefore, it will be appreciated by those skilled in the art that within the scope of the invention, a group comprising one or more mutations that can be used to detect any proto-oncogene or oncogene previously or subsequently described or known is included.

[0884] In one embodiment, the group includes 5-500 separate probe molecules, each of which is complementary to a specific target mutation. In one embodiment, the group includes 5-400 separate probe molecules, each of which is complementary to a specific target mutation. In one embodiment, the group includes 5-300 separate probe molecules, each of which is complementary to a specific target mutation. In one embodiment, the group includes 5-200 separate probe molecules, each of which is complementary to a specific target mutation. In one embodiment, the group includes 5-100 separate probe molecules, each of which is complementary to a specific target mutation. In one embodiment, the group includes 5-50 separate probe molecules, each of which is complementary to a specific target mutation.

[0885] In one embodiment, there may be more than one probe molecule specific for the same mutation. In one embodiment, there may be only one probe molecule specific for each mutation of the panel.

[0886] In one embodiment, a panel is provided, wherein the panel comprises more than one probe molecule, wherein one or more probes are complementary to EGFR mutations, one or more probes are complementary to KRAS mutations, one or more probes are complementary to ERBB2 / HER2 mutations, one or more probes are complementary to EML4-ALK mutations, one or more probes are complementary to ROS1 mutations, one or more probes are complementary to RET mutations, and one or more probes are complementary to MET mutations.

[0887] In one embodiment, a group is provided, wherein the group includes more than one probe molecule, wherein one or more probes can be complementary to EGFR mutations, one or more probes can be complementary to KRAS mutations, one or more probes can be complementary to ERBB2 / HER2 mutations, one or more probes can be complementary to EML4-ALK mutations, one or more probes can be complementary to ROS1 mutations, one or more probes can be complementary to RET mutations, and one or more probes can be complementary to MET mutations.

[0888] In one embodiment, a panel of probes selective for one or more EGFR, KRAS, BRAF, ERBB2 / HER2, EML4-ALK, ROS1, RET, MET mutations is provided.

[0889] In one embodiment, a set of probe molecules selective for EGFR mutations is provided.

[0890] In one embodiment, a panel of probe molecules selective for KRAS mutations is provided.

[0891] In one embodiment, a panel of probe molecules selective for BRAF mutations is provided.

[0892] In one embodiment, a panel of probe molecules selective for ERBB2 / HER2 mutations is provided.

[0893] In one embodiment, a panel of probe molecules selective for EML4-ALK mutations is provided.

[0894] In one embodiment, a panel of probe molecules selective for ROS1 mutations is provided.

[0895] In one embodiment, a panel of probe molecules selective for RET mutations is provided.

[0896] In one embodiment, a set of probe molecules selective for NTRK mutations is provided.

[0897] In one embodiment, a panel of probe molecules selective for ROS1 mutations is provided.

[0898] In one embodiment, a panel of probe molecules selective for MET exon 14 mutations is provided.

[0899] In one embodiment, a panel comprising more than one probe molecule selective for one or more coding sequences (CDS) is provided.

[0900] In one embodiment, a method of detecting one or more mutations using one or more of the previously described panels is provided.

[0901] In one embodiment, a method of detecting the presence or absence of one or more mutations using one or more of the previously described panels is provided.

[0902] In one embodiment, a kit is provided, comprising a set as may be described previously or subsequently, in combination with one or more agents as may be described previously or subsequently.

[0903] One skilled in the art will understand that embodiments of kits disclosing AOs include within their scope embodiments in which there are sets comprising more than one AO.

[0904] Companion Diagnostics

[0905] The methods of the present invention can be used to detect specific genetic markers in a sample, which can be used to help guide the selection of appropriate therapy. These markers can be tumor-specific mutations, or can be wild-type genomic sequences, and can be detected using tissue, blood, or any other patient sample type.

[0906] Resistance monitoring

[0907] Repeated detection of patient samples during disease treatment can allow early detection of resistance to treatment. An example of this application is non-small cell lung cancer (NSCLC), in which epidermal growth factor receptor (EGFR) inhibitors (such as gefitinib, erlotinib) are generally used as first-line treatment. During treatment, tumors may generally produce mutations (such as T790M, C797S) in the EGFR gene, and these mutations confer resistance to drugs. Early detection of these mutations can allow patients to turn to alternative therapies (such as Tagrisso).

[0908] Often, patients who are being monitored for resistance development may be too ill to undergo repeated tissue biopsies. Repeated tissue biopsies may also be expensive, invasive, and carry associated risks. Testing is best done from the blood, but very low copy numbers of the mutation of interest may be present in a reasonable blood draw. Therefore, monitoring requires sensitive testing from blood samples using the methods of the present invention, which are simple to implement and cost-effective so that they can be performed regularly.

[0909] Relapse monitoring

[0910] In this application example, patients who are declared disease-free after treatment may be monitored over time to detect recurrence of the disease. This needs to be done non-invasively and requires sensitive detection of target sequences from blood samples. By using the method of the present invention, it provides a simple and low-cost method that can be performed regularly. The targeted sequence can be a universal mutation known to be common in the disease of interest, or it can be a group of customized targets designed for a specific patient based on the detection of variants in pre-remission tumor tissue.

[0911] Minimal residual disease (MRD) monitoring

[0912] For some cancers, residual cancer cells remain in the patient's body after treatment and are a major cause of cancer and leukemia relapse. MRD monitoring and detection have several important roles: determining whether treatment has eradicated the cancer or left residual cancer, comparing the effectiveness of different treatments, monitoring a patient's remission status and detecting the recurrence of leukemia, and choosing the treatment that best meets these needs.

[0913] Screening

[0914] Population screening for early detection of disease is a long-standing goal, especially in cancer diagnosis. The challenge is twofold: identifying a panel of markers that allows reliable disease detection without too many false negatives, and developing methods that are sensitive enough and low cost enough. Compared to PCR-based tests, the methods of the present invention can be used to handle larger sets of mutations, but compared to sequencing-based diagnostics, the methods of the present invention have a simpler workflow and are less expensive.

[0915] Organ transplant rejection

[0916] When a transplanted organ is rejected by a recipient, DNA from the organ is shed into the recipient's bloodstream. Early detection of this DNA will allow early detection of rejection. This can be achieved using a customized set of donor-specific markers, or by using a set of variants known to be common in a 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 present invention disclosed herein.

[0917] Non-invasive prenatal testing (NIPT)

[0918] It has long been known that fetal DNA is present in the mother's blood, and the NIPT market has now been saturated by companies that use sequencing to identify mutations and count the number of copies of specific chromosomes to enable detection of fetal abnormalities. The methods of the invention disclosed herein have the ability to detect mutations at very low allele fractions, potentially allowing earlier detection of fetal DNA. Identifying common mutations in a given population will allow the development of assays that target mutations that may be present in maternal or fetal DNA, or allow detection of abnormalities earlier in pregnancy.

[0919] Various other aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0920] As used herein, "and / or" is considered to be a specific disclosure of each of the 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.

[0921] Unless the context indicates otherwise, the descriptions and definitions of the features listed above are not limited to any particular aspect or embodiment of the invention, and apply equally to all aspects and embodiments described.

[0922] Those skilled in the art will further appreciate that while the invention has been described by way of example with reference to several embodiments, the invention is not limited to the disclosed embodiments and that alternative embodiments may be constructed without departing from the scope of the invention as defined in the appended claims.

[0923] One skilled in the art will appreciate that reference to "partially digested strand A1" may refer to a single-stranded oligonucleotide formed by stepwise digestion of A0 in the 3'-5' direction when hybridized to a target analyte sequence until the strands dissociate due to lack of complementarity.

[0924] One skilled in the art will understand that reference to a "partially double-stranded" nucleic acid can refer to a nucleic acid in which one or more portions are double-stranded and one or more portions are single-stranded.

[0925] One skilled in the art will understand that reference to a "substantially double-stranded" nucleic acid can refer to a nucleic acid in which one or more portions are double-stranded and one or more smaller portions are single-stranded.

Claims

1. A non-diagnostic or therapeutic method for detecting a target polynucleotide sequence in a given nucleic acid analyte present in a sample, the method comprising the steps of: (a) introducing one or more nucleic acid analytes into a first reaction mixture, the first reaction mixture comprising: i. a single-stranded probe oligonucleotide A0, wherein the single-stranded probe oligonucleotide A0 comprises a 3' terminal region complementary to the target polynucleotide sequence; ii. pyrophosphatase; and iii. Ligase; wherein the analyte is annealed to the single-stranded probe oligonucleotide A0 to produce a first intermediate product, the first intermediate product is at least partially double-stranded and wherein the 3' end of A0 forms a double-stranded complex with the analyte and A0 is pyrophosphorolyzed from the 3' end in the 3'-5' direction to produce an at least partially digested strand A1 and A1 is subjected to ligation using a splint to form oligonucleotide A2, wherein the splint comprises the target polynucleotide or oligonucleotide D, and wherein undergoing ligation comprises: ligating the 3' end of A1 to the 5' end of A1 to form a circular construct; or The 3' end of A1 is connected to the 5' end of the ligation probe oligonucleotide C; (b) detecting a signal derived from a 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 inferring the presence or absence of a polynucleotide target sequence in the analyte.

2. The method according to claim 1, further characterized in that: The first reaction mixture also includes a source of pyrophosphate ions.

3. The method according to claim 1 or claim 2, further characterized in that: The first reaction mixture also includes at least one single-stranded primer oligonucleotide substantially complementary to a portion of A0 and deoxyribonucleotide triphosphates (dNTPs).

4. The method according to claim 3, further characterized in that: The first reaction mixture also includes an amplification enzyme.

5. The method according to claim 1 or claim 2, further characterized in that: 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 dNTPs.

6. The method according to claim 5, further characterized in that: The second reaction mixture also includes an amplification enzyme.

7. The method according to claim 1 or claim 2, further characterized in that: The first reaction mixture further comprises: one or more ligases; and two or more LCR probe oligonucleotides complementary to adjacent sequences on A2, wherein the 5' phosphate of one LCR probe is directly adjacent to the 3' OH of another LCR probe when the probes are successfully annealed to A2; Wherein, in the presence of A2, the two LCR probes will successfully anneal to A2 and be ligated together to form one oligonucleotide molecule, which then acts as a new target for a second round of covalent ligation, resulting in geometric amplification of the target of interest, in this case A2, and then the target of interest, in this case A2, is detected.

8. The method according to claim 1 or claim 2, further characterized in that: Prior to step (b), the product of step (a) is introduced into a second reaction mixture, the second reaction mixture comprising: one or more ligases; and two or more LCR probe oligonucleotides complementary to adjacent sequences on A2, wherein the 5' phosphate of one LCR probe is directly adjacent to the 3' OH of another LCR probe when the probes are successfully annealed to A2; Wherein in the presence of A2 the two LCR probes will successfully anneal to A2 and be ligated together to form one oligonucleotide molecule which then acts as a new target for a second round of covalent ligation, resulting in geometric amplification of the target of interest, in this case A2, and then the target of interest, in this case A2, is detected.

9. The method according to claim 1 or claim 2, further characterized in that: The first reaction mixture further comprises: an oligonucleotide complementary to a region of A2 comprising a ligation site, 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-strand specific DNA digesting enzyme; Therein, in the presence of A2, the labeled oligonucleotide is digested such that the fluorophores are separated from each other or from their corresponding quenchers, and a fluorescent signal, and thus the presence of A2, is detectable.

10. The method according to claim 1 or claim 2, further characterized in that: Prior to step (b), the product of step (a) is introduced into a second reaction mixture, the second reaction mixture comprising: an oligonucleotide complementary to a region of A2 comprising a ligation site, 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-strand specific DNA digesting enzyme; Therein, in the presence of A2, the labeled oligonucleotide is digested such that the fluorophores are separated from each other or from their corresponding quenchers, and a fluorescent signal, and thus the presence of A2, is detectable.

11. The method according to claim 1, further characterized in that: The oligonucleotide C may further include a 3' modification or an internal modification that protects it from 3'-5' exonuclease digestion.

12. The method according to claim 11, further characterized in that: The connection occurs at: during step (a); or during step (b); or Between steps (a) and (b).

13. The method according to claim 1, further characterized in that: 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.

14. The method according to claim 1 or claim 13, further characterized in that: D cannot be extended towards A1 due to 3' modification or by mismatch between the 3' end of D and the corresponding region of A1.

15. A method according to any preceding claim, further characterized in that: The first reaction mixture further comprises a 5'-3' exonuclease, and wherein the 5' end of A0 is rendered resistant to digestion by the 5'-3' exonuclease.

16. A method according to any preceding claim, further characterized in that: Prior to step (a), said nucleic acid analyte present in the sample is amplified, and wherein after amplifying said given nucleic acid analyte and before adding said first reaction mixture, said sample is further treated with a protease.

17. A method according to any preceding claim, further characterized in that: The first reaction mixture further comprises a phosphatase or a phosphohydrolase.

18. A method according to any preceding claim, further characterized in that: Prior to step (b) or during step (b), the product of the previous step is treated with pyrophosphatase.

19. A method according to any preceding claim, further characterized in that: Prior to step (b) or during step (b), the product of the previous step is treated with an exonuclease.

20. A method according to any preceding claim, further characterized in that The enzyme that pyrophosphorolyzes A0 to form partially digested chain A1 also amplifies A2.

21. The method according to any one of the preceding claims, further characterized in that: Detection is achieved using one or more oligonucleotide fluorescent conjugated dyes or molecular probes.

22. The method according to claim 21, further characterized in that: 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.

23. The method according to any one of the preceding claims, further characterized in that: More than one probe A0 is used, each probe A0 is selective for a different target sequence, and each probe A0 comprises a recognition region, and is further characterized in that the amplicon of A2 comprises the recognition region, and thus the target sequence present in the analyte is inferred by detecting the recognition region.

24. The method according to claim 23, further characterized in that: Detection of the recognition region is performed using molecular probes or by sequencing.

25. The method according to claim 21, further characterized in that: The final step of the method further comprises the following steps: i. labeling the product of step (b) using one or more oligonucleotide fluorescent binding dyes or molecular probes; ii. measuring the fluorescence signal of the product; iii. exposing the product to a set of denaturing conditions; and The polynucleotide target sequence in the analyte is identified by monitoring the change in the fluorescent signal of the product during exposure to the denaturing condition.

26. The method according to any one of claims 1 to 25, further characterized in that: The one or more nucleic acid analytes are split into more than one reaction volume, each volume having one or more probe oligonucleotides A0 introduced to detect a different target sequence.

27. The method according to any one of claims 1 to 26, further characterized in that: The different probes A0 contain a common priming site, allowing amplification using a single primer or a single set of primers.

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