Simplified polynucleotide sequence detection method

Abstract

To provide methods for improved polynucleotide detection.SOLUTION: Provided is a method of detecting a target polynucleotide sequence in a given nucleic acid analyte present in a sample, the method comprising the steps of: (a) introducing nucleic acid analytes to a first reaction mixture comprising i. a single-stranded probe oligonucleotide A0, ii. a pyrophosphorolysing enzyme, and iii. a ligase, the analyte annealing to the single-stranded probe oligonucleotide A0 to be at least partially double-stranded and in which the 3' end of A0 forms a double-stranded complex and A0 is pyrophosphorylsed in the 3'-5' direction from the 3' end to create at least a partially digested strand A1 and A1 undergoes ligation to form A2; and (b) detecting a signal derived from the products of the previous step, the products being A2 or a portion thereof, or multiple copies of A2 or multiple copies of a portion thereof, and inferring therefrom the presence or absence of the polynucleotide target sequence in the analyte.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a simplified polynucleotide sequence detection method suitable for testing for the presence of multiple diagnostic markers, including those used to identify cancer, infectious diseases, and transplant organ rejection. It is also useful for companion diagnostic tests where marker panels must be identified reliably and at low cost. Summary of the Invention

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

[0003] A second drawback is that multiplexing of PCR-based methods is in practice limited to a maximum of several tens of target sequences (often less than 10) due to the avoidance of primer-primer interactions, creating the need for a relatively narrow operating window.

[0004] Another problem is that PCR reactions cycle in an exponential manner, making target quantification difficult; small variations in reaction efficiency have a large effect on the amount of detectable material produced. Thus, even with appropriate controls and calibration in place, quantification is typically limited to an accuracy of approximately one-third to approximately three-fold.

[0005] Finally, mutations in regions targeted for study by PCR amplification may have undesirable side effects. For example, there have been cases where FDA-approved tests had to be withdrawn because the target organisms underwent mutations in gene regions targeted by the test primers, resulting in a large number of false negatives. Conversely, if a specific single nucleotide polymorphism (SNP) is targeted for amplification, the PCR method will often produce false positives if wild-type variants are present. To avoid this, very careful primer design is required, and furthermore the effectiveness of multiplexing is limited. This is particularly relevant when searching for SNP panels, such as is a common requirement in cancer testing / screening or companion diagnostics.

[0006] US2006 / 110765A1 (Wang et al.) describes enzymatic cleavage at mismatch sites, which is typically an inefficient and not very specific reaction. Furthermore, off-target hybridization of probes as disclosed in Wang et al. to similar sequences in samples produces false positive results due to the use of cleavage at mismatch sites. It is also not possible to distinguish between two different gene variants at the same or nearby positions, as they all result in cleavage and amplification of the same probe. The description of Wang et al. would therefore result in a reaction scheme with low sensitivity and specificity. In contrast, the technical effect of the method as disclosed by the present invention is to provide a rapid and efficient method with high specificity for dsDNA, which can be effectively blocked by mismatches. Furthermore, the method of the present invention is highly specific to the gene variant targeted. , allowing discrimination between different variants at the same or nearby positions.

[0007] US2009 / 239283 A1 (Liu et al.) describes the use of non-extendable 3' ends that are removed by pyrophosphorolysis, which requires the engineering of custom polymerases capable of removing the 3' blocking modification. In contrast, the present invention utilizes the native pyrophosphorolysis activity inherent in existing polymerases and does not use 3' blocking modifications. Methods such as those disclosed in Liu et al. also rely on the removal of only the terminal base from a portion of the probe to allow for subsequent amplification, and are limited to this embodiment by the use of the 3' blocking modification. In contrast, the methods disclosed in the present invention enable embodiments in which progressive removal of multiple bases from the probe is required to trigger the reaction, making the reaction substantially more robust against transient off-target annealing to either background DNA or other probes, which can result in undesired removal of the terminal base.

[0008] Summary of the Invention The present inventors have now developed a novel simplified method that builds on the inventors' experience with the pyrophosphorolysis method used in the inventors' previous patent (PCT / GB2019 / 052017) to overcome many of these limitations. In doing so, they take advantage of the double-stranded specificity of pyrophosphorolysis, a reaction that does not proceed efficiently with single-stranded oligonucleotide substrates, or double-stranded substrates that contain blocking groups or nucleotide mismatches. The novel method is faster, less complicated, and less expensive to perform than that disclosed in PCT / GB2019 / 052017. Thus, in accordance with the present invention, there is provided a method for detecting a target polynucleotide reaction in a given nucleic acid analyte, comprising: (a) i. single-stranded probe oligonucleotide A0; ii. pyrophosphoroase; and iii. Ligase introducing one or more nucleic acid analytes into a first reaction mixture comprising where A0 is pyrophosphorolyzed from the 3' end in the 3'-5' direction to generate an at least partially digested strand A1, and A1 undergoes ligation to form A2; (b) detecting a signal from the product of the previous step, where the product is A2 or a portion thereof, or multiple copies of A2 or multiple copies of a portion thereof, and inferring therefrom the presence or absence of the polynucleotide target sequence in the analyte. The method further comprises the steps of:

[0009] The analyte to which the method of the present invention may be applied is a nucleic acid, such as a naturally occurring or synthetic DNA or RNA molecule, that contains the target polynucleotide sequence(s) that one is looking for. In one embodiment, the analyte will typically be present in an aqueous solution containing the analyte and other biological material, and in one embodiment, the analyte will be present along with other background nucleic acid molecules that are not of interest for the purpose of the test. In some embodiments, the analyte will be present in small amounts compared to these other nucleic acid components. Preferably, for example, if the analyte is derived from a biological specimen containing cellular material, some or all of these other nucleic acids and extraneous biological material will have been removed using sample preparation techniques, such as filtration, centrifugation, chromatography or electrophoresis, before carrying out step (a) of the method. Suitably, the analyte is derived from a biological sample, such as blood, plasma, sputum, urine, skin or biopsy, taken from a mammalian subject, particularly a human patient. In one embodiment, the biological sample will be subjected to lysis in order to release the analyte by disrupting any cells present. In other embodiments, the analyte is present in the sample itself, already in free form; e.g., in a cell-free form circulating in blood or plasma. It may also exist as a DNA-containing molecule. [Brief description of the drawings]

[0010] [Figure 1] Protocol for a simplified polynucleotide sequence detection method. [Diagram 2]Graph comparing the level of fluorescence (corresponding to the presence of a particular target analyte sequence) detected when a 5'-3' exonuclease digestion step occurs during the pre-amplification step and when this digestion step is moved to the pyrophosphorolysis / ligation step of the protocol (as in Protocols 3-5). In this example, the 5'-3' exonuclease is lambda. [Diagram 3] We tested the method of protocol 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. All four enzymes worked very well without extensive optimization. [Figure 4] Results for detection of T790M at 1% MAF using the method of protocol 4 of the invention with four different pyrophosphorolysis (PPL) enzymes: Mako, Klenow, Bsu and Bst LF. [Diagram 5] Graph showing the level of fluorescence (corresponding to the presence of a particular 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. [Figure 6] Following protocol 4, we detected EGFR exon 20 T790M in 0.10%, 0.50% and 1% MAF. [Figure 7] Detection of EGFR exon 20 mutation T790M in 1% MAF with and without the use of exonuclease in the RCA step. [Figure 8]We investigated the effect of the PPL:RCA mixture ratio on the intensity of the signal detected for 0.5% MAF EGFR exon 20 T790M, and the results are shown in Figure 8. As can be seen, the 1:2 PPL:RCA mixture ratio produces the lowest signal intensity, but at the earliest time point. This is followed closely in time by the 1:4 PPL:RCA mixture, which has a higher signal intensity. The maximum signal intensity is seen for the 1:8 PPL:RCA mixture at the latest time point of the reaction. [Figure 9] FIG. 9 shows the results of a comparative experiment carried out according to protocol 4 using SybrGreenI (50° C. and 60° C.) and Syto82 (50° C. and 60° C.). [Figure 10] We investigated the use of two different enzymes, BST LF and BST 2.0WS, for RCA according to protocol 4. [Figure 11] We investigated the effect of different PPL enzymes on RCA reaction at different PPL:RCA mixing ratios. The results can be seen in Figure 11(A) 1:4 PPL:RCL and Figure 11(B) 1:8 PPL:RCL. All PPL enzymes affect RCA reaction at 1:4 PPL:RCA ratio, except BST LF. At 1:8 PPL:RCA ratio, all enzymes affect RCA reaction, except BST LF and Klenow. [Figure 12] Fluorescence results for Example 11 showing that the fluorescent signal appears more quickly in the reaction when oligonucleotides 3 and 4 are both present, indicating that pyrophosphorolysis and ligation of oligonucleotide 3 occurs in the first reaction mixture. [Figure 13] Detection of T790M and C797S_2389 mutations at 1% allele fraction in the same reaction. [Figure 14] Detection of three mutations simultaneously in one well: G719X_6239, G719X_6252, G719X_6253 at 0.5% allele fraction. [Figure 15]Schematic of circularization of A1 to form A2 against an analyte target sequence. A0 is progressively digested against the target in the 3'-5' direction from the 3' end of A0 to form the partially digested strand A1, shown in steps (A) and (B). This progressive digestion reveals a target region complementary to the 5' of A0 / A1, and the 5' end of A1 then hybridizes to this region. This is shown in step (C). A1 is then ligated together to form the circularized A2. Step (D). [Figure 16] Fluorescence results for Example 14 showing results from an embodiment in which pyrophosphorolysis of A0 to form A1 occurs, followed by circularization of A1 to the target sequence to form A2. [Figure 17] The single-stranded probe oligonucleotide A0 anneals to the target polynucleotide sequence to generate 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 molecules of A0 and one target polynucleotide sequence present to illustrate how A0 that is not annealed to the target does not participate in further steps of the method. In this illustrative example, the 3' end of A0 anneals to the target polynucleotide sequence while the 5' end of A0 does not anneal. The 5' end of A0 includes a 5' chemical blocking group, a common priming sequence and a barcode region. The first intermediate product, which is partially double-stranded, undergoes pyrophosphorolysis in the presence of pyrophosphorolysis enzyme in the 3'-5' direction from the 3' end of A0 to generate a partially digested strand A1, an analyte, and an undigested A0 molecule that did not anneal to the target. [Figure 18]A1 anneals to single-stranded trigger oligonucleotide B, and the A1 strand is extended in the 5'-3' direction relative to B to generate oligonucleotide A2. In this illustrative example, trigger oligonucleotide B has a 5' chemical block. Any undigested A0 anneals to trigger oligonucleotide B, but cannot be extended in the 5'-3' direction to generate a sequence that is the target for the second half of the method. In this example, A2 is primed with at least one single-stranded primer oligonucleotide to generate multiple copies of A2 or a region of A2. [Figure 19] A1 anneals to splint oligonucleotide D and is then circularized by ligation at its 3' and 5' ends. The now circularized A2 is primed with at least one single-stranded primer oligonucleotide and multiple copies of A2 or a region of A2 are generated. In this illustrative example, splint oligonucleotide D is unable to be extended relative to A1, either due to a 3' modification (chemical in this example) or through a nucleotide mismatch between the 3' end of D and the corresponding region of A2. [Figure 20] The 3' region of splint oligonucleotide D anneals to the 3' region of A1, while the 5' region of splint oligonucleotide D anneals to the 5' region of ligation probe C. Thus, a second intermediate product A2 is formed, consisting of A1, C, and optionally an intermediate region formed by extension of A1 in the 5'-3' direction and ligated to the 5' end of C. In this illustrative example, ligation probe C has a 3' chemical blocking group, so that it is also possible to digest any unligated A1 using a 3'-5' exonuclease. A2 is primed with at least one single-stranded primer oligonucleotide and multiple copies of A2 or a region of A2 are generated. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] In one aspect of the invention, there is provided a method for detecting a target polynucleotide sequence in a given nucleic acid analyte present in a sample, comprising: (a) i. single-stranded probe oligonucleotide A0; ii. pyrophosphoroase; and iii. Ligase introducing one or more nucleic acid analytes into a first reaction mixture comprising where A0 is pyrophosphorolyzed from the 3' end in the 3'-5' direction to generate an at least partially digested strand A1, and A1 undergoes ligation to form A2; (b) detecting a signal from the product of the previous step, where the product is A2 or a portion thereof, or multiple copies of A2 or multiple copies of a portion thereof, and inferring therefrom the presence or absence of the polynucleotide target sequence in the analyte. The method further comprises the steps of:

[0012] In some embodiments, the first reaction mixture further comprises a source of pyrophosphate ions. In some embodiments, the 3' end of A0 is perfectly complementary to the target polynucleotide sequence.

[0013] In some embodiments, the ligase substantially lacks single-stranded ligation activity. In some embodiments, the first reaction mixture further comprises at least one single-stranded primer oligonucleotide that is substantially complementary to a portion of A0 and deoxyribonucleotide triphosphates (dNTPs).

[0014] In some embodiments, the first reaction mixture further comprises an amplification enzyme. In some embodiments, the product of step (a) is introduced into a second reaction mixture prior to step (b), said second reaction mixture comprising at least one single-stranded primer oligonucleotide and dNTPs.

[0015] In some embodiments, the second reaction mixture further comprises an amplification enzyme. In some embodiments, one or more nucleic acid analytes may be introduced simultaneously into the first and second reaction mixtures.

[0016] In some embodiments, one or more nucleic acid analytes may be introduced sequentially into the first and second reaction mixtures. In some embodiments, the dNTPs are hot start dNTPs.

[0017] A hot-start dNTP is a dNTP that is modified at the 3' end with a heat-labile protecting group, the presence of which blocks DNA polymerase nucleotide incorporation until the nucleotide protecting group is removed using a heat activation step.

[0018] In some embodiments, during step (a), the analyte anneals to a single-stranded probe oligonucleotide A0 to generate 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.

[0019] In some embodiments, during step (a), the first intermediate product is pyrophosphorolyzed in the 3'-5' direction from the 3' end of A0 to generate a partially digested strand A1 and the analyte.

[0020] In some embodiments, the first reaction mixture further comprises a ligation probe oligonucleotide C, and the partially digested strand A1 is ligated at its 3' end to the 5' end of C to generate oligonucleotide A2.

[0021] In some embodiments, the partially digested strand A1 is circularized through the linkage of its 3' and 5' ends to generate oligonucleotide A2. In some embodiments, the linkage of A1 is: During step (a); or During step (b); or Between steps (a) and (b) occurs in.

[0022] In some embodiments, the first reaction mixture further comprises a 5'-3' exonuclease, and the 5' end of A0 is rendered resistant to 5'-3' exonuclease digestion.

[0023] In some embodiments, after amplification of the nucleic acid analyte of interest and prior to the addition of the first reaction mixture (step (a)), the sample is further treated with a proteinase. In some embodiments, the first reaction mixture further comprises a phosphatase or a phosphohydrolase.

[0024] In some embodiments, the product of the previous step is treated with pyrophosphatase prior to or during step (b). In some embodiments, prior to or during step (b), the product of the previous step is treated with an exonuclease.

[0025] In some embodiments, oligonucleotide C further comprises a 3' or internal modification that protects against 3'-5' exonuclease digestion. In some embodiments, oligonucleotide C further comprises a 5' modification that protects against 5'-3' exonuclease digestion.

[0026] In some embodiments, the first or second reaction mixture further comprises a splint oligonucleotide D. In some embodiments, D comprises a region of the oligonucleotide that is complementary to the 3' end of A1 and a region that is complementary to either the 5' end of oligonucleotide C or the 5' end of A1.

[0027] In some embodiments, D is unable to undergo extension relative to A1, either by a 3' modification or through a nucleotide mismatch between the 3' end of D and the corresponding region of A1.

[0028] In some embodiments, the enzyme that carries out pyrophosphorolysis of A0 to form the partially digested chain A1 also amplifies A2. In some embodiments, detection is accomplished using one or more oligonucleotide fluorescent conjugated dyes or molecular probes.

[0029] In some embodiments, the signal increase over time resulting from A2 amplicon production is used to infer the concentration of the target sequence in the analyte. In some embodiments, multiple probes A0 are used, each selective for a different target sequence and each containing an identifying region, and the amplicon derived from A2 contains this identifying region, and thus the target sequence present in the analyte is further characterized by being inferred through detection of the identifying region(s).

[0030] In some embodiments, detection of the identified region(s) is performed using molecular probes or through sequencing. In some embodiments, the final step of the method is: i. labeling the product of step (b) with one or more oligonucleotide fluorescent conjugated dyes or molecular probes; ii. measuring the fluorescent signal of the product; iii. exposing the product to a set of denaturing conditions; and Identifying polynucleotide target sequences in the analyte by monitoring changes in the fluorescent signal of the product during exposure to denaturing conditions. The method further comprises the steps of:

[0031] In some embodiments, one or more nucleic acid analytes are divided into multiple reaction volumes, with each volume having one or more probe oligonucleotides A0 introduced to detect a different target sequence.

[0032] In some embodiments, the different probes A0 contain a common priming site, allowing a single primer or a single set of primers to be used for amplification of the A2 region.

[0033] In some embodiments, a method for detecting a target polynucleotide sequence in a given nucleic acid analyte present in a sample comprises: (a) amplifying a predetermined nucleic acid analyte present in a sample; (b) i. single-stranded probe oligonucleotide A0; ii. pyrophosphoroase; and iii. Ligase introducing the product of step (a) into a first reaction mixture comprising where A0 is pyrophosphorolyzed from the 3' end in the 3'-5' direction to generate an at least partially digested strand A1, and A1 undergoes ligation to form A2; (c) detecting a signal from the product of the previous step, where the product is A2 or a portion thereof, or multiple copies of A2 or multiple copies of a portion thereof, and inferring therefrom the presence or absence of the polynucleotide target sequence in the analyte. The method further comprises the steps of:

[0034] 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 may be applied may be prepared from the above-mentioned biological sample by a series of preliminary steps designed to amplify the analyte and separate the analyte from background genomic DNA, which is typically present in significant excess. The method is generally applicable to the production of single-stranded target analytes and is therefore useful in situations other than those where it is combined with or further comprises parts of the method of the first aspect of the present invention. Thus, a method is provided for preparing at least one single-stranded analyte of a nucleic acid comprising a target polynucleotide region, characterized by the steps of (1) subjecting a biological sample comprising the analyte(s) and optionally background genomic DNA to an amplification cycle to produce an amplicon of the analyte(s). In a preferred embodiment, amplification is performed using polymerase chain reaction (PCR) in the presence of a polymerase, nucleoside triphosphates and at least one corresponding primer pair, where one of the primers includes a 5'-3' exonuclease blocking group, and (2) optionally digesting the product of step (1) with an exonuclease having 5'-3' exonuclease activity. In one embodiment, the method may further comprise the steps of (3) reacting the product of step (2) with a proteinase to destroy the polymerase, and then (4) deactivating the proteinase by heating the product of step (3) to a temperature above 50°C.

[0035] In one preferred embodiment, steps (1)-(4) are performed prior to step (a) of the method of the first aspect of the invention, resulting in an integrated method for detecting a target sequence from a biological sample. In another embodiment, the biological sample has undergone cell lysis prior to carrying out step (1).

[0036] In some embodiments of step (1), the nucleoside triphosphates are a mixture of the four deoxynucleoside triphosphates characteristic of naturally occurring DNA. In a preferred embodiment, the mixture of deoxynucleoside triphosphates includes deoxyuridine triphosphate (dUTP) instead of deoxythymidine triphosphate (dTTP), and step (1) is further carried out in the presence of the enzyme dUTP-DNA glycolase (UDG) to remove any contaminating amplicons from the previous assay. In yet another embodiment, a high fidelity polymerase, such as one sold under the trademark Phusion® or Q5, is used in step (1). In yet another embodiment, the polymerase may be KAPA HiFi Uracil+ DNA polymerase.

[0037] High-fidelity DNA polymerases have several safeguards to protect against both the creation and amplification of errors during DNA copying. These enzymes have a significant binding preference for correct nucleoside triphosphates over incorrect nucleoside triphosphates during polymerization. When an incorrect nucleotide binds to the polymerase active site, incorporation is slowed down due to a suboptimal structure of the active site complex. This delay time increases the chance that the incorrect nucleotide dissociates before the polymerase proceeds, thereby allowing the process to start again with the correct nucleoside triphosphate. If an incorrect nucleotide is inserted, a proofreading DNA polymerase provides an additional line of defense. The perturbation caused by the mispaired base is detected, and the polymerase moves the 3' end of the growing DNA strand into the proofreading 3'→5' exonuclease domain. Here, the incorrect nucleotide is removed by the 3'→5' exonuclease activity, at which point the strand is again transferred back to the polymerase domain where polymerization can continue.

[0038] In some embodiments, the nucleoside triphosphates are a mixture of synthetic or modified deoxynucleoside triphosphates. In some embodiments, the nucleoside triphosphates are a mixture of the four deoxynucleoside triphosphates and synthetic or modified deoxynucleotide triphosphates.

[0039] In some embodiments, step (1) is carried out using a limited amount of primers and an excess of amplification cycles. This means that a fixed amount of amplicon is produced regardless of the initial amount of analyte. Thus, the need for analyte quantification before the subsequent steps is avoided. In another embodiment of step (1), which has the advantage of eliminating the need for step (2), amplification is carried out in the presence of a primer pair, where one of the two primers is present in excess of the other, and when one primer is fully utilized, this results in the production of a single-stranded amplicon.

[0040] In a preferred embodiment of step (2), the 5' primer is blocked with an exonuclease blocking group selected from phosphorothioate linkages, inverted bases, DNA spacers and other oligonucleotide modifications commonly known in the art. In another embodiment, the other primer of the pair has a phosphate group at the 5' end.

[0041] In some embodiments, in step (3), the proteinase used is proteinase K, and the process is carried out by heating at a temperature of 80 to 100° C. for up to 30 minutes. In one embodiment, in step (3), the proteinase used is proteinase K, and step (3) is carried out by heating to a temperature of 55° C. for 5 minutes, and step (4) is carried out by heating to a temperature of 95° C. for 10 minutes. In another embodiment, at some point after step (2), the reaction medium is treated with a phosphatase or phosphohydrolase to remove any residual nucleoside triphosphates that may be present.

[0042] In some embodiments, the target polynucleotide sequence in the analyte is a gene or chromosomal region in the DNA or RNA of a cancerous tumor cell and will be characterized by the presence of one or more mutations; for example, mutations in the form of one or more single nucleotide polymorphisms (SNPs). Thus, the present invention will be useful in monitoring and / or treating disease recurrence. Patients who have been declared disease-free after treatment may be monitored over time to detect disease recurrence. This needs to be done non-invasively and requires sensitive detection of the target sequence from blood samples. Similarly, for some cancers, there are residual cancer cells remaining in the patient after treatment. The levels of these cells (or cell-free DNA) present in the patient's blood can be monitored using the present invention to detect disease recurrence or failure of current therapy and the need to switch to alternative therapy.

[0043] In some embodiments, detection of target polynucleotide sequences allows repeated testing of patient samples during disease treatment, and allows early detection of the development of resistance to therapy.For example, epidermal growth factor receptor (EGFR) inhibitors, such as gefitinib, erlotinib, are commonly used as first-line treatment for non-small cell lung cancer (NSCLC).During treatment, tumors will often develop mutations in the EGFR gene (e.g., T790M, C797S), which confer resistance to therapy.Early detection of these mutations allows patients to switch to alternative therapies.

[0044] In some embodiments, the target polynucleotide sequence in the analyte will be in a gene or chromosomal region within DNA or RNA of fetal origin and will be characterized by the presence of one or more mutations, e.g., mutations in the form of one or more single nucleotide polymorphisms (SNPs).Thus, the present invention can be used to detect mutations at very low allele rates at earlier stages of pregnancy than other available testing techniques.

[0045] In another embodiment, the target polynucleotide sequence may be a gene or genomic region derived from an otherwise healthy individual from which genetic information is obtained, which may aid in generating valuable companion diagnostic information and enable medical or therapeutic conclusions to be drawn across one or more defined groups within the human population.

[0046] In yet another embodiment, the target polynucleotide sequence may be characteristic of an infectious disease, or resistance of an infectious disease to treatment with a particular therapy; for example, a polynucleotide sequence characteristic of a bacterial or viral gene or chromosomal region, or a mutation therein that confers resistance to a therapy.

[0047] In some embodiments, the target polynucleotide sequence may be characteristic of donor DNA. When a transplanted organ is rejected by a patient, DNA from the organ is shed into the patient's bloodstream. Early detection of this DNA would allow early detection of rejection. This can be achieved by using a custom panel of donor-specific markers, or by using a panel of variants known to be common in the population, some present in the donor and some present in the recipient. Thus, routine monitoring of organ recipients over time is possible with the claimed method.

[0048] In yet another embodiment, various versions of the method using different combinations of probes (see below) can be used in parallel to simultaneously screen analytes for multiple target sequences; for example, cancer sources, cancer indicators, or multiple sources of infection.In this approach, the amplified products obtained by parallel application of the method are contacted with a detection panel consisting of 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 linking oligonucleotide in combination with one or more chemical and biological probes that are selective for target polynucleotide sequences, or the use of sequencing to identify amplified probe regions.

[0049] In some embodiments, the single-stranded probe oligonucleotide A0 comprises a priming region and a 3' end that is complementary to the target polynucleotide sequence to be detected. By this means, a first intermediate product that is at least partially double-stranded is generated. In one embodiment, this step is carried out in the presence of excess A0 and in an aqueous medium containing the analyte and any other nucleic acid molecules.

[0050] During step (a), the double-stranded region of the first intermediate product is pyrophosphorolyzed in the 3'-5' direction from the 3' end of its A0 strand. As a result, the A0 strand is progressively digested to produce a partially digested strand; hereafter referred to as A1. If the probe oligonucleotide erroneously hybridizes to a non-target sequence, the pyrophosphorolysis reaction will stop at any mismatch and prevent the subsequent steps of the method from proceeding. In another embodiment, this digestion is continued until A1 lacks sufficient complementarity to form a stable duplex with the analyte or a target region therein. At this point, the various strands then separate by melting, thereby generating a single-stranded form of A1. Under typical pyrophosphorolysis conditions, this separation occurs when there are between 6 and 20 complementary nucleotides between the analyte and A0.

[0051] In another embodiment, digestion continues until A1 lacks sufficient complementarity with the analyte or target region therein for pyrophosphorolysis enzyme to bind or for the pyrophosphorolysis reaction to continue. This typically occurs when there are between 6-20 complementary nucleotides between the analyte and the probe. In some embodiments, this occurs when there are between 6-40 complementary nucleotides remaining.

[0052] In another embodiment, where splint oligonucleotide D has complementarity to the 5' and 3' ends of A1 (see below), digestion continues until the length of complementarity between A1 and the target is reduced to the point where it is energetically favorable for oligo 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 region of complementarity between oligo D and the 3' end of A1, although longer complementarity between A1 and the analyte molecule is also possible due to the favorability of intramolecular hybridization of oligo D, which may already be hybridized to the 5' end of A1.

[0053] In another embodiment, where ligation of A1 is performed using the analyte molecule as a splint (see FIG. 16), digestion continues until the 5' end of A1 is capable of hybridizing to the analyte molecule such that the 3' and 5' ends of A1 are adjacent and separated only by a nick, at which point they are ligated together by a ligase and digestion can no longer proceed.

[0054] Suitably, pyrophosphorolysis is carried out in a reaction medium in the presence of at least one polymerase exhibiting pyrophosphorolytic activity and a source of pyrophosphate ions at a temperature in the range of 20-90° C. Further information regarding pyrophosphorolysis reactions as applied to polynucleotide digestion can be found, for example, in J. Biol. Chem. 244 (1969) pp. 3019-3028 or in our earlier patent applications.

[0055] In some embodiments, the pyrophosphorolysis step is driven by the presence of an excess source of polypyrophosphate, suitable sources include compounds containing three or more phosphorus atoms.

[0056] In some embodiments, the first reaction mixture includes an excess source of polypyrophosphate. In some embodiments, the pyrophosphorolysis step is driven by the presence of an excess source of modified pyrophosphate. Suitable modified pyrophosphates include those with other atoms or groups substituted in place of the bridging oxygens, or pyrophosphates (or polypyrophosphates) that contain substituents or modifying groups on the other oxygens. Those of skill in the art will appreciate that there are many such examples of modified pyrophosphates suitable for use in the present invention, a non-limiting selection of which include:

[0057] [ka]

[0058] It is. In some embodiments, the first reaction mixture includes an excess of a source of modified polypyrophosphate.

[0059] In a preferred embodiment, the source of pyrophosphate ions is PNP, PCP or tripolyphosphate (PPPi). Further, non-limiting examples of sources of pyrophosphate ions for use in the pyrophosphorolysis step (b) can be found in WO2014 / 165210 and WO00 / 49180.

[0060] In some embodiments, the source of excess modified pyrophosphate can also be represented as YH, where Y is represented by the general formula (XO)P(=B)-(ZP(=B)(OX)). n -, where n is an integer from 1 to 4; each Z- is independently selected from -O-, -NH-, or -CH2-; each B is independently either O or S; and each X group is independently selected from -H, -Na, -K, an alkyl, alkenyl, or heterocyclic group, with the proviso that when both Z and B correspond to -O- and n is 1, at least one X group is not H.

[0061] In some embodiments, Y has the general formula (XO)P(=B)-(ZP(=B)(OX)) n -, where n is 1, 2, 3 or 4. In another embodiment, the Y group corresponds to the general formula (XO)2P(=O)-ZP(=O)(OH)-, where X One of the groups is -H. In yet another preferred embodiment, Y corresponds to the general formula (XO)2P(=O)-ZP(=O)(OX)--, where at least one of the X groups is selected from methyl, ethyl, allyl, or dimethylallyl.

[0062] In another embodiment, Y corresponds to the general formula (HO)P(=O)-ZP(=O)(OH)-, where Z is -NH- or -CH-, or to either (XO)P(=O)-ZP(=O)(OX)-, where all X groups are either -Na or -K and Z is either -NH- or -CH-.

[0063] In another embodiment, Y corresponds to the general formula (HO)2P(=B)-OP(=B)(OH)-, where each B group is independently either O or S, with at least one being S.

[0064] Specific examples of preferred embodiments of Y include those of the formula (X1-O)(HO)P(=O)-ZP(=O)(O-X2), where 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.

[0065] In some embodiments, when a molecular probe is to be used for detection, the probe oligonucleotide A0 is designed to contain an oligonucleotide identification region 5' to the region complementary to the target sequence, and the molecular probe used is designed to anneal to this identification region. In one embodiment, only the 3' region of A0 is capable of annealing to the target; i.e., any other region lacks sufficient complementarity with the analyte for a stable duplex to exist at the temperature at which the pyrophosphorolysis step is performed. Here and throughout, by the term "sufficient complementarity" it is meant that the region of complementarity is longer than 10 nucleotides in length, so long as the given region has complementarity with the given region on the analyte.

[0066] In a further aspect of the method of the invention, an alternative embodiment is provided in which the pyrophosphorolysis step of any of the previous embodiments is replaced with an exonuclease digestion step using a double-stranded specific exonuclease. Those skilled in the art will appreciate that double-stranded specific exonucleases include, among others, those that read in the 3'-5' direction, e.g., exoIII, and those that read in the 5'-3' direction, e.g., lambda exo.

[0067] In some embodiments of the invention where the exonuclease digestion step utilizes a double strand specific 5'-3' exonuclease, it is the 5' end of A0 that is complementary to the target analyte, and the common priming sequence and blocking group are located 3' to the region complementary to the target. In further embodiments where a molecular probe is to be used for detection, the probe oligonucleotide A0 is designed to contain an oligonucleotide identification region 3' to the region complementary to the target sequence, and the molecular probe used is designed to anneal to this identification region.

[0068] In embodiments of the invention in which the exonuclease digestion step utilises a double-strand specific 5'-3' exonuclease, an exonuclease having 3' to 5' exonuclease activity may optionally be added to the reaction mixture for the purpose of leaving any material comprising A0 and the partially digested strand A1 intact whilst digesting any other nucleic acid molecules present. Suitably, this resistance to exonucleolysis is achieved as described elsewhere in this application.

[0069] In one preferred embodiment of the invention, the 5' end of A0 or an internal site 5' to the priming region is rendered resistant to exonucleolysis. By this means, and after or simultaneously with the pyrophosphorolysis step, an exonuclease with 5'-3' exonuclease activity may optionally be added to the reaction medium in order to digest any other nucleic acid molecules present while leaving intact any material comprising A0 and the partially digested strand A1. Suitably, this resistance to exonucleolysis is achieved by introducing one or more blocking groups into the oligonucleotide A0 at the required points. In one embodiment, these blocking groups may be selected from phosphorothioate linkages and other backbone modifications commonly used in the art, C3 spacers, phosphate groups, modified bases, etc.

[0070] In some embodiments, the identification region will comprise or be embedded within a barcode region that has a unique sequence and is adapted to be indirectly identified using a sequence-specific molecular probe applied to the amplified components A2, or directly identified by sequencing of these components. Examples of molecular probes that may be used include, but are not limited to, molecular beacons, TaqMan® probes, Scorpion® probes, and the like.

[0071] In some embodiments, the A2 strand or a desired region thereof is made to undergo amplification so that many copies, typically millions, are made. This is accomplished by priming A2 and any subsequent amplicons derived therefrom with a single-stranded primer oligonucleotide, provided, for example, in the form of a forward / reverse or sense / antisense pair, capable of annealing to a complementary region thereon. The primed strand then becomes the origin for amplification. Amplification methods include, but are not limited to, thermal cycling and isothermal methods, such as polymerase chain reaction, recombinase polymerase amplification and rolling circle amplification; the last of these is applicable when A2 is circularized. By any of these means, many amplicon copies of a region of A2 and, in some cases, its sequence complement, can be rapidly generated. The exact methodology for carrying out any of these amplification methods is well known to those of ordinary skill in the art, and the exact conditions and temperature regimes used are readily available in the general literature, to which reference may be made. In particular, in the case of polymerase chain reaction (PCR), the methodology generally involves extending a primer oligonucleotide against the A2 strand in the 5'-3' direction using a polymerase and a source of various single nucleoside triphosphates until a complementary strand is produced; dehybridizing the double-stranded product produced to regenerate the A2 strand and the complementary strand; repriming both the A2 strand and its amplicon, and then repeating these extension / dehybridization / repriming steps multiple times to increase the concentration of the A2 amplicon to a level that can be reliably detected.

[0072] In another embodiment, the first or second reaction mixture further comprises components of a hybridization chain reaction (HCR). In some embodiments, the first reaction mixture includes a ligated probe oligonucleotide C with a 5' phosphate, a splint oligonucleotide D complementary to the 3' end of A1 and the 5' end of C, and the partially digested strand A1 is ligated at its 3' end to the 5' end of C to form oligonucleotide A2. In some embodiments, the second reaction mixture further includes a hairpin oligonucleotide 1 (HO1) and a hairpin oligonucleotide 2 (HO2), each of which includes a fluorophore and a quencher such that if each oligonucleotide remains in the hairpin configuration, the fluorophore and quencher will contact each other. HO1 is designed such that A2 will anneal to HO1, opening the "hairpin" structure and separating the fluorophore from the quencher. Now the "open" HO1 is able to anneal to HO2, opening the "hairpin" structure and separating the fluorophore from the quencher. Separate from the quencher.

[0073] In some embodiments, multiple hairpin oligonucleotides are present such that the presence of one A2 can trigger a chain reaction that opens the hairpin oligonucleotide, resulting in the production of a detectable fluorescent signal. This methodology is known in the literature as hybridization chain reaction (HCR).

[0074] In some embodiments, the fluorophore of the fluorophore-quencher pair is selected from, but not limited to, the fluorescein family, the carboxyrhodamine family, the cyanine family, and the rhodamine family of dyes. Other families of dyes that can be used include, for example, polyhalofluorescein family dyes, hexachlorofluorescein family dyes, coumarin family dyes, oxazine family dyes, thiazine family dyes, squaraine family dyes, chelated lanthanide family dyes, the dye family available under the trade name Alexa Fluor J from Molecular Probes, the dye family available under the trade name Atto from ATTO-TEC (Siegen, Germany), and the dye family available under the trade name Bodipy J 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), tetrapropano-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 commercially available, for example, from Perkin-Elmer (Foster City, Calif.), Molecular Probes, Inc. (Eugene, Oregon), and Amersham GE Healthcare (Piscataway, New Jersey).

[0075] In some embodiments, the quencher of the fluorophore-quencher pair may 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), anthraquinones, malachite green, nitrothiazole, and nitroimidazole compounds. Exemplary non-fluorescent quenchers that dissipate energy absorbed from a fluorophore include those sold under the trade name Black Hole by Biosearch Technologies, Inc. (Navato, Calif.). TM Available under the trade name Eclipse, of Epoch Biosciences, Bothell, WA. TM . those available under the trade name Qx1J from Anaspec, Inc., San Jose, Calif.; those available under the trade names ZEN and TAO from Integrated DNA Technologies, Coralville, Iowa; and those available under the trade name Iowa Black from Integrated DNA Technologies, Coralville, Iowa. TM Includes those available under:

[0076] In some embodiments, the fluorophore of the fluorophore-quencher pair is a fluorescein. In some embodiments, the pigments may be 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinimidyl 1-pyrenebutyrate, and 4-acetamido-4'-isothiocyanatostilbene-2-,2'-disulfonic acid derivatives.

[0077] In some embodiments, the fluorophore of the fluorophore-quencher pair may be LC-Red640, LC-Red705, Cy5, Cy5.5, lisamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriaminepentaacetic acid, or other chelates of lanthanide ions (e.g., europium or terbium).

[0078] In some embodiments, the present invention utilizes fluorescently labeled oligonucleotides that are dual quenched. The inclusion of a second internal quencher shortens the distance between the dye and the quencher and, in concert with the first quencher, provides greater overall dye quenching, reducing background and increasing signal detection. The second and first quenchers can be any of the quenchers described above.

[0079] In some embodiments, the first reaction mixture contains 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 strand A1 is ligated at its 3' end to the 5' end of C to form oligonucleotide A2.

[0080] In some embodiments, the 5' and 3' ends of A1 are linked together to form a circularized A2. In some embodiments, A1 is circularized to a ligation probe oligonucleotide C to form A2.

[0081] In some embodiments, A1 is circularized to the target sequence to form A2. In some embodiments, A1 is cyclized as described above or below to form A2.

[0082] In some embodiments, A2 is formed from partially digested chain A1, as described above or below. In some embodiments, the second reaction mixture comprises: - oligonucleotide A comprising a substrate arm, a partial catalytic core and a sensor arm; - an oligonucleotide B comprising a substrate arm, a partial catalytic core and a sensor arm; and - A substrate containing a fluorophore-quencher pair further comprising; Here, the sensor arms of oligonucleotides A and B are complementary to the flanking regions of A2 such that in the presence of A2, oligonucleotides A and B combine to form a catalytic multicomponent nucleic acid enzyme (MNAzyme). In this embodiment, the MNAzyme forms only in the presence of A2 and cleaves a substrate that contains a fluorophore-quencher pair such that a detectable fluorescent signal is generated.

[0083] In some embodiments, the fluorophore-quencher pair is as described above. In some embodiments, the first and second reaction mixtures are combined. In some embodiments, the first and second reaction mixtures are combined such that pyrophosphorolysis, ligation, and production of a detectable fluorescent signal occur without the addition of further reagents.

[0084] In another embodiment, the second reaction mixture further comprises one or more DNAzymes. In some embodiments, the second reaction mixture further comprises a nucleic acid construct that is partially double-stranded, wherein: - one strand contains at least one RNA base, at least one fluorophore, and a region of this strand is complementary to a region of A2, and this strand may be referred to as the "substrate" strand; - the other strand comprises at least one quencher, and one region of this strand is complementary to a region of A2 adjacent to the region to which the substrate strand is complementary, such that in the presence of A2, the partially stranded nucleic acid construct becomes substantially more double stranded; Here, in the process of becoming substantially more double-stranded, the substrate strand of the double-stranded nucleic acid construct is cleaved at an RNA base such that at least one quencher on the "other" strand is no longer in sufficient proximity to at least one fluorophore on the substrate strand, resulting in fluorescence.

[0085] In other words, a partially double-stranded nucleic acid construct will have a double-stranded portion of greater size in the presence of A2. In some embodiments, the fluorophore-quencher pair may be as described above.

[0086] In some embodiments, additional reagents, such as appropriate buffers and / or ions, are present in the second reaction mixture. In some embodiments, the second reaction mixture comprises Mg 2+ It further comprises ions.

[0087] In some embodiments, the second reaction mixture comprises Zn 2+ It further comprises ions. In some embodiments, the second reaction mixture comprises X 2+ ion, and X is a metal.

[0088] In some embodiments, the second reaction mixture comprises one or more X 2+ ion, and X is a metal. In another embodiment, the first reaction mixture further comprises reagents for ligase chain reaction (LCR).

[0089] In such embodiments, the first reaction mixture comprises: - one or more ligases; and - two or more LCR probe oligonucleotides that are complementary to adjacent sequences on A2, where the 5' phosphate of one LCR probe is directly adjacent to the 3' OH of the other LCR probe when the probes are successfully annealed to A2. Further includes:

[0090] In some embodiments, in the presence of A2, the two LCR probes successfully anneal to A2 and are ligated together to form one oligonucleotide molecule, which then serves as a new target for a second round of covalent ligation, leading to geometric amplification of the target of interest, in this case A2. The ligated product or amplicon is complementary to A2 and serves as a target in the next cycle of amplification. Thus, specific Exponential amplification of the target DNA sequence is achieved through repeated cycles of denaturation, hybridization, and ligation in the presence of excess LCR probes, which infers the presence of A2, and thus the target polynucleotide sequence.

[0091] In some embodiments, in the presence of A2, the two PCR probes successfully anneal to A2 and are ligated together to form one oligonucleotide molecule, which then serves as a new target for a second round of covalent ligation, leading to geometric amplification of the target of interest, in this case A2, which is then detected.

[0092] In some embodiments, the ligated oligonucleotide molecules are detected in real time using an intercalating dye or molecular probe. In some embodiments, the ligated oligonucleotide molecules are detected using gel electrophoresis.

[0093] Those skilled in the art will recognize that there are many techniques that allow for the detection of ligated oligonucleotide molecules. In some embodiments, the dNTPs are hot start dNTPs.

[0094] In some embodiments, one or more ligases are thermostable. In some embodiments, one or more of the ligases are naturally occurring. In another embodiment, one or more ligases are engineered.

[0095] In some embodiments, the one or more ligases are selected from any of the ligases disclosed above or subsequently. In some embodiments, one or more polymerases are thermostable.

[0096] In some embodiments, the one or more polymerases are selected from any of the polymerases disclosed above or subsequently. In some embodiments, the one or more polymerases are naturally occurring.

[0097] In some embodiments, one or more polymerases are engineered. In some embodiments, the one or more polymerases are the same as those used for pyrophosphorolysis.

[0098] In some embodiments, one or more enzymes of the present invention are hot-start enzymes. In some embodiments, one or more enzymes of the present invention are thermostable.

[0099] In another embodiment, the product of step (a) is introduced prior to step (b) into a second reaction mixture, which further comprises reagents for ligase chain reaction (LCR). In this embodiment, the second reaction mixture comprises -one or more ligases; - optionally one or more polymerases; - two or more LCR probe oligonucleotides that are complementary to adjacent sequences on A2, such that the 5' phosphate of one LCR probe is directly adjacent to the 3' OH of the other LCR probe when the probes are successfully annealed to A2; - optionally, one or more primers for amplification of successfully ligated LCR probes; and -Optionally dNTPs Includes.

[0100] In some embodiments, in the presence of A2, the two LCR probes are successfully annealed to A2 and ligated together to form one oligonucleotide molecule, which then serves as the new target for the second round of covalent ligation, leading to the geometric amplification of the target of interest, in this case A2. The ligated product, or amplicon, is complementary to A2 and serves as the target in the next cycle of amplification. Thus, the exponential amplification of specific target DNA sequence is achieved through repeated cycles of denaturation, hybridization, and ligation in the presence of excess LCR probe. This infers the presence of A2, and therefore the presence of target polynucleotide sequence.

[0101] In some embodiments, in the presence of A2, the two PCR probes successfully anneal to A2 and are ligated together to form one oligonucleotide molecule, which then serves as a new target for a second round of covalent ligation, leading to geometric amplification of the target of interest, in this case A2, which is then detected.

[0102] In some embodiments, the ligated oligonucleotide molecules are detected in real time using an intercalating dye or molecular probe. In some embodiments, the ligated oligonucleotide molecules are detected using gel electrophoresis.

[0103] Those skilled in the art will recognize that there are many techniques that allow for the detection of ligated oligonucleotide molecules. In some embodiments, the dNTPs are hot start dNTPs.

[0104] In some embodiments, one or more ligases are thermostable. In some embodiments, one or more of the ligases are naturally occurring. In another embodiment, one or more ligases are engineered.

[0105] In some embodiments, the one or more ligases are selected from any of the ligases disclosed above or subsequently. In some embodiments, one or more polymerases are thermostable.

[0106] In some embodiments, the one or more polymerases are selected from any of the polymerases disclosed above or subsequently. In some embodiments, the one or more polymerases are naturally occurring.

[0107] In another embodiment, one or more polymerases are engineered. In some embodiments, the one or more polymerases are the same as those used for pyrophosphorolysis.

[0108] In some embodiments, the second reaction mixture further comprises a pyrophosphatase in addition to the one or more polymerases. In some embodiments, one or more enzymes of the present invention are hot-start enzymes.

[0109] In some embodiments, one or more enzymes of the present invention are thermostable. In another embodiment, the first reaction mixture comprises: - an oligonucleotide complementary to a region of A2 containing a linking site, the fluorescence of which is different from that of the other the oligonucleotide comprising one or multiple fluorophores positioned to be quenched either by proximity to said oligonucleotide or to one or more fluorescent quenchers; -Double-strand specific DNA digesting enzyme further comprising; In the presence of A2, the fluorophores are separated from each other and from the corresponding quenchers and the labeled oligonucleotide is digested such that the fluorescent signal, and thus the presence of A2, is detectable.

[0110] In some embodiments, the fluorophore and quencher may be as described above. In some embodiments, the double-strand specific DNA digestive enzyme is an exonuclease. In other embodiments, it is a polymerase with proofreading activity. In other embodiments, the second reaction mixture comprises a mixture of one or more of: an exonuclease or a polymerase with proofreading activity.

[0111] In some embodiments, the double-strand-specific DNA digestive enzyme is a hot-start enzyme. In some embodiments, the double-strand-specific DNA digestive enzyme has reduced activity at the temperature at which the pyrophosphorolysis reaction of the method occurs.

[0112] In some embodiments, the double-strand-specific DNA digestive enzyme has no activity at the temperature at which the pyrophosphorolysis reaction of the method occurs. In another embodiment, the second reaction mixture comprises: - an oligonucleotide complementary to a region of A2 containing the linking site, said oligonucleotide comprising one or multiple fluorophores arranged such that their fluorescence is quenched either by proximity to each other or to one or more fluorescence quenchers; -Double-strand specific DNA digesting enzyme further comprising; In the presence of A2, the fluorophores are separated from each other and from the corresponding quenchers and the labeled oligonucleotide is digested such that the fluorescent signal, and thus the presence of A2, is detectable.

[0113] In some embodiments, the fluorophore and quencher may be as described above. In some embodiments, the double-strand specific DNA digestive enzyme is an exonuclease. In other embodiments, it is a polymerase with proofreading activity. In other embodiments, the second reaction mixture comprises a mixture of one or more of: an exonuclease or a polymerase with proofreading activity.

[0114] In some embodiments, the double-strand-specific DNA digestive enzyme is a hot-start enzyme. In some embodiments, the double-strand-specific DNA digestive enzyme has reduced activity at the temperature at which the pyrophosphorolysis reaction of the method occurs.

[0115] In some embodiments, the double-strand-specific DNA digestive enzyme has no activity at the temperature at which the pyrophosphorolysis reaction of the method occurs. In some embodiments, the first or second reaction mixture further comprises one or more partially double-stranded DNA constructs, each construct containing one or more fluorophores and one or more quenchers. In some embodiments, when the construct is partially double-stranded, sufficient quenching of the one or more fluorophores is The one or more fluorophores and one or more quenchers are positioned in sufficient proximity so that quenching occurs.

[0116] In some embodiments, the construct loops back on itself. It is a single strand of DNA that contains a region of self-complementarity (on itself). In some embodiments, the construct comprises one primer of a primer pair.

[0117] In some embodiments, the first or second reaction mixture further comprises the other primer of the primer pair. In some embodiments, a portion of the single-stranded section of the construct hybridizes to A2 and is extended to A2 by DNA polymerase. In some embodiments, the other primer of the primer pair then hybridizes to the extended construct. This primer is then extended to the construct to displace the self-complementary region. Thus, the one or more fluorophores and the one or more dyes are sufficiently separated for a fluorescent signal to be detected, indicating the presence of A2 in the reaction mixture.

[0118] In such embodiments, the construct may be known as a Sunrise primer. In some embodiments, the construct comprises two separate DNA strands. In some embodiments, a portion of the single-stranded section of the construct hybridizes to A2 and is extended by DNA polymerase toward A2. In some embodiments, the other primer of the primer pair then hybridizes to the extended construct. This primer is then extended toward the construct in the direction of the double-stranded section, displacing the shorter DNA strand, and thus the one or more fluorophores and one or more dyes are sufficiently separated for a fluorescent signal to be detected, indicating the presence of A2 in the reaction mixture.

[0119] In such embodiments, the construct may be known as a Molecular Zipper. Those skilled in the art will recognize that for both the Sunrise primer and the Molecular Zipper, one or more fluorophore and one or more quencher pairs can be located at various positions within each respective construct. The important feature is that each pair is located close enough to each other that in the absence of A2, i.e., when extension and strand displacement do not occur, no fluorescent signal is emitted.

[0120] In some embodiments, prior to step (a) of the method, any RNA templates present in the sample are used to form corresponding DNA sequences. In some embodiments, this is accomplished through the use of reverse transcriptase and appropriate nucleotides.

[0121] In some embodiments, the formation of DNA from any RNA template present occurs simultaneously with any pre-amplification via PCR of nucleic acids present in the sample. In some embodiments, the formation of DNA from any RNA template present occurs in a step separate from any pre-amplification via PCR of nucleic acids present in the sample.

[0122] In some embodiments of any of the methods described above or subsequently, the RNA present in the sample is not converted to a corresponding DNA sequence. In such embodiments, A0 undergoes pyrophosphorolysis of the RNA sequence to form a partially digested strand A1, and the method then proceeds as described above or subsequently.

[0123] In some embodiments of any of the methods described above or subsequently, one or more reaction mixtures may be combined. In some embodiments, the first reaction mixture further comprises a ligation probe oligonucleotide C, and the partially digested strand A1 is ligated at its 3' end to the 5' end of C, while in other embodiments, A1 is circularized through ligation of its 3' and 5' ends; In each case oligonucleotide A2 is formed.

[0124] In one embodiment, the linkage of A1 is: During step (a); or During step (b); or Between steps (a) and (b) occurs in.

[0125] In one embodiment, A1 is optionally extended in the 5'-3' direction prior to ligation. In some embodiments, this optional extension and ligation is performed to the target oligonucleotide, while in other embodiments, they are performed through the addition of an additional splint oligonucleotide D to which A1 anneals prior to extension and / or ligation. In one embodiment, D comprises an oligonucleotide region complementary to the 3' end of A1 and a region complementary to either the 5' end of oligonucleotide C or the 5' end of A1. In another embodiment, D is non-extendable relative to A1 due to a 3' end modification or through a nucleotide mismatch between the 3' end of D and the corresponding region of A1.

[0126] In some embodiments, ligation probe C has a 5' region complementary to at least a portion of the 5' end region of splint oligonucleotide D or to the target oligonucleotide. By such means, a second intermediate product is formed, in which A2 strand is composed of A1, C and optionally an intermediate region formed by extension of A1 in the 5'-3' direction to meet the 5' end of C. In such embodiments, the primers used in step (c) (see below) are selected to amplify at least the region of A2 that includes the site where ligation of A1 to C occurs. In this embodiment, the inventors have found it suitable to include a 3' blocking group on C so that any unligated A1 can be digested prior to amplification using a 3'-5' exonuclease. Suitable polymerases that can be used for extension prior to ligation include, but are not limited to, Hemo KlenTaq, Mako and Stoffel fragment.

[0127] 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 is able to continue and is not overpowered by the forward polymerization reaction.

[0128] In some embodiments, prior to or during step (b), the product of the previous step is treated with a pyrophosphatase to hydrolyze pyrophosphate ions, prevent further pyrophosphorylation reactions from occurring, and support the forward polymerization reaction.

[0129] In some embodiments, prior to or during step (b), the product of the previous step is treated with an exonuclease. In some embodiments, the enzyme that pyrophosphorolysis A0 to form the partially digested chain A1 also amplifies A2. One of skill in the art will recognize that many such enzymes exist.

[0130] Amplicons are detected and the resulting information is used to identify the polynucleotide sequence of the original analyte. The presence or absence of a target sequence and / or associated characteristics of the target sequence can be inferred by this means. For example, a target sequence characteristic of a cancerous tumor cell can be detected by reference to the specific SNP being sought. In another embodiment, a target sequence characteristic of a virus or bacterial genome (including novel mutations thereof) can be detected. Many methods of detecting amplicons or identifying regions can be used, including, for example, oligonucleotide-binding dyes, sequence-specific molecular probes, such as fluorescently labeled molecular beacons or hairpin probes. Alternatively, direct sequencing of the A2 amplicon can be performed using one of the direct sequencing methods used or reported in the art. When using oligonucleotide-binding dyes, fluorescently labeled beacons or probes, it is preferred to detect the amplicon using an arrangement that includes a source of stimulating electromagnetic radiation (laser, LED, lamp, etc.) and a light detection device arranged to detect the emitted fluorescence and generate a signal comprising a data stream therefrom that can be analyzed by a microprocessor or computer using a specifically designed algorithm.

[0131] In some embodiments, detection is accomplished using one or more oligonucleotide fluorescent binding dyes or molecular probes. In these embodiments, the increase in signal over time resulting from A2 amplicon production is used to infer the concentration of target sequence in the analyte. In one embodiment, the final step of the method is: i. labeling the product of step (b) with one or more oligonucleotide fluorescent conjugated dyes or molecular probes; ii. measuring the fluorescent signal of the product; iii. exposing the product to a set of denaturing conditions; and Identifying polynucleotide target sequences in the analyte by monitoring changes in the fluorescent signal of the product during exposure to denaturing conditions. The method further comprises the steps of:

[0132] In some embodiments, multiple probes A0 are used, each selective for a different target sequence and each containing an identifying region and further characterized by the inclusion of the identifying region in the amplicon of A2, and thus the target sequence present in the analyte is inferred through detection of the identifying region(s). In such embodiments, detection of the identifying region(s) is performed with a molecular probe or through sequencing.

[0133] In some embodiments, one or more nucleic acid analytes are divided into multiple reaction volumes, with each volume having one or more probe oligonucleotides A0 introduced to detect a different target sequence.

[0134] In some embodiments in which different probes A0 are used, the different probes A0 contain one or more common priming sites, allowing a single primer or a single primer set to be used for amplification.

[0135] In another aspect of the invention, there is provided a method for identifying a target polynucleotide sequence in a given nucleic acid analyte characterized by the steps of any of the previous aspects of the invention, wherein multiple copies of A2 or a region of A2 are labeled with one or more oligonucleotide fluorescent binding dyes or molecular probes. The fluorescent signals of these multiple copies are measured, and the multiple copies are exposed to a set of denaturing conditions. The target polynucleotide sequence is identified by monitoring the change in the fluorescent signal of the multiple copies during exposure to the denaturing conditions.

[0136] In some embodiments, the temperature is changed, e.g., to a point where the duplex begins to dissociate. Denaturing conditions may be provided by increasing the pH of the solution, such that the conditions are acidic or alkaline, or by adding additives or agents such as strong acids or bases, concentrated inorganic salts, or organic solvents such as alcohols.

[0137] In another aspect of the invention, there is provided the use of the above method for screening a mammalian subject, particularly a human patient, for the presence of an infectious disease, cancer, or for the purposes of generating companion diagnostic information.

[0138] In a further aspect of the present invention, there is provided a control probe for use in the method as described above. The embodiments of the present invention include those in which the presence of a specific target sequence or sequences is revealed by the generation of a fluorescent signal. In such embodiments, there will necessarily be a level of signal generated from non-target DNA present in the sample. For a given sample, this background signal will have a later onset than the "true" signal, but this onset may vary between samples. Accurate detection of the presence of low concentrations of a target sequence or sequences therefore relies on knowledge of what signal is expected in its absence. While references are available for the intended sample, this is not true for a true "blind" sample from a patient. A control probe (E0) is utilized to determine the expected background signal profile for each assay probe. The control probe targets a sequence that is expected not to be present in the sample, and the signal generated from this probe may then be used to infer the expected rate of signal generation from the sample in the absence of the target sequence.

[0139] therefore: a. repeating the steps of the method, either subsequently or simultaneously, with a separate aliquot of the sample or in the same aliquot and using a second detection channel, using a second single-stranded probe oligonucleotide E0 having a 3'-end region that is at least partially mismatched to the target sequence; b. Estimate the background signal expected to be generated from A in the absence of any target analyte in the sample; and c. Inferring the presence or absence of a polynucleotide target sequence in the analyte through a comparison of the expected background signal inferred in (a) with the actual signal observed in the presence of the target analyte. A method for detecting a target polynucleotide sequence in a given nucleic acid analyte is provided according to any of the methods described above characterized by the steps:

[0140] In some embodiments, the control probe (E0) and A0 are added to separate portions of the sample, while in other embodiments, E0 and A0 are added to the same portion of the sample and the respective signals are measured using different detection channels (e.g., different colored dyes). The signal generated by E0 may then be used to estimate and correct for the background signal expected to be generated by A0 in the absence of the polynucleotide target sequence in the sample. For example, correcting for background signal may involve subtracting the signal observed from E0 from that observed from A0, or through calibration of the signal observed from A0 with a calibration curve of the relative signals generated by A0 and E0 under various conditions.

[0141] In some embodiments, a single E0 may be used to calibrate all of the assay probes that may be produced. In some embodiments, a separate E0 may be used to calibrate each amplicon of the sample DNA generated in the first amplification step. Each amplicon contains multiple mutations / mutations of interest. A single E0 may contain the target sequence, but will be sufficient to calibrate all of the assay probes to a single amplicon.

[0142] In further embodiments, a separate E0 may be used for each target sequence. For example, when targeting a C>T mutation, an E0 may be designed to target a C>G mutation at the same site that is not known to occur in patients. The signal profile generated by E0 under various conditions may be evaluated in calibration reactions, and these data may be used to infer the expected signal from an assay probe targeting a C>T mutation in the absence of the mutation.

[0143] Several embodiments of the methods of the present invention can be seen in Figures 17-20. In Figure 17, a single-stranded probe oligonucleotide A0 anneals to a target polynucleotide sequence to generate 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 molecules of A0 and one target polynucleotide sequence present to illustrate how A0 that did not anneal to the target does not participate in further steps of the method. In this illustrative example, the 3' end of A0 anneals to the target polynucleotide sequence, while the 5' end of A0 does not anneal. The 5' end of A0 includes a 5' chemical blocking group, a common priming sequence, and a barcode region.

[0144] The first intermediate product, which is partially double-stranded, undergoes pyrophosphorolysis in the 3'-5' direction from the 3' end of A0 in the presence of pyrophosphorolysis enzyme to generate partially digested strand A1, the analyte, and undigested A0 molecules that did not anneal to the target.

[0145] In Figure 18, A1 is annealed to single-stranded trigger oligonucleotide B, and the A1 strand is extended in the 5'-3' direction relative to B to generate oligonucleotide A2. In this illustrative example, trigger oligonucleotide B has a 5' chemical block. Any undigested A0 anneals to trigger oligonucleotide B, but is unable to extend in the 5'-3' direction relative to B to generate a sequence that is a target for later portions of the method. In this example, A2 is primed with at least one single-stranded primer oligonucleotide, and multiple copies of A2 or a region of A2 are generated.

[0146] In Figure 19, A1 is annealed to splint oligonucleotide D and then circularized by ligation of its 3' and 5' ends. The now circularized A2 is primed with at least one single-stranded primer oligonucleotide and generates multiple copies of A2 or a region of A2. In this illustrative example, splint oligonucleotide D is unable to extend onto A1 due to a 3' modification (chemical in this example) or through a nucleotide mismatch between the 3' end of D and the corresponding region of A2.

[0147] In Figure 20, the 3' region of splint oligonucleotide D anneals to the 3' region of A1, while the 5' region of splint oligonucleotide D anneals to the 5' region of ligation probe C. Thus, a second intermediate product A2 is formed, comprised of A1, C, and optionally an intermediate region formed by extension of A1 in the 5'-3' direction to meet the 5' end of C. In this illustrative example, ligation probe C has a 3' chemical blocking group such that any unligated A1 can be digested using a 3'-5' exonuclease.

[0148] A2 is primed with at least one single-stranded primer oligonucleotide and multiple copies of A2 or a region of A2 are generated. The specificity of the method of the present invention may be improved by the introduction of a blocking oligonucleotide. For example, a blocking oligonucleotide may be introduced to hybridize to at least a portion of the wild-type DNA to promote annealing of A0 only to the target polynucleotide sequence and not to the wild-type. Alternatively or additionally, a blocking oligonucleotide may be used to improve the specificity of the polymerase chain reaction (PCR) to prevent the amplification of any wild-type sequence present. A common technique used is to design an oligonucleotide that anneals between the PCR primers and cannot be displaced or digested by the PCR polymerase. The oligonucleotide is designed to anneal to the non-target (usually healthy) sequence while being mismatched (often by a single base) to the target (mutant) sequence. This mismatch results in different melting temperatures for the two sequences, and the oligonucleotide is designed to dissociate from the target sequence while remaining annealed to the non-target sequence at the PCR extension temperature.

[0149] Blocking oligonucleotides often may have modifications to prevent digestion by the exonuclease activity of PCR polymerase or to increase the melting temperature difference between target and non-target sequences.

[0150] Incorporation of locked nucleic acids (LNAs) or other melting temperature altering modifications into blocking oligonucleotides can significantly increase the difference in melting temperature of the oligonucleotide for target and non-target sequences.

[0151] Thus, an embodiment of the invention is provided that uses blocking oligonucleotides. The blocking oligonucleotides must be resistant to pyrophosphorolysis (PPL) reactions to ensure that they are not digested and displaced. This can be achieved in several different ways, for example, either through mismatches at the 3' end or through modifications such as phosphorothioate linkages or spacers.

[0152] In those embodiments or aspects of the invention using blocking oligonucleotides, the method of detecting a target polynucleotide sequence in a given nucleic acid analyte is characterized by annealing a single-stranded blocking oligonucleotide to at least a subset of the non-target polynucleotide sequences prior to or during the same step in which the analyte target sequence is annealed to a 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.

[0153] In some embodiments, blocking oligonucleotides are made to be resistant to pyrophosphorolysis through a mismatch at the 3' end. In other embodiments, blocking oligonucleotides are made to be resistant through the presence of a 3' blocking group. In other embodiments, blocking oligonucleotides are made to be resistant through the presence of a spacer or other internal modification. In further embodiments, blocking oligonucleotides include both modifications that increase the melting temperature and are rendered resistant to pyrophosphorolysis.

[0154] References herein to a "phosphatase enzyme" refer to any enzyme or functional fragment thereof capable of hydrolytically removing a nucleoside triphosphate produced by the methods of the invention, including any enzyme or functional fragment thereof capable of cleaving a phosphate monoester into a phosphate ion and an alcohol.

[0155] As used herein, "pyrophosphatase enzyme" refers to any enzyme or functional fragment thereof that has the ability to catalyze the conversion of one ion of pyrophosphate into two phosphate ions. This also includes inorganic pyrophosphatases and inorganic diphosphatases. A non-limiting example is thermostable inorganic pyrophosphate (TIPP).

[0156] In some embodiments, modifications of any of the above or below described embodiments are provided in which the use of pyrophosphatase is optional. In one embodiment, A1 is circularized to the analyte target sequence. In this embodiment, the target region revealed by progressive digestion of A0 in the 3'-5' direction from the 3' end of A0 to form A1 is complementary to the 5' end of A0 / A1. In this embodiment, a ligase may be used to join the 3' and 5' ends of A1 to form the circularized oligonucleotide A2. This is shown, for example, in FIG. 15. In one embodiment, the 5' end of A0 / A1 is complementary to the target over a region 5-50 nucleotides in length. In one embodiment, this is 5-25 nucleotides in length. In one embodiment, this is 5-20 nucleotides in length. In one embodiment, this is 5-15 nucleotides in length. In one embodiment, this is 5-12 nucleotides in length. In one embodiment, this is 5-10 nucleotides in length.

[0157] In one embodiment, a method for diagnosing or monitoring cancer in a patient, comprising: -Extract DNA or RNA from the plasma or serum of the patient's blood; - one or more DNA or RNA analytes: i. single-stranded probe oligonucleotide A0; ii. pyrophosphoroase; and iii. Ligase into a first reaction mixture comprising: wherein the analyte anneals to single-stranded probe oligonucleotide A0 to produce a first intermediate product that is at least partially double-stranded and the 3' end of A0 forms a double-stranded complex, and A0 is pyrophosphorolyzed from the 3' end in the 3'-5' direction to produce an at least partially digested strand A1, and A1 undergoes ligation to form A2; - detecting a signal derived from the product of the previous step, where the product is A2 or a portion thereof, or multiple copies of A2 or a portion thereof, and inferring therefrom the presence or absence of cancer in the patient. The method further comprises the steps of:

[0158] In one embodiment, a method for monitoring cancer in a patient, comprising: -Extract DNA or RNA from the plasma or serum of the patient's blood; - one or more DNA or RNA analytes: i. single-stranded probe oligonucleotide A0; ii. pyrophosphoroase; and iii. Ligase into a first reaction mixture comprising: wherein the analyte anneals to single-stranded probe oligonucleotide A0 to produce a first intermediate product that is at least partially double-stranded and the 3' end of A0 forms a double-stranded complex, and A0 is pyrophosphorolyzed from the 3' end in the 3'-5' direction to produce an at least partially digested strand A1, and A1 undergoes ligation to form A2; - detecting a signal derived from the product of the previous step, where the product is A2 or a part thereof, or multiple copies of A2 or multiple copies of a part thereof, and from which the patient is Predicting progression or remission of cancer in The method further comprises the steps of:

[0159] In one embodiment, a method for monitoring a response to one or more cancer therapies in a patient, comprising: - Extracting DNA or RNA from plasma or serum of blood of patients undergoing one or more cancer treatments; - one or more DNA or RNA analytes: i. single-stranded probe oligonucleotide A0; ii. pyrophosphoroase; and iii. Ligase into a first reaction mixture comprising: wherein the analyte anneals to single-stranded probe oligonucleotide A0 to produce a first intermediate product that is at least partially double-stranded and the 3' end of A0 forms a double-stranded complex, and A0 is pyrophosphorolyzed from the 3' end in the 3'-5' direction to produce an at least partially digested strand A1, and A1 undergoes ligation to form A2; - detecting a signal derived from the product of the previous step, where the product is A2 or a portion thereof, or multiple copies of A2 or multiple copies of a portion thereof, and inferring therefrom the patient's response to one or more cancer therapies. The method further comprises the steps of:

[0160] In one embodiment, a method for screening a patient comprising: -Extract DNA or RNA from the plasma or serum of the patient's blood; - one or more DNA or RNA analytes: i. single-stranded probe oligonucleotide A0; ii. pyrophosphoroase; and iii. Ligase into a first reaction mixture comprising: wherein the analyte anneals to single-stranded probe oligonucleotide A0 to produce a first intermediate product that is at least partially double-stranded and the 3' end of A0 forms a double-stranded complex, and A0 is pyrophosphorolyzed from the 3' end in the 3'-5' direction to produce an at least partially digested strand A1, and A1 undergoes ligation to form A2; - detecting a signal derived from the product of the previous step, where the product is A2 or a portion thereof, or multiple copies of A2 or a portion thereof, and inferring therefrom the presence or absence of one or more cancers in the patient. The method further comprises the steps of:

[0161] In one embodiment, a method for non-invasive prenatal testing (NIPT) comprising: -Extraction of DNA or RNA from maternal plasma or serum of pregnant patients; - one or more DNA or RNA analytes: i. single-stranded probe oligonucleotide A0; ii. pyrophosphoroase; and iii. Ligase into a first reaction mixture comprising: wherein the analyte anneals to single-stranded probe oligonucleotide A0 to produce a first intermediate product that is at least partially double-stranded and the 3' end of A0 forms a double-stranded complex, and A0 is pyrophosphorolyzed from the 3' end in the 3'-5' direction to produce an at least partially digested strand A1, and A1 undergoes ligation to form A2; - detecting a signal derived from the product of the previous step, where the product is A2 or a part thereof, or multiple copies of A2 or a part thereof, and from which one or more to predict the presence or absence of fetal abnormalities or more The method further comprises the steps of:

[0162] In one embodiment, a method for screening a patient comprising: -Extract DNA or RNA from the plasma or serum of the patient's blood; - one or more DNA or RNA analytes: i. single-stranded probe oligonucleotide A0; ii. pyrophosphoroase; and iii. Ligase into a first reaction mixture comprising: wherein the analyte anneals to single-stranded probe oligonucleotide A0 to produce a first intermediate product that is at least partially double-stranded and the 3' end of A0 forms a double-stranded complex, and A0 is pyrophosphorolyzed from the 3' end in the 3'-5' direction to produce an at least partially digested strand A1, and A1 undergoes ligation to form A2; - detecting a signal derived from the product of the previous step, where the product is A2 or a portion thereof, or multiple copies of A2 or a portion thereof, and inferring therefrom the presence or absence of one or more cancers in the patient. The method further comprises the steps of:

[0163] In one embodiment, a method for monitoring a transplant recipient patient, comprising: - Extraction of DNA or RNA from plasma or serum of transplant recipient patient blood; - one or more DNA or RNA analytes: i. single-stranded probe oligonucleotide A0; ii. pyrophosphoroase; and iii. Ligase into a first reaction mixture comprising: wherein the analyte anneals to single-stranded probe oligonucleotide A0 to produce a first intermediate product that is at least partially double-stranded and the 3' end of A0 forms a double-stranded complex, and A0 is pyrophosphorolyzed from the 3' end in the 3'-5' direction to produce an at least partially digested strand A1, and A1 undergoes ligation to form A2; - detecting a signal derived from the product of the previous step, where the product is A2 or a part thereof, or multiple copies of A2 or multiple copies of a part thereof, and inferring therefrom the presence or absence of DNA or RNA derived from the transplanted organ; The method further comprises the steps of:

[0164] In some embodiments of the invention, there is provided a kit for use in a method for detecting a target polynucleotide sequence in a given nucleic acid analyte present in a sample, comprising: (a) a single-stranded probe oligonucleotide A0 capable of forming a first intermediate product with a target polynucleotide sequence, the intermediate product being at least partially double-stranded; (b) ligase; (c) a pyrophosphorolytic enzyme capable of digesting the first intermediate product in the 3'-5' direction from the A0 end to form a partially digested strand A1; (d) at least one single-stranded primer oligonucleotide substantially complementary to a portion of A0; (e) an amplifying enzyme; and (f) Suitable buffers The kit further comprises:

[0165] In one embodiment, the kit further comprises a source of pyrophosphate ions. Suitable source(s) of pyrophosphate ions are described above. In some embodiments, the kit further comprises suitable positive and negative controls.

[0166] In some embodiments, the kit may further comprise one or more control probes (E0), as described above. In some embodiments, the kit may further comprise one or more blocking oligonucleotides, as described above.

[0167] In some embodiments, the kit may further comprise one or more control probes (E0) and one or more blocking oligonucleotides. In some embodiments, the 5' end of A0 may be rendered resistant to 5'-3' exonuclease digestion, and the kit may further comprise a 5'-3' exonuclease.

[0168] In some embodiments, the kit may further comprise a ligation probe oligonucleotide C. In some embodiments, the kit may further comprise a splint oligonucleotide D.

[0169] In some embodiments, the kit may include both C and D. Ligation probe C may contain a 3' or internal modification that protects the probe from 3'-5' exonuclease digestion.

[0170] D may include an oligonucleotide region that is complementary to the 3' end of A1, and a region that is complementary to either the 5' end of oligonucleotide C or the 5' end of A1. In some embodiments, D may be unable to undergo extension relative to A1, either by a 3' modification or through a mismatch between the 3' end of D and the corresponding region of A1 or C.

[0171] In some embodiments, the kit may further comprise dNTPs, a polymerase, and buffers suitable for the initial amplification of a target polynucleotide sequence present in a sample. In some embodiments, the kit may further comprise a high-fidelity polymerase that incorporates dUTP, dUTP and uracil-DNA N-glycosylase (UDG).

[0172] In some embodiments, the kit may further comprise a phosphatase or a phosphohydrolase. In some embodiments, the kit may further comprise a pyrophosphatase. The pyrophosphatase may be hot start.

[0173] In some embodiments, the kit may further comprise a proteinase. In some embodiments, the kit may further comprise one or more oligonucleotide binding dyes or molecular probes.

[0174] In some embodiments, the kit may further comprise multiple A0's, each selective for a different target sequence and each containing an identifying region. In some embodiments, the amplification enzyme and the pyrophosphorylase of (e) are the same.

[0175] The kit may further include a purification device and reagents for isolating and / or purifying a portion of the polynucleotide according to the processes as described herein. Suitable reagents are well known in the art and include gel filtration columns and wash buffers. Further suitable reagents include magnetic beads and silica columns.

[0176] In one embodiment of the present invention: A fluid path between the first region, the second region, and the third region. A device comprising at least: The first region includes one or more wells, each well containing: dNTPs; at least one single-stranded primer oligonucleotide; An amplification enzyme for the initial amplification of DNA present in the sample including; and The second region includes one or more wells, each well containing: a single-stranded probe oligonucleotide A0 capable of forming a first intermediate product with a target polynucleotide sequence, said intermediate product being at least partially double-stranded; A pyrophosphorylase capable of digesting the first intermediate product in the 3'-5' direction from the end of A0 to generate the partially digested strand A1. including; and The third region includes one or more wells, each well containing: dNTPs; Buffers; Amplification enzymes; Means for detecting signals derived from A2 or a portion thereof, or from multiple copies of A2 or a portion thereof including; and 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; The device is provided.

[0177] In some embodiments, the means for detecting a signal is located within one or more wells of the third region. In some embodiments, the means for detecting a signal is located in a third region of the device.

[0178] In some embodiments, the means for detecting a signal is located in an adjacent region of the device. In some embodiments, the dNTPs in each well of the first region may be dUTP, dGTP, dATP and dCTP, and each well may further comprise a high-fidelity polymerase that incorporates dUTP and uracil-DNA N-glycosylase (UDG).

[0179] In some embodiments, the dNTPs in each well of the third region may be dUTP, dGTP, dATP and dCTP, and each well may further comprise a high-fidelity polymerase that incorporates dUTP and uracil-DNA N-glycosylase (UDG).

[0180] In some embodiments, each well of the second region may further comprise a source of pyrophosphate ions. In some embodiments, the 5' end of A0 may be rendered resistant to 5'-3' exonuclease digestion, and the wells of the second region may be rendered resistant to 5'-3' exonuclease digestion. It may further comprise Ze.

[0181] In some embodiments, each well of the second or third region may further comprise a ligase and a ligation probe oligonucleotide C or a splint oligonucleotide D.

[0182] Ligation probe C may contain a 3' or internal modification that protects the probe from 3'-5' exonuclease digestion. Splint oligonucleotide D may contain a region of oligonucleotide complementary to the 3' end of A1 and a region complementary to either the 5' end of oligonucleotide C or the 5' end of A1.

[0183] D may be incapable of undergoing extension relative to A1, either by a 3' modification or through a mismatch between the 3' end of D and the corresponding region of A1 or C. In some embodiments, the dNTPs may be a hot start and each well of the second region may further comprise a phosphatase or a phosphohydrolase.

[0184] In some embodiments, each well of the second region may further comprise pyrophosphatase. In some embodiments, each well of the third region may further comprise pyrophosphatase.

[0185] In some embodiments, the pyrophosphatase may be hot start. In some embodiments, each well of the third region may further comprise one or more oligonucleotide-conjugated dyes or molecular probes.

[0186] In some embodiments, each well of the second region may contain at least one or more different A0s that are selective for a target sequence that includes the identification region. In some embodiments, the amplification enzyme and the pyrophosphorylase in the second region can be the same.

[0187] In some embodiments, there may be a fourth region comprising one or more wells, where each well may contain a proteinase, and said fourth region may be located between the first and second regions.

[0188] In some embodiments, the second and third regions of the device are configured such that the wells of the second region: dNTPs; Buffers; Amplification enzyme; and Means for detecting signals derived from A1 or a portion thereof, or from multiple copies of A1 or a portion thereof may be further combined to include:

[0189] In some embodiments, the means for detecting a signal is located within one or more wells of the second region. In some embodiments, the means for detecting a signal is located in a second region of the device.

[0190] In some embodiments, the means for detecting a signal comprises a Located. In some embodiments, the first region may be fluidly connected to the sample container through a fluid interface.

[0191] In some embodiments, heating and / or cooling elements may be present in one or more regions of the device. In some embodiments, heating and / or cooling may be applied to one or more regions of the device.

[0192] In some embodiments, each region of the device may independently contain at least 100 or 200 wells. In some embodiments, each region of the device may independently comprise between about 100-300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 or more wells. The wells may be of any shape and their locations may be arranged in any format or pattern on the support.

[0193] In some embodiments, the well support may be constructed from metal (such as, but not limited to, gold, platinum, or nickel alloys), ceramic, glass, or other PCR-compatible polymeric materials, or composite materials. The well support includes a number of wells.

[0194] In some embodiments, the wells may be formed as blind holes or through holes in the well support. The wells may be produced in the well support by, for example, laser drilling (e.g., excimer or solid-state laser), ultrasonic embossing, hot embossing lithography, electroforming of nickel molds, injection molding, and injection compression molding.

[0195] In some embodiments, the individual well volumes may range from 0.1 to 1500 nl. In one embodiment, it is 0.5 to 50 nl. Each well may have a volume of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 , 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 nL.

[0196] In some embodiments, the well dimensions may have any shape, such as circular, elliptical, square, rectangular, oval, hexagonal, octagonal, conical, and other shapes known to those of skill in the art. In some embodiments, the well shapes may have varying cross-sectional areas along an axis, for example, a square hole may taper from a first size to a second size that is a fraction of the first size.

[0197] In some embodiments, the well dimensions may be square with approximately equal diameter and depth. In some embodiments, the walls defining the well may be non-parallel.

[0198] In some embodiments, the walls defining the well may converge to a point. The well dimensions may be derived from the total volumetric capacity of the well support. In some embodiments, the depth of the wells may range from 25 μm to 1000 μm.

[0199] In some embodiments, the wells may have a depth of 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 μm.

[0200] In some embodiments, the well diameter may range from about 25 μm to about 500 μm. In some embodiments, the wells may have a width of 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 μm.

[0201] In some embodiments, a portion of one or more regions of the device may be modified to promote or prevent fluid adhesion. Surfaces defining the wells may be coated with a hydrophilic substance (or modified to be hydrophilic) and thus promote fluid retention.

[0202] In some embodiments, a portion of one or more regions of the device may be coated with (or modified to be hydrophobic) and thus prevent fluid retention. One skilled in the art will appreciate that other surface treatments may be used to facilitate drainage of excess fluid, such that fluid is preferably retained within the wells, but not on the upper surface.

[0203] In some embodiments, the wells of the well support may be patterned to have a simple geometric pattern of aligned rows and columns, or a diagonally or hexagonally arranged pattern, hi one embodiment, the wells of the well support may be patterned to have a complex geometric pattern, such as a chaotic or isogeometric design pattern.

[0204] In some embodiments, the wells may be geometrically separated from one another and / or may feature a large depth-to-width ratio to help prevent cross-contamination of reagents. 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 of the regions of the device. Such auxiliary regions may be fluidly connected to one or more of the regions of the device through one or more membranes, valves, and / or pressure-cleavable substrates (i.e., materials that break when subjected to a predetermined amount of pressure from a fluid within the auxiliary region or within an adjacent portion of the fluid pathway), such as metal foils or thin films.

[0205] In some embodiments, the fluid pathway of the device may include an extended torturous portion. The torturous path between the inlet passage of the fluid pathway and one or more of the regions of the device may be useful for controlling and handling the fluid process. The torturous path may help reduce the formation of gas bubbles that may interfere with the flow of oil through the fluid pathway.

[0206] In some embodiments, the device may further comprise a gas-permeable membrane that allows gas to exit the wells of one or more regions of the device while not allowing fluid to pass through. The gas-permeable membrane may be attached to the well support of the device by a gas-permeable adhesive. In one embodiment, the membrane is made of polydimethylsiloxane ( The membrane may be constructed from PDMS and have a thickness ranging from 20 to 1000 μm. In some embodiments, the membrane may have a thickness ranging from 100 to 200 μm.

[0207] In some embodiments, all or a portion of the well support may contain a conductive metal portion (e.g., gold) to allow heat transfer from the metal to the well. In one embodiment, the interior surface of the well may be coated with a metal to allow heat transfer.

[0208] In some embodiments, after the appropriate reagents have filled the wells of one or more regions of the device, an insulating oil or thermally conductive liquid may be applied to the device to prevent crosstalk.

[0209] In some embodiments, the wells in one or more regions of the device may be shaped like a cone, tapering from a larger diameter to a smaller diameter. Conical wells with sloping walls allow the use of non-contact deposition methods for reagents (e.g., inkjet). The conical shape also aids drying and has been found to prevent bubbles and leaks when a gas-permeable membrane is present.

[0210] In some embodiments, the wells of one or more regions of the device may be filled by advancing (e.g., by pressure) a sample fluid along a fluid path of the device. As the fluid passes over the wells of one or more regions of the device, each well fills with fluid that is initially held within the well through surface tension. As previously described, portions of the well supports of the device may be coated with hydrophilic / hydrophobic materials as desired to promote complete and uniform filling of the wells as the sample fluid passes through.

[0211] In some embodiments, the wells of one or more regions of the device may be "capped" with oil after filling. This may then help reduce evaporation when the well support is subjected to thermal cycling. In one embodiment, after oil capping, an aqueous solution may be filled into one or more regions of the device to improve thermal conductivity.

[0212] In some embodiments, the stationary aqueous solution may be pressurized in one or more regions of the device to stop the movement of the fluid and any bubbles. In some embodiments, oil, such as mineral oil, may be used to isolate one or more regions of the wells of the device and provide thermal conductivity. However, any thermally conductive liquid may be used, such as a fluorinated liquid (e.g., 3M FC-40). In this disclosure, reference to oil should be understood to include such alternatives as one skilled in the art would recognize as applicable.

[0213] In some embodiments, the device may further comprise one or more sensor assemblies. In some embodiments, one or more sensor assemblies may include a charge-coupled device (CCD) / complementary metal oxide semiconductor (CMOS) detector coupled to a fiber optic faceplate (FOFP). The filter may be layered on top of the FOPF and may be placed against or adjacent to the well support. In one embodiment, the filter may be layered (bonded) directly on top of the CCD with the FOPF placed on top.

[0214] In some embodiments, one or more regions of the device have up to 100% humidity, or at least sufficient humidity to prevent evaporation during thermal cycling. To achieve this, a hydration fluid, such as distilled water, may be heated in the first region or one of the auxiliary regions.

[0215] In some embodiments, after loading of the device is complete, the well supports may be heated by an external device in thermal contact with the device to perform thermal cycling for PCR.

[0216] In some embodiments, non-contact methods of heating may be used, such as RFID, Curie point, inductive or microwave heating. These and other non-contact methods of heating will be well known to those skilled in the art. During thermal cycling, the device may be monitored for chemical reactions through the sensor arrangements described above.

[0217] In some embodiments, the reagents deposited in one or more of the wells of one or more regions of the device are deposited in a predetermined arrangement. In some embodiments: providing a sample fluid to a fluid pathway of the device, where the device comprises a fluid pathway between at least a first region, a second region, and a third region, the first, second, and third regions independently comprising one or more wells; filling the second region with the amplified fluid from the first region such that one or more wells of the second region are coated with the amplified fluid; draining the amplified fluid from the second region such that the one or more wells remain wetted with at least some of the amplified fluid; filling the third region with the fluid discharged from the second basin such that one or more wells of the third region are coated with the fluid; and Draining the fluid from the third chamber such that one or more of the wells remain wetted with at least some of the fluid. A method is provided, comprising the steps of:

[0218] In some embodiments of the method, the fluid pathway may be valve-free. In some embodiments of the method, the evacuated second region may be filled with a hydrophobic material.

[0219] In some embodiments of the method, the evacuated third region may be filled with a hydrophobic material. In some embodiments of the method, the hydrophobic material may be provided from an oil chamber in fluid communication with the second and third regions.

[0220] In some embodiments of the method, the sample fluid may be directed along the fluid path in a tortuous manner. In some embodiments, the method may further include applying heating and cooling cycles to one or more of the first, second, or third regions.

[0221] Those skilled in the art will understand that when the term "predict" is used, for example, "predicting the presence or absence of a particular sequence" refers to 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.

[0222] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. As used herein, "and / or" refers to two specified terms, either with or without the other. For example, "A and / or B" is to be construed as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein.

[0223] Unless otherwise indicated, the feature descriptions and definitions provided above are not limited to any particular aspect or embodiment of the present invention and apply equally to all aspects and embodiments described.

[0224] Those skilled in the art will further appreciate that while the present invention has been described by way of example with reference to certain embodiments, the invention is not limited to the disclosed embodiments and that alternative embodiments may be constructed without departing from the scope of the present invention as defined in the appended claims. EXAMPLES

[0225] Example 1 - Simplified Protocol For purposes of this and the following sections, embodiments of the invention are illustrated and referred to as Protocols 1 through 5, respectively.

[0226] Figure 1 provides an overview of the different protocols. The table below gives an overview of the time it takes to execute each protocol:

[0227] [Table 1]

[0228] In one embodiment, TIPP is not present in any one of the protocols, methods, kits and or devices of the present invention. In one embodiment, 5'-3' exonucleases are not present in any one of the protocols, methods, kits and or devices of the present invention.

[0229] The following table gives an overview of the enzymes used in each protocol:

[0230] [Table 2]

[0231] In one embodiment, the presence of pyrophosphatase is optional. In one embodiment, the presence of a 5'-3' exonuclease is optional. In one embodiment, the presence of UDG is optional.

[0232] [Table 3]

[0233] As can be seen, we have reduced the total number of enzymes required and therefore the cost and complexity of the method. Surprisingly, we have discovered that moving the 5'-3' exonuclease addition from the pre-amplification step of the protocol to the pyrophosphorolysis / ligation step (as in protocols 3-5) results in a higher fluorescent signal (corresponding to the detection of a specific target analyte sequence), as shown in Figure 2.

[0234] Example 2: Pyrophosphorolysis (PPL) enzyme We have tested the method of protocol 3 of the present invention using a range of different PPL enzymes, the results of which can be seen in Figure 3. Figure 3(A) shows detection of 1% MAF T790M using Mako, Klenow and Bsu. Figure 3(B) shows detection of 0.5% MAF T790M using Bst LF at a range of different PPi concentrations.

[0235] We have tested the method of protocol 4 of the invention with a range of different PPL enzymes, the results of which can be seen in FIG. Example 3: Protocol 1 vs. Protocol 4 We detected exon 19 del_6223 at 0.5%, 0.10% and 0.05% MAF using both protocol 1 and protocol 4, and the results can be seen in Figure 5. As can be seen, the fluorescence peak is larger when protocol 4 is used.

[0236] Example 4: Protocol 4 - Sensitivity Following protocol 4, we detected EGFR exon 20 T790M mutation in 0.10%, 0.50% and 1% MAF, as shown in FIG. 6 .

[0237] Example 5: Protocol 4 - Is an exonuclease digestion step necessary during RCA? The present inventors have shown that the exonuclease digestion step during RCA is not essential. However, detectable signals are detected later during RCA when the exonuclease digestion step is omitted. Figure 7 shows the detection of EGFR exon 20 T790M at 1% MAF with and without the presence of exonuclease during the RCA step.

[0238] Example 6: Protocol 4 - PPL:RCA Mix Ratio We investigated the effect of the PPL:RCA mixture on the signal intensity detected for 0.5% MAF EGFR exon 20 T790M, and the results are shown in Figure 8. As can be seen, the 1:2 PPL:RCA mixture produces the lowest signal intensity, which is at the earliest time point. The highest signal intensity is seen for the 1:8 PPL:RCA mixture at the latest time point of the reaction.

[0239] Example 7: Protocol 4 - Dye Selection We investigated whether the dyes used during RCA could be optimized. Figure 9 shows the results of a comparative experiment carried out according to protocol 4 using SybrGreenI (50°C and 60°C) and Syto82 (50°C and 60°C). The Syto82 dye allows RCA to be performed at a lower temperature of 50°C, while SybrGreenI requires a higher temperature of 60°C. For protocol 5, which omits the addition of proteinase K to the reaction mixture, a lower RCA temperature is required. For detection using the method of the present invention, temperatures higher than 50°C are required for the amplification enzymes used to prepare at least one single-stranded analyte of nucleic acid containing the target polynucleotide region to work. The use of SybrGreenI requires a reaction temperature of 60°C, and therefore proteinase K must be added at some point in the method to inactivate the amplification enzymes prior to RCA.

[0240] The lower RCA temperature may allow the method of the invention to be performed in a plate reader instead of qPCR. The reaction utilizing Syto82 is more rapid as can be seen in FIG. 9, and the total fluorescence is lower for Syto82, although this can be mitigated by using a higher concentration of the Syto82 dye.

[0241] Example 8: Protocol 4 - BST LF vs. BST 2.0WS We investigated the use of two different enzymes, BST LF and BST 2.0WS, for RCA according to protocol 4 to detect the 0.5% MAF EGFR exon 20 T790M mutation. The results are shown in Figure 10, from which it is clear that BST It can be seen that the reaction is the fastest with 2.0WS. BST 2.0 WS is designed to incorporate dUTP, which aids in the reaction rate. BST LF and BST There is negligible difference in the total signal intensity achieved between BST 2.0WS. According to the description provided by New England Biolabs (NEB), BST 2.0WS should be more stable and active only at temperatures above 45° C.

[0242] Example 9: Effect of PPL enzyme on signal detection We investigated 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. All PPL enzymes, except BST, were found to be effective at 1:4 PPL:RCA. PPL:RCA ratio influences RCA response. At a 1:8 PPL:RCA ratio, BST and Klenow have no effect on RCA response.

[0243] Example 10: Pyrophosphorolysis and ligation specificity for single-base mismatches A single-stranded first oligonucleotide (SEQ ID NO:1) was prepared having the following nucleotide sequence:

[0244] [ka]

[0245] A single-stranded ligation oligonucleotide 2 (SEQ ID NO:2) was prepared having the following nucleotide sequence:

[0246] [ka]

[0247] where A, C, G, and T represent nucleotides carrying the associated characteristic nucleobases of DNA; / 5Phos / indicates the 5'-terminal phosphate. * indicates a phosphorothioate bond.

[0248] A set of single-stranded oligonucleotides 3-4 (SEQ ID NOs:3-4) was also prepared having, in the 5' to 3' direction, the following nucleotide sequences:

[0249] [ka]

[0250] where oligonucleotide 3 contains a 17-base region complementary to the 17 bases at the 3' end of oligonucleotide 1, and oligonucleotide 4 contains the same region with a single-base mismatch at position 3. SEQ ID NOs: 3 and 4 are portions of the human EGFR gene with and without the C797S mutation, respectively.

[0251] A first reaction mixture was then prepared having a composition corresponding to that obtained from the following formulation: 0.5 μL 20x buffer pH 7.0 0.25 μL 5x buffer pH 8.0 0.25 μL 5xHF buffer 0.2 μL Oligonucleotide 1, 1000 nM 0.3 μL Oligonucleotide 2, 1000 nM 1 μL oligonucleotide 2 (500 nM) or a mixture of oligos 2 and 3 (500 and 0.5 nM, respectively) 0.3U Klenow fragment exo-(NEB) 0.01 μL inorganic pyrophosphate, 10 mM 0.0132U apyrase (e.g. NEB) 1U E. coli DNA ligase (e.g. NEB) Up to 10μL of water Here, the 20x buffer contained the following mixture: 200 μL Tris acetate, 1M, pH 7.0 342.5 μL aqueous magnesium acetate, 1M 120 μL aqueous potassium acetate, 5M 50 μL Triton X-100 detergent (10%) Up to 1mL of water Here, the 5x buffer contained the following mixture: 50 μL Acetate Trizma, 1M, pH 8.0 25 μL aqueous magnesium acetate, 1M 25 μL aqueous potassium acetate, 5M 50 μL Triton X-100 detergent (10%) Up to 1mL of water The mixture was then incubated at 45° C. for 15 minutes to effect circularization through pyrophosphorolysis of oligonucleotide 1 followed by ligation, and the resulting product mixture was used in an amplification reaction (Example 11).

[0252] Example 11: Amplification of circularization probes A pair of single-stranded oligonucleotide primers 1 (SEQ ID NO:5) and 2 (SEQ ID NO:6) were prepared having the following nucleotide sequences:

[0253] [ka]

[0254] where A, C, G, and T represent nucleotides bearing the associated characteristic nucleobases of DNA. A second reaction mixture was then prepared, having a composition corresponding to that obtained from the following formulation: 3 μL 10x Thermopol buffer 3.2U BST 2.0WS 0.32 μL Oligonucleotide 1, 10 μM 0.32 μL Oligonucleotide 2, 10 μM 1.125 μL Syto82, 30 μM 0.165U inorganic pyrophosphatase 1.2μL dNTP mix, 10mM 1.25 μL of reaction mixture of Example 10 11.25μL water Here, the 10x Thermopol buffer contained the following mixture: 200 μL Tris-HCl, pH=8.8, 1M 100μL NH4)2SO4, 1M 100 μL mM KCl, 1M 20mM MgSO4, 1M 10 μL Triton® X-100, 10% Up to 1mL of water The reaction mixture was then incubated at 50° C. for 40 minutes, and the resulting reaction products were then analyzed by real-time fluorescence, the results of which are shown in FIG.

[0255] From this analysis, the fluorescent signal appeared more rapidly in the reaction when both oligonucleotides 3 and 4 were present, indicating that pyrophosphorolysis and ligation of oligonucleotide 3 had occurred in the first reaction mixture.

[0256] Example 12: Multicolor detection using Sunrise primers 1. Target Oligo Dilution WT oligo dilution consists of the following components: 0.5xA7 buffer 0.5x Phusion U Buffer 200 nM WT oligonucleotide (SEQ ID NO: 7) Total volume: 5 μL T790M and C797S 1% AF mutant oligo mix: 0.5xA7 buffer 0.5x Phusion U Buffer 100 nM WT oligonucleotide (SEQ ID NO: 7) 2 nM T790M oligonucleotide (SEQ ID NO:8) 2nM C797S_2389 oligo (SEQ ID NO:9) Total volume: 5 μL

[0257] [ka]

[0258] 1xA7 composition Tris acetate pH=8.0 10mM Potassium acetate 25mM Magnesium acetate 5mM Triton-X 0.01% Phusion U Buffer The PhusionU buffer composition is not publicly available.

[0259] 2. PPL Mixtures corresponding to the following were prepared: 1XBFF1 37.5U / mL Mako DNA polymerase (3'→5' exo-) 100U / mL E. coli ligase 1.2U / mL apyrase 0.6mM PPi 20nM T790M probe 20nM C797S_2389 probe 30nM T790M splint oligonucleotide 30nM C797S_2389 splint oligonucleotide 5 μL of point 1 WT or 1% AF mutant dilution Total volume 10 μL The mixture was then incubated at 41° C. for 30 minutes.

[0260] 1xBFF1 composition Tris acetate pH=7.0 10mM Potassium acetate 30mM Magnesium acetate 17.125mM Triton-X 0.01%

[0261] [ka] During the ceremony, * indicates a phosphorothioate bond.

[0262] 3. TIPP Mixtures corresponding to the following were prepared: 1xA7 66.6U / mL TIPP 10 μL of the mixture from point 2 Total volume 20 μL The mixture was then incubated at 25°C for 5 min and at 95°C for 5 min.

[0263] 4. Consolidation Mixtures corresponding to the following were prepared: 1xA7 100U / mL E. coli ligase 20 μL of the mixture from point 3 10nM T790M splint oligonucleotide 10nM C797S_2389 splint oligonucleotide Total volume 30 μL The mixture was then incubated at 37°C for 10 min and at 95°C for 10 min.

[0264] [ka]

[0265] 5. Exonuclease Treatment Mixtures corresponding to the following were prepared: 1xA7 100U / mL E. coli ligase 30 μL of the mixture from point 4 625U / mL Exonuclease III 62.5U / mL T5 exonuclease Total volume 40 μL The mixture was then incubated at 30°C for 5 min and at 95°C for 5 min.

[0266] 6. RCA Mixtures corresponding to the following were prepared: 1x Thermopol buffer (53.2 mM Tris-HCl, 26.6 mM (N H4) 2SO4, 26.6 mM KCl, 5.32 mM MgSO4, 0.266% Triton® X-100, pH 8.8) 0.2 μM primer mix 1 0.4 μM reverse primer 533.3U / mL BST LF 0.4mM dNTP 10 μL of reaction mixture from point 5 Total volume 15 μL

[0267] [ka]

[0268] The mixture was then incubated at 60° C. for 90 minutes. Fluorescence measurements were taken every minute. Cq was obtained based on the automatic threshold provided by the Bio-rad instrument. The results can be seen in FIG. 13.

[0269] Example 13: Multicolor detection using Molecular Zipper 1. Target Oligo Dilution WT oligo dilution consists of the following components: 0.5xA7 buffer 0.5xQ5U buffer 100 nM WT oligonucleotide (SEQ ID NO: 17) Total volume: 1.25 μL G719X_6239, G719X_6252, G719X_6253 0.5% AF mutant oligonucleotide mix: 0.5xA7 buffer 0.5xQ5U buffer 100 nM WT oligonucleotide (SEQ ID NO: 17) 0.5 nM G719X_6239 oligonucleotide (SEQ ID NO: 18) 0.5 nM G719X_6252 oligonucleotide (SEQ ID NO: 19) 0.5 nM G719X_6253 oligonucleotide (SEQ ID NO: 20) Total volume: 1.25 μL

[0270] [ka]

[0271] 1xA7 composition Tris acetate pH=8.0 10mM Potassium acetate 25mM Magnesium acetate 5mM Triton-X 0.01% Q5U Buffer The Q5U buffer composition is not publicly available.

[0272] 2. Pyrophosphorolysis (PPL) and Ligation Mixtures corresponding to the following were prepared: 1XBFF1 10U / mL Klenow (exo-) 100U / mL E. coli ligase 1.2U / mL apyrase 100U / mL Lambda Exo 0.25mM PPi 6.6 nM G719X_6239 probe oligonucleotide (SEQ ID NO: 21) 6.6 nM G719X_6252 probe oligonucleotide (SEQ ID NO: 22) 6.6 nM G719X_6253 probe oligonucleotide (SEQ ID NO: 23) 30 nM splint oligonucleotide (SEQ ID NO:24) 1.25 μL of the mixture from point 2 Total volume 10 μL The mixture was then incubated at 45° C. for 15 minutes.

[0273] 1xBFF1 composition Tris acetate pH=7.0 10mM Potassium acetate 30mM Magnesium acetate 17.125mM Triton-X 0.01%

[0274] [ka] During the ceremony, * indicates a phosphorothioate bond.

[0275] 3. Detect-RCA Mixtures corresponding to the following were prepared: 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-100, pH 8.8) 0.28 μM dye primer mix 1 0.56 μM quencher primer 1 0.28 μM quencher primer 2 0.84 μM reverse primer 568.8U / mL BST 2.0 WarmStart 14.67U / mL TIPP 1.06mM dNTP 1.25 μL of reaction mixture from point 2 Total volume 11.25 μL Dye primer mix 1 consisted of

[0276] [ka]

[0277] The mixture was then incubated for 150 minutes at 58° C. Fluorescence measurements were taken every minute and the results can be seen in FIG. Example 14: Pyrophosphorolysis and ligation to target 1. Oligonucleotide Dilution Preparation Oligonucleotide dilutions were prepared in 0.5xA7 and 0.5xQ5 buffers: WT oligonucleotide 200nM + / - mutant oligonucleotide 500pM Total volume 1.25 μL

[0278] [ka]

[0279] 2. Pyrophosphorolysis and Ligation A PPL mixture was prepared consisting of: 1xBFF1 10U / mL Klenow (exo-) 100U / mL E. coli ligase 1.2U / mL apyrase 100U / mL Lambda Exo 0.25mM PPi 20nM Probe A0 1.25 μL of oligo from point 1 Total volume 10 μL

[0280] [ka]

[0281] During the ceremony, * is a phosphorothioate bond. 1xBFF1 composition Tris acetate pH=7.0 10mM Potassium acetate 30mM Magnesium acetate 17.125mM Triton-X 0.01% 1xA7 composition Tris acetate pH=8.0 10mM Potassium acetate 25mM Magnesium acetate 5mM Triton-X 0.01% Q5 Buffer The Q5 buffer composition is not publicly available. The resulting mixture was incubated at 45° C. for 15 minutes.

[0282] 3. Detect-RCA An RCA mixture was prepared consisting of: 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-100, pH 8.8) 0.28 μM primer mix 284.4U / mL BST 2.0 WarmStart 14.67U / mL TIPP 1.06mM dNTP Syto82 dye 3μM 1.25 μL of reaction from point 2 Total volume 11.25 μL Primer Mixture:

[0283] [ka]

[0284] The resulting mixture was incubated at 50° C. for 70 minutes. Fluorescence readings were taken every minute and the results can be seen in FIG. Example 15: Further selected applications and embodiments of the present invention KRAS detection The KRAS gene controls cell proliferation and, when mutated, this negative signaling is disrupted and cells are allowed to continue to proliferate, often leading to cancer. Single amino acid substitutions, and in particular single base substitutions, are responsible for the activating mutations associated with a variety of cancers: lung adenocarcinoma, mucinous adenocarcinoma, pancreatic ductal carcinoma and colorectal cancer. KRAS mutations have been used as prognostic biomarkers, for example in lung cancer.

[0285] Driver mutations in KRAS are associated with up to 20% of human cancers and there are targeted therapies in development against this mutation and its associated disease(s), a non-limiting list of some such therapies can be found in the table below:

[0286] [Table 4]

[0287] The presence of KRAS mutations has been found to reflect a very poor response to the EGFR inhibitors, panitumumab (Vectibix) and cetuximab (Erbitux). Activating mutations in the gene encoding KRAS occur in 30%-50% of colorectal cancers, and studies have shown that patients whose tumors express this mutated form of the KRAS gene do not respond to panitumumab and cetuximab. Although the presence of a wild-type KRAS gene does not guarantee that a patient will respond to these drugs, studies have shown that cetuximab has significant efficacy in metastatic colorectal cancer patients with wild-type KRAS tumors. Lung cancer patients who are positive for KRAS mutations (wild-type EGFR) have an estimated response rate of 5% or less to the EGFR antagonists, erlotinib or gefitinib, compared to a 60% response rate in patients without KRAS mutations.

[0288] Early detection of the emergence of KRAS mutations (activating or overexpressing), a frequent driver of acquired resistance to cetuximab therapy (anti-EGFR therapy) in colorectal cancer, allows for treatment modifications (e.g. early initiation of mitogen-activated protein kinase kinase (MEK) inhibitors) that delay or reverse resistance, and thus it is preferred that the methods of the invention allow for rapid and inexpensive detection of a patient's KRAS status.

[0289] A non-limiting list of mutations are: G12D, G12A, G12C, G13D, G12V, G12S, G12R, A59T / E / G, Q61H, Q61K, Q61R / L, K117N and A146P / T / V.

[0290] A further non-limiting list of mutations is shown in the table below:

[0291] [Table 5]

[0292] BRAF detection BRAF is a human gene that encodes a protein called B-Raf that is involved in sending signals inside cells that are involved in directing cell growth. BRAF has been shown to be mutated in several human cancers. B-Raf is a member of the Raf kinase family of growth signaling protein kinases and plays a role in regulating the MAP kinase / ERK signaling pathway, which among other things affects cell division.

[0293] Certain other inherited BRAF mutations cause birth defects. More than 30 mutations in the BRAF gene associated with human cancers have been identified. In 90% of cases, a thymine is substituted with an adenine at nucleotide 1799. This leads to a substitution of a valine (V) with a glutamic acid (E) at codon 600 in the activating segment (herein referred to as V600E) that is found in human cancers. This mutation is widespread: -Colorectal cancer -melanoma -Papillary thyroid cancer -Non-small cell lung cancer -Ameloblastoma is observed.

[0294] A non-limiting list of other mutations that have been found are: R461I, I462S, G463E, G463V, G465A, G465E, G465V, G468A, G468E, N580S, E585K, D593V, F594L, G595R, L596V, T598I, V599D, V599E, V599K, V599R, V600K and A727V.

[0295] Drugs have been developed to treat cancers driven by BRAF mutations; vemurafenib and dabrafenib have been approved by the FDA for the treatment of late-stage melanoma. For metastatic melanoma, the response rate to treatment with vemurafenib was 53%, compared with 7-12% for the previous chemotherapy agent of choice, dacarbazine.

[0296] ERBB2 / HER2 detection 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 invasive forms of breast cancer. Overexpression of the ERBB2 gene is also known to occur in 30% of ovarian, gastric, lung adenocarcinomas, and invasive forms of uterine cancer and salivary duct carcinomas. Structural modifications that cause ligand-independent firing of the receptor in the absence of overexpression have also been identified.

[0297] There are many targeted therapies approved and in development for this mutation and its associated disease(s), a non-limiting list of some of these therapies can be found in the table below:

[0298] [Table 6]

[0299] HER2 testing is routinely performed in breast cancer patients to assess prognosis, monitor response to treatment, and determine suitability for targeted therapy (such as trastuzumab). Because trastuzumab is expensive and associated with significant side effects (cardiac toxicity), it is important that only HER2+ patients are selected to receive this agent, and it is therefore advantageous that the method of the present invention allows for rapid and inexpensive detection of a patient's HER2 status.

[0300] In one embodiment, the methods of the invention are used to detect the presence or absence of an ERRB2 exon 20 insertion mutation. A further non-limiting list of ERBB2 mutations is provided in the table below:

[0301] [Table 7]

[0302] EML4-ALK detection 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 leads to the production of the protein EML4-ALK, which appears 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.

[0303] There are many targeted therapies approved and in development for this mutation and its associated disease(s), a non-limiting list of some of these therapies can be found in the table below:

[0304] [Table 8]

[0305] The EML4-ALK gene fusion is responsible for approximately 5% of non-small cell lung cancer (NSCLC), which accounts for approximately 9,000 new cases per year in the United States and approximately 45,000 cases worldwide.

[0306] There are many variants of EML4-ALK, all of which have the essential coiled-coil domain in the EML4 N-terminal portion and the kinase domain of ALK exon 20, which is necessary for transforming activity. Fusions of EML4 exon 13 and ALK exon 20 (variant 1: V1) (the detection of which can be seen in Figure 20), EML4 exon 20 and ALK exon 20 (V2), and EML4 exon 6 and ALK exon 20 (V3) are some of the more common variants. The clinical significance of these different variants is only becoming clear recently.

[0307] V3 has emerged as a suitable marker for the selection of patients likely to have shorter progression-free survival (PFS) after non-tyrosine kinase inhibitor (TKI) treatment, such as chemotherapy and radiotherapy. There is further evidence that V3 is associated with shorter PFS in patients receiving first and second generation lines of treatment, as well as worse overall survival (OS), compared to EML4-ALK V1 and V2.

[0308] It has also been found that V3-positive patients develop resistance to first and second lines of therapy through the development of resistance mutations, and this is probably driven by incomplete tumor cell suppression, as wild-type V3 has a higher IC50. Detection of unfavorable V3 may be used to select patients who require more aggressive monitoring and treatment strategies. It appears that administration of third-generation lorlanitib to patients with V3 may give longer PFS than those with V1, and therefore it is preferred that the method of the present invention allows for the rapid and inexpensive detection of which mutations a patient may have.

[0309] The methods of the present invention further 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.

[0310] For example, G1202R is a solvent-front mutation that causes interference with drug binding and inhibits high levels of ALK inhibitors against first- and second-generation ALK inhibitors. It is therefore preferred that the method of the invention allows for the identification of patients who are likely to carry this mutation and who may benefit from initiation of treatment with third generation therapy rather than first or second.

[0311] Further non-limiting examples of EML4-ALK mutations are shown in the table below:

[0312] [Table 9]

[0313] EGFR detection 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.

[0314] EGFR mutations occur in EGFR exons 18 to 21, and mutations in exons 18, 19, and 21 indicate suitability for treatment with EGFR-TKIs (tyrosine kinase inhibitors). Mutations in exon 20 (with the exception of some mutations) indicate that the tumor is EGFR-TKI-resistant and not suitable for treatment with EGFR-TKIs.

[0315] The two most common EGFR mutations are a short in-frame deletion in exon 19, and a point mutation in exon 21 at nucleotide 2573 (CTG to CGG) resulting in a substitution of leucine by 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 may be used to predict which patients will respond to TKIs.

[0316] Therefore, it is preferred that the method of the present invention allows the identification of patients who carry these mutations and may benefit from initiating treatment with a TKI.Those skilled in the art will recognize that the method of the present invention allows the identification of a range of EGFR mutations, a non-exhaustive list of which is: G719X, Ex19Del, S768I, Ex20Ins and L861Q.

[0317] A further non-limiting list of mutations is shown in the table below:

[0318] [Table 10-1]

[0319] [Table 10-2]

[0320] ROS1 ROS1 is a receptor tyrosine kinase (encoded by the gene ROS1) with structural similarity to the anaplastic lymphoma kinase (ALK) protein; it is encoded by the c-ros oncogene.

[0321] A non-limiting list of ROS1 mutations is provided in the table below:

[0322] [Table 11]

[0323] RET proto-oncogene The RET proto-oncogene encodes a receptor tyrosine kinase for a member of the glial cell line-derived neurotrophic factor (GDNF) family of extracellular signaling molecules.

[0324] A non-limiting list of RET mutations is provided in the table below:

[0325] [Table 12]

[0326] MET exon 14 MET exon 14 skipping occurs at a frequency of approximately 5% of NSCLCs and is seen in both squamous and adenocarcinoma histology.

[0327] A non-limiting list of MET mutations is provided in the table below:

[0328] [Table 13]

[0329] NTRK proto-oncogene NTRK gene fusion leads to abnormal proteins called TRK fusion proteins, which can cause cancer cell proliferation. NTRK gene fusions can be found in several types of cancer, including brain, head and neck, thyroid, soft tissue, lung, and colon cancer. Also called neurotrophic tyrosine receptor kinase gene fusion.

[0330] A non-limiting list of NTRK mutations is provided in the table below:

[0331] [Table 14]

[0332] panel In one embodiment of the present invention, a panel is provided that includes a number of probe molecules (A0), each A0 being complementary to a target mutation. The mutations may be selected from any of the mutations described above or below or known. Thus, one skilled in the art will recognize that within the scope of the present invention are panels that may be useful in detecting one or more mutations to any of the proto-oncogenes or oncogenes described above or below or known.

[0333] In one embodiment, the panel comprises between 5 and 500 individual probe molecules, each complementary to a specific target mutation. In one embodiment, the panel comprises between 5 and 400 individual probe molecules, each complementary to a specific target mutation. In one embodiment, the panel comprises between 5 and 300 individual probe molecules, each complementary to a specific target mutation. In one embodiment, the panel comprises between 5 and 200 individual probe molecules, each complementary to a specific target mutation. In one embodiment, the panel comprises between 5 and 100 individual probe molecules, each complementary to a specific target mutation. In one embodiment, the panel comprises between 5 and 50 individual probe molecules, each complementary to a specific target mutation.

[0334] In one embodiment, there may be multiple probe molecules specific for the same mutation, hi one embodiment, there may be only one probe molecule specific for each mutation in the panel.

[0335] In one embodiment, a panel is provided comprising a number of probe molecules, 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.

[0336] In one embodiment, a panel comprising a number of probe molecules, wherein one or more of the probes may be complementary to an EGFR mutation, one or more of the probes may be complementary to a KRAS mutation, and one or more of the probes may be complementary to an E. one or more probes may be complementary to RBB2 / HER2 mutations, one or more probes may be complementary to EML4-ALK mutations, one or more probes may be complementary to ROS1 mutations, one or more probes may be complementary to RET mutations, and one or more probes may be complementary to MET mutations.

[0337] In one embodiment, a panel of probes selective for one or more of EGFR, KRAS, BRAF, ERBB2 / HER2, EML4-ALK, ROS1, RET, MET mutations is provided.

[0338] In one embodiment, a panel of probe molecules that are selective for EGFR mutations is provided. In one embodiment, a panel of probe molecules that are selective for KRAS mutations is provided.

[0339] In one embodiment, a panel of probe molecules that are selective for BRAF mutations is provided. In one embodiment, a panel of probe molecules that are selective for ERBB2 / HER2 mutations is provided.

[0340] In one embodiment, a panel of probe molecules that are selective for EML4-ALK mutations is provided. In one embodiment, a panel of probe molecules that are selective for ROS1 mutations is provided.

[0341] In one embodiment, a panel of probe molecules that are selective for RET mutations is provided. In one embodiment, a panel of probe molecules that are selective for NTRK mutations is provided.

[0342] In one embodiment, a panel of probe molecules that are selective for ROS1 mutations is provided. In one embodiment, a panel of probe molecules that are selective for MET exon 14 mutations is provided.

[0343] In one embodiment, a panel is provided that comprises a number of probe molecules that are selective for one or more coding sequences (CDS). In one embodiment, a method is provided in which one or more of the above-described panels are used to detect one or more mutations.

[0344] In one embodiment, a method is provided in which one or more of the above described panels are used to detect the presence or absence of one or more mutations. In one embodiment, a kit is provided comprising a panel, which may be as described above or below, in combination with one or more reagents, which may be as described above or below. One of skill in the art will recognize that the embodiments of the kit disclosing A0 include within their scope embodiments in which there is a panel comprising multiple A0s.

[0345] Companion Diagnostics The methods of the present invention may be used to detect specific genetic markers in a sample, These markers may be tumor-specific mutations or wild-type genomic sequences and may be detected using tissue, blood or any other patient sample type.

[0346] Resistance monitoring Repeated testing of patient samples during the treatment of a disease may allow early detection of developing resistance to therapy. An example of this application is in non-small cell lung cancer (NSCLC), where epidermal growth factor receptor (EGFR) inhibitors (e.g. gefitinib, erlotinib) are commonly used as first-line treatment. During treatment, tumors often develop mutations in the EGFR gene (e.g. T790M, C797S), which confer resistance to the drug. Early detection of these mutations may allow conversion of patients to alternative therapies (e.g. Tagrisso).

[0347] Typically, patients monitored for resistance onset may be too ill to perform repeat tissue biopsies, which may also be costly, invasive, and have associated risks. Although testing from blood is preferred, in a reasonable blood sample, the mutation of interest may only be present at very low copy numbers. Thus, monitoring requires highly sensitive testing from blood samples using the method of the present invention, which is simple to perform and cost-effective so that it can be performed regularly.

[0348] Recurrence monitoring In this application, patients who are declared disease-free after treatment may be monitored over time to detect disease recurrence. This needs to be done non-invasively and requires highly sensitive detection of target sequences from blood samples. Using the method of the present invention provides a simple and low-cost method that can be performed routinely. The targeted sequences may be gene mutations known to be common in the disease of interest, or may be custom panels of targets designed for a specific patient based on the detection of variants in tumor tissue before remission.

[0349] Minimal Residual Disease (MRD) Surveillance For some cancers, where there are residual cancer cells remaining in the patient after treatment, this is the main cause of cancer and leukemia recurrence. MRD surveillance and testing has several important roles: determining whether the treatment has eradicated the cancer or if traces remain, comparing the effectiveness of different treatments, monitoring patient remission status, and detecting leukemia recurrence, and selecting the treatment that will best meet these needs.

[0350] screening Population screening for early detection of disease has been a long-standing goal, especially in cancer diagnosis.The difficulty is twofold: identifying a marker panel that allows reliable detection of disease without too many false negatives, and developing a method with sufficient sensitivity and low enough cost.Using the method of the present invention, it is possible to address a larger mutation panel than PCR-based tests, but with a much simpler workflow and lower cost than sequencing-based diagnostic methods.

[0351] Organ transplant rejection When a transplanted organ is rejected by the recipient, DNA from the organ is shed into the recipient's bloodstream. Early detection of this DNA allows for early detection of rejection. This may be accomplished using a custom panel of donor-specific markers, or by using a panel of variants known to be common in the population, some of which are present in the donor and some of which are present in the recipient. Routine monitoring of organ recipients over time may be possible with the low cost and simple workflow of the invention disclosed herein.

[0352] Non-Invasive Prenatal Testing (NIPT) It has been known for a long time that fetal DNA exists in maternal blood, and the NIPT market is currently quite saturated with companies that use sequencing to identify mutations and count the copy number of specific chromosomes, allowing fetal abnormality detection.The method of the present invention as disclosed herein has the ability to detect mutations with very low allele ratios, potentially allowing earlier detection of fetal DNA.The identification of common mutations in a given population will allow targeting mutations that may exist in either maternal or fetal DNA, or developing assays that allow abnormality detection at earlier stages of pregnancy.

[0353] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. "And / or" is to be construed herein as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is to be construed as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein.

[0354] Unless the context indicates otherwise, the feature descriptions and definitions provided above are not limited to any particular aspect or embodiment of the present invention and apply equally to all aspects and embodiments described.

[0355] Those skilled in the art will further recognize that while the present invention has been described by way of example and with reference to certain embodiments, it is not limited to the disclosed embodiments and that alternative embodiments can be constructed without departing from the scope of the present invention as defined in the appended claims.

[0356] One of skill in the art will understand that reference to "partially digested strand A1" can refer to a single-stranded oligonucleotide that, when hybridized to a target analyte sequence, is formed by progressive digestion of A0 in the 3'-5' direction until the strands dissociate due to lack of complementarity.

[0357] One of skill 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.

[0358] Those 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. 1. A method for detecting a target polynucleotide sequence in a given nucleic acid analyte present in a sample, comprising: (a) i. Single-stranded probe oligonucleotide A ０ ; ii. pyrophosphorylase; and iii. Ligase introducing one or more nucleic acid analytes into a first reaction mixture comprising where the analyte is a single-stranded probe oligonucleotide A ０ anneals to and is at least partially double-stranded; and ０ The 3' end of A forms a double-stranded complex, and ０ is pyrophosphorolysis in the 3'-5' direction from the 3' end to form at least a partially digested strand A. １ and A １ is connected to A ２ Forming (b) detecting a signal from the product of the previous step, where the product is A ２ or a part thereof, or A ２ or a portion thereof, from which the presence or absence of a polynucleotide target sequence in an analyte can be inferred. The method comprises the steps of:

2. 10. The method as claimed in claim 1, further characterized in that the first reaction mixture further comprises a source of pyrophosphate ions.

3. The first reaction mixture is ０ 3. The method as claimed in claim 1 or claim 2, further characterized by further comprising at least one single-stranded primer oligonucleotide substantially complementary to a portion of and deoxyribonucleotide triphosphates (dNTPs).

4. 4. The method as claimed in claim 3, further characterized in that the first reaction mixture further comprises an amplification enzyme.

5. The method as claimed in 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), said second reaction mixture comprising at least one single-stranded primer oligonucleotide and dNTPs.

6. 6. The method as claimed in claim 5, further characterized in that the second reaction mixture further comprises an amplification enzyme.

7. The first reaction mixture comprises: one or more ligases; and A ２ Two or more LCR probe oligonucleotides complementary to the above adjacent sequences, ２ the 5' phosphate of one LCR probe is immediately adjacent to the 3' OH of the other LCR probe when successfully annealed to said probe oligonucleotide and A ２ In the presence of ２ and are ligated together to form one oligonucleotide molecule, which then serves as the new target for a second round of covalent ligation to the target of interest, in this case A. ２ leading to an exponential amplification of A method as claimed in claim 1 or claim 2.

8. The product of step (a) is introduced into a second reaction mixture prior to step (b), said second reaction mixture comprising: one or more ligases; and A ２ Two or more LCR probe oligonucleotides complementary to the above adjacent sequences, ２ the 5' phosphate of one LCR probe is immediately adjacent to the 3' OH of the other LCR probe when successfully annealed to said probe oligonucleotide and further characterized by including A ２ In the presence of ２ and are ligated together to form one oligonucleotide molecule, which then serves as the new target for a second round of covalent ligation to the target of interest, in this case A. ２ leading to an exponential amplification of A method as claimed in claim 1 or claim 2.

9. The first reaction mixture comprises: A linking moiety that includes one or multiple fluorophores arranged such that the fluorescence of the fluorophores is quenched either by the proximity of the fluorophores to one another or by the proximity of one or more fluorescence quenchers. ２ an oligonucleotide complementary to a region of Double-strand specific DNA digestive enzyme and A ２ In the presence of A, the labeled oligonucleotide is digested, separating the fluorophores from each other or from their corresponding quenchers, and generating a fluorescent signal, and thus A. ２ The presence of A method as claimed in claim 1 or claim 2.

10. The product of step (a) is introduced into a second reaction mixture prior to step (b), said second reaction mixture comprising: A linking moiety that includes one or multiple fluorophores arranged such that the fluorescence of the fluorophores is quenched either by the proximity of the fluorophores to one another or by the proximity of one or more fluorescence quenchers. ２ an oligonucleotide complementary to a region of Double-strand specific DNA digestive enzyme [0033] The invention is further characterized as comprising: A ２ In the presence of A, the labeled oligonucleotide is digested, separating the fluorophores from each other or from their corresponding quenchers, and generating a fluorescent signal, and thus A. ２ The presence of A method as claimed in claim 1 or claim 2.

11. Partially digested chain A １ is circularized through ligation of the 3' and 5' ends to give oligonucleotide A ２ The method as claimed in any one of claims 1 to 6, further characterized by producing

12. The first reaction mixture further comprises a ligation probe oligonucleotide C and a partially digested strand A. １ is linked at its 3' end to the 5' end of C, ２ 12. The method as claimed in any one of claims 1 to 6 or claim 11, further characterized by producing

13. 13. The method as claimed in claim 12, further characterized in that oligonucleotide C further comprises a 3' or internal modification that protects against 3'-5' exonuclease digestion.

14. The concatenation is: During step (a); or During step (b); or Between steps (a) and (b) 14. The method as claimed in any one of claims 11 to 13, further characterized in that:

15. A method as claimed in any one of claims 1-6 or 11-14, further characterized in that the first or second reaction mixture further comprises a splint oligonucleotide D.

16. D is A １ and the 5' end of oligonucleotide C or A １ 16. The method as claimed in claim 15, further characterized by comprising a region complementary to either of the 5' ends of:

17. D is modified by 3' or the 3' end of D and A ２ through a mismatch between the corresponding regions of １ 17. A method as claimed in claim 15 or claim 16, further characterized in that the method is unable to undergo elongation relative to

18. The first reaction mixture further comprises a 5'-3' exonuclease, and ０ A method as claimed in any preceding claim, further characterised in that the 5' end of the nucleic acid sequence is rendered resistant to 5'-3' exonuclease digestion.

19. A method as claimed in any preceding claim, wherein prior to step (a), the nucleic acid analytes present in the sample undergo amplification, and further characterized by further treating the sample with a proteinase after amplification of the desired nucleic acid analytes and prior to addition of the first reaction mixture.

20. A method as claimed in any preceding claim, further characterized in that the first reaction mixture further comprises a phosphatase or a phosphohydrolase.

21. A method as claimed in any preceding claim, further characterized by treating the product of the previous step with pyrophosphatase prior to or during step (b).

22. A method as claimed in any preceding claim, further characterized by treating the product of the previous step with an exonuclease prior to or during step (b).

23. A ０ to obtain the partially digested chain A. １ The enzyme that forms A ２ 3. A method as claimed in any one of the preceding claims, further characterized by amplifying

24. 2. A method as claimed in any one of the preceding claims, further characterised in that detection is achieved using one or more oligonucleotide fluorescent binding dyes or molecular probes.

25. A ２ 25. The method as claimed in claim 24, further characterized by using the increase in signal over time resulting from amplicon production of the target sequence to infer the concentration of the target sequence in the analyte.

26. A number of probes A, each selective for a different target sequence and each containing an identification region. ０ and A ２ The amplicon and thus the presence of a target sequence in the analyte is inferred through detection of the identification region(s).

27. 27. The method as claimed in claim 26, further characterized in that the detection of the identified region(s) is performed using a molecular probe or through sequencing.

28. The final step of the method is: i. labeling the product of step (b) with one or more oligonucleotide fluorescent conjugated dyes or molecular probes; ii. measuring the fluorescent signal of the product; iii. exposing the product to a set of denaturing conditions; and Identifying polynucleotide target sequences in the analyte by monitoring changes in the fluorescent signal of the product during exposure to denaturing conditions.

25. The method as claimed in claim 24, further characterized by comprising the further step of:

29. One or more nucleic acid analytes are divided into multiple reaction volumes, with each volume containing one or more probe oligonucleotides A introduced to detect a different target sequence. ０ The method as claimed in claims 1 to 28, further characterized in that it comprises:

30. Different probe A ０ A method as claimed in claims 1 to 29, further characterized in that it comprises a common priming site, allowing a single primer or a single set of primers to be used for amplification.

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