Low-bias sequential multiplex amplification assays

Abstract

Provided herein is technology relating to multiplex pre-amplification of multiple different target nucleic acids from a sample, such as an environmental or biological sample, particularly, but not limited to, methods, compositions, kits, and related uses for detecting and evaluating mixtures of target nucleic acids, for example. The technology provides methods, compositions, and kits for performing highly multiplex pre-amplification for assaying samples containing multiple different target nucleic acids, followed by highly multiplex detection assays, such as PCR assays, next generation sequencing assays, and PCR-flap assays, using low bias amplification conditions.

Description

[Technical field]

[0001] Sequence Listing The text of the computer readable sequence listing submitted herewith, entitled "39883-601_SEQUENCE_LISTING", created on April 27, 2023, with a file size of 108,041 bytes, is hereby incorporated by reference in its entirety.

[0002] Provided herein is technology related to amplification-based detection of nucleic acids, particularly, but not limited to, methods and compositions for sequential steps of multiplex amplification to balance amplification from different target nucleic acids present in a reaction mixture. The technology further provides methods for using the integrated signal from sequential multiplex amplification of multiple marker genes in a single fluorescent channel without distinguishing the signal from any single marker gene that is combined. [Background technology]

[0003] Methods for the detection and quantification of nucleic acids are important in many areas of molecular biology, especially in molecular diagnostics.At the DNA level, such methods are used to determine, for example, the presence or absence of variant alleles, the copy number of amplified gene sequences in the genome, and the amount, presence, or absence of methylation throughout a gene or at specific loci within a gene.Furthermore, methods for the quantification of nucleic acids are used to determine the amount of mRNA as a measure of gene expression.

[0004] Among several different analytical methods for detecting and quantifying nucleic acid or nucleic acid sequences, the polymerase chain reaction (PCR) variant has become the most powerful and widespread technology, the principle of which is disclosed in U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188. Preamplification of target nucleic acid (e.g., genomic DNA, cDNA, etc.) in low target samples can be used to enrich DNA in the sample before splitting the sample for further analysis of specific targets. For example, whole genome amplification using simple primers (e.g., random hexamers) has been used to increase the amount of essentially all DNA in a sample in a manner that is not specific to any particular target of interest. (Sigma-Aldrich's GenomePlex systems, Arneson, et al., Cold Spring Harb. Protoc.; 2008; doi:10.1101 / pdb.prot4920).

[0005] Another approach is to amplify one or more regions of particular interest in a semi-targeted manner, generating a mixture of amplified fragments (amplicons) containing different mutations or loci that are subjected to further analysis. Successive rounds of amplification using the same primers tend to have a high background of non-specific amplification, resulting in artifacts, e.g., artificial recombination molecules, high non-specific background, and biased amplification of different intended targets. Therefore, such preamplification PCR is typically performed under special conditions, e.g., using limited number of cycles and / or low concentrations of primers (e.g., 10-20 times lower than in standard PCR), to avoid increasing background non-specific amplification, since it has been shown in negative control reactions that the use of concentrations of each primer above about 160 nM in multiplex preamplification increases the amplification background (see, e.g., Andersson, et al., Expert Rev. Mol. Diagn. Early online, 1-16 (2015)).

[0006] After the first round of amplification in multiplex PCR, the preamplified DNA is typically diluted and aliquoted into a new amplification reaction for quantitative or qualitative PCR analysis using conditions typical of standard PCR, e.g., more concentrated reagents and more cycles; the second amplification is generally performed using a different primer pair, e.g., "nested" primers that anneal to sites within the preamplified fragment rather than annealing to the original primer sites at both ends of the amplicon.

[0007] Some uses of amplification involve the measurement or analysis of multiple mutations or marker nucleic acids in a sample. Multiplex amplification of multiple different specific target sequences is typically performed using a relatively standard PCR reagent mixture, such as, for Amplitaq DNA polymerase, a mixture containing 50 mM KCl, 1.5-2.5 mM MgCl. 2 and a Tris-HCl buffer of about pH 8.5 is used. If a second amplification is performed, the primers are typically present in limiting amounts (see Andersson, supra). For subsequent assays, the amplified DNA is diluted or purified and then a small aliquot is added to an additional amplification reaction.

[0008] It is known that amplifying previously amplified DNA segments in a targeted manner again, for example amplifying an aliquot or dilution of the amplicon product of a target-specific PCR, is prone to undesired artifacts, for example products with a high background of undesired DNA. Therefore, analysis of target nucleic acids using successive rounds of specific PCR is typically performed under special conditions, for example using different primer pairs in successive reactions. For example, in "nested PCR", a first round of amplification is performed to generate a first amplicon, and a second round of amplification is performed using a primer pair in which one or both primers anneal to a site inside the region defined by the first primer pair, i.e., the second primer pair is considered to be "nested" inside the first primer pair. In this way, background amplification products from the first PCR that do not contain the correct internal sequence are not further amplified in the second reaction. Other strategies to reduce undesired effects include using very low concentrations of primers in the first amplification. Changing reaction conditions between the first and second amplifications (or other detection assays) is often done by either purifying the DNA from the first amplification reaction or by using sufficient dilution so that negligible amounts of reaction components are carried over into the subsequent reaction. Summary of the Invention

[0009] During the course of development of the methods described herein, it was determined that complex combinations of marker nucleic acids can be both pre-amplified and then amplified for real-time detection without having to individually optimize the concentrations of different individual primer pairs to uniformly equalize amplification efficiency. Conditions are provided that reduce amplification bias between different co-amplified targets in a complex multiplexed pre-amplification mixture.

[0010] Surprisingly, the use of pre-amplification reaction conditions that reduce amplification bias between multiplexed targets allows for more complex multiplexing in subsequent PCR assays, such as PCR-flap assay reactions, eliminating the need to use differently labeled probes or FRET cassettes to separately detect and measure each different target amplified in a subsequent detection reaction, and allows, for example, analysis based on a composite signal without the need to measure each signal separately.

[0011] This technology does not require the use of nested primers or nested primer pairs in the preamplification of whole genome and / or PCR-flap assay reaction.Surprisingly, targeted preamplification can be multiplexed using the same combination of primer pairs used in the second round, which is the highly multiplexed amplification of the same target set (or a subset of target loci).In a preferred embodiment, the subsequent multiplex detection assay comprises PCR-flap assay reaction, such as QuARTS and LQAS / TELQAS flap assay reaction, which combines PCR target amplification with FEN-1-mediated flap cleavage for signal amplification.

[0012] The methods, compositions, systems, devices, and kits disclosed herein each have several aspects, no single one of which is solely responsible for their desirable properties. Without limiting the scope of the claims, some prominent features will now be briefly discussed. Numerous other embodiments are also contemplated, including embodiments having fewer, additional, and / or different components, steps, features, objects, benefits, and advantages. The components, aspects, and steps may be arranged and ordered differently. After considering this discussion, and especially after reading the section entitled "Detailed Description of the Invention," one will be able to appreciate how the features of the devices and methods disclosed herein provide advantages over other known devices and methods.

[0013] The present technology provides the following non-limiting embodiments.

[0014] 1. A method for analyzing a mixture containing a plurality of target nucleic acids, comprising: a) treating a sample suspected of containing a plurality of different target nucleic acids in a preamplification reaction mixture to generate a multiplex preamplified mixture; the pre-amplification reaction mixture comprises at least four different target-specific primer pairs for generating amplified regions from at least four different target nucleic acids (if present in the sample), and at least one reference primer pair for generating amplified regions from a reference nucleic acid; said treating, wherein each of said primers in said at least four different target-specific primer pairs and in said at least one reference primer pair is at essentially the same concentration; b) adding a portion of said multiplex pre-amplified mixture to a multiplex PCR assay reaction mixture, said multiplex PCR assay reaction mixture comprising: i) an additional amount of each of the at least four different target-specific primer pairs and the reference primer pair, wherein the primers in the additional amount of the at least four different target-specific primer pairs and the at least one reference primer pair are added in essentially equal amounts; ii) at least four different target-specific probe oligonucleotides, each of which specifically hybridizes to a different one of the amplified regions (if amplified in step a) from the at least four different target nucleic acids, the at least four different target-specific probe oligonucleotides comprising a first label; and iii) adding a reference probe flap oligonucleotide that specifically hybridizes to the amplified region from the reference nucleic acid, the reference probe flap oligonucleotide comprising a second label; And c) performing a PCR assay using the multiplex PCR assay reaction mixture, wherein the reference nucleic acid region and each of the at least four different target regions (if amplified in step a)) are amplified in the PCR assay reaction mixture, target-specific probe oligonucleotides and the reference probe oligonucleotides specifically hybridize to the target regions and the reference nucleic acid regions amplified in the multiplex PCR assay reaction mixture, are cleaved to release first and second labels, and measure the released first and second labels.

[0015] 2. The method of embodiment 1, wherein the preamplification reaction mixture comprises a low bias amplification buffer.

[0016] 3. The method of embodiment 2, wherein said multiplex PCR assay reaction mixture comprises a low bias amplification buffer, preferably the same low bias amplification buffer used in said pre-amplification reaction mixture.

[0017] 4. The method according to any one of embodiments 1 to 3, wherein in step b), the at least four different target-specific probe oligonucleotides and the reference probe flap oligonucleotide are present in the multiplex PCR assay reaction mixture at essentially the same concentration.

[0018] 5. The method of any one of embodiments 1 to 4, wherein the first label comprises a first 5' flap sequence, and the first 5' flap sequence is not substantially complementary to any of the amplified regions from the at least four different target nucleic acids.

[0019] 6. The method of embodiment 5, wherein the second label comprises a second 5' flap sequence, the second 5' flap sequence being different from the first 5' flap sequence and not substantially complementary to the amplified region from the reference nucleic acid.

[0020] 7. The method of embodiment 5 or embodiment 6, wherein the PCR assay reaction mixture further comprises a first FRET cassette labeled with a first fluorophore and / or a second FRET cassette labeled with a second fluorophore, wherein the first FRET cassette comprises a sequence complementary to the first 5' flap sequence and the second FRET cassette comprises a sequence complementary to the second 5' flap sequence.

[0021] 8. The method of any one of embodiments 1 to 7, wherein said PCR assay reaction mixture further comprises a flap endonuclease, preferably a FEN-1 endonuclease, preferably an archaeal FEN-1 endonuclease.

[0022] 9. The method of any one of embodiments 1-8, wherein the first label comprises a first FRET system comprising a first fluorophore, the second label comprises a second FRET system comprising a second fluorophore, and fluorescence from the first fluorophore and the second fluorophore is measured during the PCR assay.

[0023] 10. The at least four different target-specific primer pairs are at least five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine ... 10. The method of any one of embodiments 1-9, comprising 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different target-specific primer pairs.

[0024] 11. The at least four different target-specific probe oligonucleotides are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 11. The method of any one of embodiments 1-10, comprising 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different target-specific probe oligonucleotides.

[0025] 12. The method of any one of embodiments 1-11, wherein the concentrations of the at least four different target-specific primer pairs and the reference primer pair in the PCR assay reaction mixture are essentially the same as the concentrations of the at least four different target-specific primer pairs and the reference primer pair in the pre-amplification reaction mixture.

[0026] 13. The low bias amplification buffer comprises a 3-(n-morpholino)propanesulfonic acid (MOPS) buffer and at least about 6 mM, preferably 6.1 mM, 6.2 mM, 6.5 mM, 6.4 mM, 6.5 mM, 6.6 mM, 6.7 mM, 6.8 mM, 6.9 mM, 7.0 mM, 7.1 mM, 7.2 mM, 7.3 mM, 7.4 mM, 7.5 mM, 7.6 mM, 7.7 mM, 7.8 mM, 7.9 mM, 8.0 mM, 8.1 mM, 8.2mM, 8.3mM, 8.4mM, 8.5mM, 8.6mM, 8.7mM, 8.8mM, 8.9mM, 9.0mM, 9.1mM, 9.2mM, 9.3mM, 9.4mM, 9.5mM, 9.6mM, 9.7mM , 9.8mM, 9.9mM, 10.0mM, 10.1mM, 10.2mM, 10.3mM, 10.4mM, 10.5mM, 10.6mM, 10.7mM, 10.8mM, 10.9mM, or 11.0mM Mg ++The method according to any one of embodiments 1 to 12, comprising:

[0027] 14. The low bias amplification buffer contains about 7.5 mM Mg ++ 14. The method of embodiment 13, comprising:

[0028] 15. The pre-amplification reaction mixture comprises at least one additional target-specific primer pair for generating an amplified region from an additional target nucleic acid (if present in the sample), the amplified region being different from those from the at least four different target nucleic acids and from those from the reference nucleic acid, and the multiplex PCR assay reaction mixture comprises: i) an additional amount of said at least one additional target-specific primer pair essentially the same as said additional amount of said at least four different target-specific primer pairs; and ii) at least one additional target-specific probe oligonucleotide that specifically hybridizes to an amplified region from said at least one additional target nucleic acid (if amplified in step a), said at least one additional target-specific probe oligonucleotide having a third label, said third label being different from said first and second labels.

[0029] 16. The method of embodiment 15, wherein the third label comprises a third 5' flap sequence, the third 5' flap sequence being different from the first 5' flap sequence and the second 5' flap sequence and not substantially complementary to the amplified region from the additional target nucleic acid.

[0030] 17. The method of embodiment 16, wherein the PCR assay reaction mixture further comprises a third FRET cassette labeled with a third fluorophore, the third FRET cassette comprising a sequence complementary to the third 5' flap sequence.

[0031] 18. The at least one additional target-specific primer pair is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different additional target nucleic acids (in said sample). 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different additional target-specific primer pairs; and The multiplex PCR assay reaction mixture may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 11 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 additional target specific probe oligonucleotides, leotide is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 18. The method of any one of embodiments 14-17, wherein the amplified region (if amplified in step a)) from 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different additional target nucleic acids specifically hybridizes to the amplified region (if amplified in step a)).

[0032] 19. A method for analyzing a sample for multiple target nucleic acids in a PCR-flap assay, comprising: a) treating a nucleic acid sample in a preamplification reaction mixture to produce a multiplex preamplified mixture, the pre-amplification reaction mixture comprises at least four different target-specific primer pairs for generating amplified regions from at least four different target nucleic acids (if present in the sample), and at least one reference primer pair for generating amplified regions from a reference nucleic acid; said treating, wherein each of said primers in said at least four different target-specific primer pairs and in said at least one reference primer pair is at essentially the same concentration; b) adding a portion of said multiplex pre-amplified mixture to a multiplex PCR-flap assay reaction mixture, i) an additional amount of each of the at least four different target-specific primer pairs and the reference primer pair, wherein the primers in the additional amount of the at least four different target-specific primer pairs and the at least one reference primer pair are added in essentially equal amounts; ii) at least four different target-specific flap probe oligonucleotides, each of which specifically hybridizes to a different one of the amplified regions (if amplified in step a)) from said at least four different target nucleic acids, at least four different target-specific flap probe oligonucleotides, each one of said at least four different target-specific flap probe oligonucleotides comprising a first 5' flap sequence, said first 5' flap sequence being substantially not complementary to any of said amplified regions from said at least four different target nucleic acids; iii) a reference flap probe oligonucleotide that specifically hybridizes to the amplified region from the reference nucleic acid, the reference flap probe oligonucleotide having a second 5' flap sequence, the second 5' flap sequence being different from the first 5' flap sequence and not substantially complementary to the amplified region from the reference nucleic acid; iv) a first FRET cassette labeled with a first fluorophore, the first FRET cassette comprising a sequence complementary to the first 5' flap sequence; v) a second FRET cassette labeled with a second fluorophore, the second FRET cassette comprising a sequence complementary to the second 5' flap sequence; and vi) PCR-flap assay buffer; And c) performing a PCR-flap assay using the multiplex PCR-flap assay reaction mixture, wherein the reference nucleic acid and each of the at least four different target regions (if amplified in step a) are amplified in the PCR-flap assay reaction mixture, and measuring fluorescence from the first fluorophore and the second fluorophore.

[0033] 20. The method of embodiment 19, wherein the preamplification reaction mixture comprises a low bias amplification buffer.

[0034] 21. The method according to embodiment 19 or embodiment 20, wherein in step b), the at least four different target-specific flap probe oligonucleotides and the reference flap probe oligonucleotide present in the multiplex PCR-flap assay reaction mixture are at essentially the same concentration.

[0035] 22. The method of any one of embodiments 1-21, wherein treating the nucleic acid in the pre-amplification reaction mixture comprises thermal cycling the pre-amplification reaction mixture for less than 20 thermal cycles, preferably less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, or less than 5 thermal cycles.

[0036] 23. The method of any one of embodiments 19 to 21, wherein the concentrations of the at least four different target-specific primer pairs and the reference primer pair in the PCR-flap assay reaction mixture are essentially the same as the concentrations of the at least four different target-specific primer pairs and the reference primer pair in the pre-amplification reaction mixture.

[0037] 24. The method of any one of the preceding claims, further comprising diluting at least a portion of said multiplex pre-amplified mixture prior to step b).

[0038] 25. The pre-amplification reaction mixture comprises at least one additional target-specific primer pair for generating an amplified region from an additional target nucleic acid (if present in the sample), the amplified region being different from those from the at least four different target nucleic acids and from those from the reference nucleic acid, and the multiplex PCR-flap assay reaction mixture comprises: i) an additional amount of said at least one additional target-specific primer pair essentially the same as said additional amounts of said at least four different target-specific primer pairs; ii) at least one additional target-specific flap probe oligonucleotide that specifically hybridizes to an amplified region from the at least one additional target nucleic acid (if amplified in step a), the at least one additional target-specific flap probe oligonucleotide having a third 5' flap sequence, the third 5' flap sequence being different from the first and second 5' flap sequences and not substantially complementary to the amplified region from the at least one additional target nucleic acid; and iii) A method according to any one of embodiments 19 to 24, further comprising a third FRET cassette labeled with a third fluorophore, the third FRET cassette comprising a sequence complementary to the third 5' flap sequence.

[0039] 26. The at least one additional target-specific primer pair is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different additional target nucleic acids (in said sample). 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different additional target-specific primer pairs; and The multiplex PCR-flap assay reaction mixture may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 23 , 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 additional target specific flap probe oligonucleotides, The target oligonucleotide is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 26. The method of any one of embodiments 19 to 25, wherein the nucleic acid sequence specifically hybridizes to an amplified region (if amplified in step a)) from 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different additional target nucleic acids.

[0040] 27. The method of any one of embodiments 1 to 26, wherein the sample suspected of containing the plurality of different target nucleic acids is prepared from an animal or human sample comprising one or more of soil, water, a sample comprising one or more of plant material, stool, tissue, sputum, mucus, blood or a blood product (selected from plasma, serum, whole blood), organ waste, and urine.

[0041] 28. The method of embodiment 27, wherein the sample suspected of containing the plurality of different target nucleic acids comprises cell-free DNA isolated from plasma.

[0042] 29. The method of embodiment 28, wherein the sample suspected of containing a plurality of different target nucleic acids comprises cDNA prepared from RNA isolated from plasma.

[0043] 30. The method of embodiment 28 or embodiment 29, wherein the pre-amplification reaction mixture of step a) has a total volume, and the sample suspected of containing the plurality of different target nucleic acids is prepared from at least 1 mL of plasma and is at least 20-50% of the total volume of the pre-amplification reaction mixture of step a).

[0044] 31. A method for analyzing a sample for at least 10 different target nucleic acids in a single PCR-flap assay reaction, comprising: a) treating nucleic acid comprising a plurality of different target nucleic acids in a preamplification reaction mixture comprising a PCR-flap assay buffer to generate a multiplex preamplified mixture; said treating, wherein said pre-amplification reaction mixture comprises at least 10 different target-specific primer pairs for generating amplified regions from at least 10 different target nucleic acids in said sample and at least one reference primer pair for generating amplified regions from a reference nucleic acid, and each of said primers within said at least 10 different target-specific primer pairs and within said at least one reference primer pair are at essentially the same concentration in said pre-amplification reaction mixture; b) adding a portion of said multiplex pre-amplified mixture to a multiplex PCR-flap assay reaction mixture, i) an additional amount of each of the at least 10 different target-specific primer pairs and the reference primer pair, wherein the primers in the additional amount of the at least 10 different target-specific primer pairs and the at least one reference primer pair are at essentially the same concentration in the multiplex PCR-flap assay reaction mixture; ii) at least 10 different target-specific flap oligonucleotides, each target-specific flap oligonucleotide specifically hybridizing to a different one of the amplified regions from said at least 10 different target nucleic acids; the at least ten different target-specific flap oligonucleotides divided into a first group and a second group, each of the flap oligonucleotides in the first group comprising a first 5' flap sequence and each of the flap oligonucleotides in the second group comprising a second 5' flap sequence; iii) a reference flap oligonucleotide that specifically hybridizes to the amplified region from the reference nucleic acid, the reference flap oligonucleotide having a third 5' flap sequence, the third 5' flap sequence being different from the first 5' flap sequence and the second 5' flap sequence; iv) a first FRET cassette labeled with a first fluorophore, the first FRET cassette comprising a sequence complementary to the first 5' flap sequence; v) a second FRET cassette labeled with a second fluorophore, the second FRET cassette comprising a sequence complementary to the second 5' flap sequence; vi) a third FRET cassette labeled with a third fluorophore, the third FRET cassette comprising a sequence complementary to the third 5' flap sequence; and vi) PCR-flap assay buffer; And c) performing a PCR-flap assay using the multiplex PCR-flap assay reaction mixture, wherein the reference nucleic acid and each of the at least 10 different target regions are amplified in the PCR-flap assay reaction mixture, and measuring fluorescence from the first fluorophore, the second fluorophore, and the third fluorophore.

[0045] 32. The method of embodiment 31, wherein performing the PCR-flap assay with the multiplex PCR-flap assay reaction mixture comprises thermal cycling the multiplex PCR-flap assay reaction mixture for at least 25 thermal cycles, preferably more than 30, more than 31, more than 32, more than 33, more than 34, more than 35, more than 36, more than 37, more than 38, more than 39, more than 40, more than 41, more than 42, more than 43, more than 44, or more than 45 thermal cycles.

[0046] 33. The method of embodiment 31 or embodiment 32, wherein fluorescence from the first fluorophore, the second fluorophore, and the third fluorophore is measured during thermal cycling.

[0047] 34. A composition comprising: a) a group of oligonucleotides, i) a first set of at least four different target-specific primer pairs for generating amplified regions from a first group of at least four different target nucleic acids; ii) at least one reference primer pair for generating an amplified region from a reference nucleic acid; wherein each of said primers within said at least four different target-specific primer pairs and within said at least one reference primer pair are at essentially the same concentration; iii) a first set of at least four different target-specific flap oligonucleotides, each target-specific flap oligonucleotide specifically hybridizing to a different one of the amplified regions from the group of at least four different target nucleic acids; a first set of at least four different target-specific flap oligonucleotides, each one of the at least four different flap oligonucleotides comprising a first 5' flap sequence; iv) a reference flap oligonucleotide that specifically hybridizes to the amplified region from the reference nucleic acid, the reference flap oligonucleotide having a second 5' flap sequence; the reference flap oligonucleotide, wherein the second 5' flap sequence is different from the first 5' flap sequence; v) a first FRET cassette labeled with a first fluorophore, the first FRET cassette comprising a sequence that is complementary to the first 5' flap sequence and that is not substantially complementary to the second 5' flap sequence; and vi) a second FRET cassette labeled with a second fluorophore, the second FRET cassette comprising a sequence that is complementary to the second 5' flap sequence and that is not substantially complementary to the first 5' flap sequence; and b) dNTPs in the mixture.

[0048] 35. The first set of at least four different target-specific flap oligonucleotides is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 35. The composition of embodiment 34, comprising 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different flap oligonucleotides.

[0049] 36. The first set of at least four different target-specific primer pairs is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53. , 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different primer pairs.

[0050] 37.vii) A second set of different target-specific primer pairs for generating amplified regions from a second group of different target nucleic acids, a second set of different target-specific primer pairs, each of said primers in said set being in essentially the same amount or concentration as the primers in said first set of target-specific primer pairs; viii) a second set of different target-specific flap oligonucleotides, each target-specific flap oligonucleotide specifically hybridizing to a different one of the amplified regions from a second group of different target nucleic acids, a second set of different target-specific flap oligonucleotides, each of the flap oligonucleotides in the second set of target-specific flap oligonucleotides comprising a third 5' flap sequence; and ix) a third FRET cassette labeled with a third fluorophore, the third FRET cassette comprising a sequence complementary to the third 5' flap sequence and substantially not complementary to either the first 5' flap sequence or the second 5' flap sequence.

[0051] 38. The second set of different target-specific flap oligonucleotides is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 38. The composition of embodiment 37, comprising 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different flap oligonucleotides.

[0052] 39. The second set of different target-specific primer pairs is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 108, 109, 109, 101, 102, 103, 104, 105, 106, 1 39. The composition of embodiment 37 or embodiment 38, comprising 3, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different primer pairs.

[0053] 40.x) DNA polymerases, preferably thermostable DNA polymerases; xi) a flap endonuclease, preferably a FEN-1 endonuclease, preferably an archaeal FEN-1 endonuclease, and xii) a low bias amplification buffer.

[0054] 41.xiii) The composition of any one of embodiments 37 to 40, further comprising a portion of a multiplex pre-amplified mixture amplified in a low bias amplification buffer using at least four different target-specific primer pairs and at least one reference primer pair at essentially the same concentration, said multiplex pre-amplified mixture comprising amplified regions of a first group of said at least four different target nucleic acids and an amplified region of said reference nucleic acid.

[0055] 42. a) i) a first set of at least four different target-specific primer pairs for generating amplified regions from a first group of at least four different target nucleic acids in said sample; ii) at least one reference primer pair for generating an amplified region from a reference nucleic acid in said sample; wherein each of said primers within said at least four different target-specific primer pairs and within said at least one reference primer pair are in essentially the same amount or concentration; iii) a first set of at least four different target-specific flap oligonucleotides, each target-specific flap oligonucleotide specifically hybridizing to a different one of the amplified regions from the group of at least four different target nucleic acids; a first set of at least four different target-specific flap oligonucleotides, each one of the at least four different flap oligonucleotides comprising a first 5' flap sequence; iv) a reference flap oligonucleotide that specifically hybridizes to the amplified region from the reference nucleic acid, the reference flap oligonucleotide having a second 5' flap sequence; the reference flap oligonucleotide, wherein the second 5' flap sequence is different from the first 5' flap sequence; v) a first FRET cassette labeled with a first fluorophore, the first FRET cassette comprising a sequence that is complementary to the first 5' flap sequence and that is not substantially complementary to the second 5' flap sequence; and vi) a mixture comprising a population of oligonucleotides comprising a second FRET cassette labeled with a second fluorophore, the second FRET cassette comprising a sequence complementary to the second 5' flap sequence and not substantially complementary to the first 5' flap sequence.

[0056] 43. The first set of at least four different target-specific flap oligonucleotides is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 43. The kit of embodiment 42, comprising 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different flap oligonucleotides.

[0057] 44. The first set of at least four different target-specific primer pairs is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53. , 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different primer pairs.

[0058] 45. The mixture, vii) a second set of different target-specific primer pairs for generating amplified regions from a second group of different target nucleic acids in the sample, a second set of different target-specific primer pairs, each of said primers in said set being in essentially the same amount or concentration as the primers in said first set of target-specific primer pairs; viii) a second set of different target-specific flap oligonucleotides, each target-specific flap oligonucleotide specifically hybridizing to a different one of the amplified regions from a second group of different target nucleic acids, a second set of different target-specific flap oligonucleotides, each of the flap oligonucleotides in the second set of target-specific flap oligonucleotides comprising a third 5' flap sequence; and ix) A kit according to any one of embodiments 42 to 44, further comprising a third FRET cassette labeled with a third fluorophore, the third FRET cassette comprising a sequence that is complementary to the third 5' flap sequence and is not substantially complementary to either the first 5' flap sequence or the second 5' flap sequence.

[0059] 46. ​​The second set of different target-specific flap oligonucleotides is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 46. ​​The kit of embodiment 45, comprising 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different flap oligonucleotides.

[0060] 47. The second set of different target-specific primer pairs is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 108, 109, 109, 101, 102, 103, 104, 105, 106, 1 47. The kit of embodiment 45 or embodiment 46, comprising 3, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different primer pairs.

[0061] 48.b) a DNA polymerase, preferably a thermostable DNA polymerase; c) a flap endonuclease, preferably a FEN-1 endonuclease, preferably an archaeal FEN-1 endonuclease; d) a low bias amplification buffer; and e) dNTPs.

[0062] 49.f) in a second mixture, a portion of said group of oligonucleotides, i) a first set of at least four different target-specific primer pairs for generating amplified regions from a first group of at least four different target nucleic acids in a sample; ii) said at least one reference primer pair for generating an amplified region from a reference nucleic acid in said sample; 49. The kit of any one of embodiments 42 to 48, further comprising a portion of the group of oligonucleotides, wherein each of the primers in the at least four different target-specific primer pairs and in the at least one reference primer pair is in essentially the same amount or concentration.

[0063] 50. A kit according to any one of embodiments 42 to 49, wherein the mixture of a) is in a dry form or in the form of a solution.

[0064] 51. The kit according to embodiment 49 or 50, wherein the mixture of f) is in a dry form or in the form of a solution.

[0065] 52. The low bias amplification buffer comprises a 3-(n-morpholino)propanesulfonic acid (MOPS) buffer and Mg in the PCR reaction mixture. ++ and preferably 6.1 mM, 6.2 mM, 6.5 mM, 6.4 mM, 6.5 mM, 6.6 mM, 6.7 mM, 6.8 mM, 6.9 mM, 7.0 mM, 7.1 mM, 7.2 mM, 7.3 mM, 7.4 mM, 7.5 mM, 7.6 mM, 7.7 mM, 7.8 mM, 7.9 mM, 8.0 mM, 8.1 mM, 8.2 mM, 8.3 mM, 8.4 mM, 8.5 mM, 8.6mM, 8.7mM, 8.8mM, 8.9mM, 9.0mM, 9.1mM, 9.2mM, 9.3mM, 9.4mM, 9.5mM, 9.6mM, 9.7mM, 9.8mM, 9.9mM, 10.0mM, 10.1mM, 10.2mM, 10.3mM, 10.4mM, 10.5mM, 10.6mM, 10.7mM, 10.8mM, 10.9mM, or 11.0mM Mg ++ The concentration of Mg ++ The kit according to any one of embodiments 48 to 51, comprising:

[0066] 53. The low bias amplification buffer reduces the amount of Mg in the PCR reaction mixture. ++ The final concentration of Mg is about 7.5 mM. ++ The concentration of Mg ++ 53. The method of embodiment 52, comprising:

[0067] definition To facilitate understanding of the present technology, several terms and phrases are defined below. Further definitions are set forth throughout the detailed description.

[0068] Throughout the specification and claims, the following terms have the meanings expressly associated therewith, unless the context clearly dictates otherwise. As used herein, the phrase "in one embodiment" may refer to the same embodiment, but not necessarily to the same embodiment. Additionally, as used herein, the phrase "in another embodiment" may refer to a different embodiment, but not necessarily to a different embodiment. Thus, as described below, various embodiments of the invention can be readily combined without departing from the scope or spirit of the invention.

[0069] Additionally, as used herein, the term "or" is an inclusive "or" operator and is synonymous with the term "and / or" unless the context clearly dictates otherwise. The term "based on" is not exclusive and acknowledges that a relationship may be based on additional unlisted factors unless the context clearly dictates otherwise. Additionally, throughout this specification, the meanings of "a," "an," and "the" include plural referents. The meaning of "in" includes "in" and "on."

[0070] The transitional phrase "consisting essentially of" used in the claims of this application limits the claims to the specified materials or steps and to those materials or steps that "do not materially affect the basic and novel characteristic(s)" of the claimed invention, as stated in In re Herz, 537 F.2d 549,551-52,190 USPQ 461,463 (CCPA 1976). For example, a composition "consisting essentially of" a recited element may contain unrecited contaminants at levels that, if present, do not alter the function of the recited composition, as compared to a pure composition, i.e., a composition "consisting essentially of" the recited components.

[0071] Conditional expressions, such as "can," "could," "might," or "may," are generally intended to convey that certain features, elements, and / or steps are included in certain embodiments but not in other embodiments, unless specifically stated otherwise or understood otherwise within the context in which they are used. Thus, such conditional expressions are generally not intended to imply that features, elements, and / or steps are in any way required by one or more embodiments, or that one or more embodiments necessarily include logic, with or without user input or direction, for determining whether those features, elements, and / or steps are included or should be performed in any particular embodiment.

[0072] Conjunctive language such as "at least one of X, Y, and Z," unless specifically stated otherwise, is generally understood to have other meanings depending on the context in which it is used, and conveys that an item, term, etc., can be X, Y, or Z. Thus, such conjunctive language is generally not intended to imply that a particular embodiment requires the presence of at least one of X, at least one of Y, and at least one of Z.

[0073] As used herein, expressions of degree, such as "approximately," "about," "generally," and "substantially," refer to a value, amount, or characteristic that approaches a stated value, amount, or characteristic that still performs a desired function or achieves a desired result.

[0074] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA, or for the production of a polypeptide or a precursor thereof. A functional polypeptide can be encoded by a full-length coding sequence or by any portion of that coding sequence, so long as the desired activity or functional property of that polypeptide (e.g., enzymatic activity, ligand binding, signal transduction, etc.) is retained. The term "portion" when used in reference to a gene refers to a fragment of that gene. Fragments can range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, "a nucleotide sequence comprising at least a portion of a 'gene'" can include a fragment of a gene or the entire gene.

[0075] The term "gene" also encompasses the coding region of a structural gene, which includes sequences located adjacent to the coding region at both the 5' and 3' ends, e.g., spanning a distance of about 1 kb on either end, and which corresponds to the length of the full-length mRNA (e.g., including coding, regulatory, structural and other sequences). Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated or untranslated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated or untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. In some organisms (e.g., eukaryotes), genomic forms or clones of a gene contain the coding region interrupted by non-coding sequences called "introns" or "intervening regions" or "intervening sequences". Introns are gene segments that are transcribed into nuclear RNA (hnRNA) and may contain regulatory elements such as enhancers. Introns are removed or "excised" from the nuclear or primary transcript and therefore are absent in the messenger RNA (mRNA) transcript, which functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0076] In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3' ends of the sequences present in the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the untranslated sequences present in the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers, which control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct the termination of transcription, post-transcriptional cleavage, and polyadenylation.

[0077] The term "wild type" when referring to a gene refers to a gene that has the characteristics of a gene isolated from a naturally occurring source. The term "wild type" when referring to a gene product refers to a gene product that has the characteristics of a gene product isolated from a naturally occurring source. The term "wild type" when referring to a protein refers to a protein that has the characteristics of a naturally occurring protein. The term "naturally occurring" when applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and has not been intentionally modified by the hand of man in a laboratory is naturally occurring. A wild type gene is often the gene or allele that is most frequently observed in a population and is therefore arbitrarily designated the "normal" or "wild type" form of the gene. In contrast, the terms "modified" or "mutated" when referring to a gene or gene product refer to a gene or gene product that exhibits modifications (e.g., altered characteristics) in sequence and / or functional properties, respectively, when compared to a wild type gene or gene product. It is noted that naturally occurring mutants can be isolated and identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.

[0078] The term "allele" refers to genetic variations, including, but not limited to, variants and mutations, polymorphic and single nucleotide polymorphic loci, frameshifts, and splice variants. Alleles may occur naturally in a population or may arise during the lifetime of any particular individual in that population.

[0079] Thus, the terms "variant" and "mutant" when used in reference to a nucleotide sequence refer to a nucleic acid sequence that differs from another, usually a related, nucleic acid sequence by one or more nucleotides. A "mutation" is a difference between two different nucleotide sequences, typically one of which is a reference sequence.

[0080] As used herein, "methylation" refers to methylation of cytosine at the C5 or N4 position of cytosine, at the N6 position of adenine, or other types of nucleic acid methylation. In vitro amplified DNA is usually unmethylated, since typical in vitro DNA amplification methods do not preserve the methylation pattern of the amplified template. However, "unmethylated DNA" or "methylated DNA" can also refer to amplified DNA whose original template was unmethylated or methylated, respectively.

[0081] Thus, as used herein, "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl moiety on a nucleotide base, where the methyl moiety is not present in recognized typical nucleotide bases.For example, cytosine does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at the 5th position of its pyrimidine ring.Thus, cytosine is not a methylated nucleotide, but 5-methylcytosine is a methylated nucleotide.In another example, thymine contains a methyl moiety at the 5th position of its pyrimidine ring, but since thymine is a typical nucleotide base of DNA, for the purposes of this specification, thymine is not considered to be a methylated nucleotide when present in DNA.

[0082] As used herein, a "methylated nucleic acid molecule" refers to a nucleic acid molecule that contains one or more methylated nucleotides.

[0083] As used herein, the "methylation state," "methylation profile," and "methylation status" of a nucleic acid molecule refer to the presence or absence of one or more methylated nucleotide bases in a nucleic acid molecule. For example, a nucleic acid molecule that contains a methylated cytosine is considered to be methylated (e.g., the methylation state of the nucleic acid molecule is methylated). A nucleic acid molecule that does not contain any methylated nucleotides is considered to be unmethylated. In some embodiments, a nucleic acid may be characterized as "unmethylated" if it is not methylated at a particular locus (e.g., at a particular single CpG dinucleotide locus) or at a particular combination of loci, even if it is methylated at other loci within the same gene or molecule.

[0084] The methylation state of a particular nucleic acid sequence (e.g., a genetic marker or DNA region described herein) can indicate the methylation state of every base within the sequence, or can indicate the methylation state of a subset of bases (e.g., one or more cytosines) within the sequence, or can indicate information about the local methylation density within the sequence, with or without providing precise information about the internal sequence location where methylation occurs.

[0085] As used herein, the terms "marker gene", "biomarker" and "marker" are used interchangeably and refer to DNA, RNA, or protein (or other sample component) that is associated with the condition of a subject or environment from which the sample was obtained. A biomarker may indicate, for example, a viral or bacterial strain present in the environment or subject, cancer or other gene-related disease, whether or not the marker region is within the coding region of the DNA. Markers may include, for example, regulatory regions, flanking regions, intergenic regions, etc. Similarly, the term "marker" as used with respect to any component of a sample, e.g., protein, RNA, carbohydrate, small molecule, etc., refers to a component that can be assayed (e.g., measured or otherwise characterized) in the sample and that is associated with the condition of the subject or sample from the subject. The term "methylation marker" refers to a gene or DNA whose methylation state is associated with a condition, e.g., cancer.

[0086] The methylation state of a nucleotide locus in a nucleic acid molecule refers to the presence or absence of a methylated nucleotide at a particular locus in the nucleic acid molecule. For example, the methylation state of a cytosine at the 7th nucleotide in a nucleic acid molecule is methylated if the nucleotide present at the 7th nucleotide in the nucleic acid molecule is 5-methylcytosine. Similarly, the methylation state of a cytosine at the 7th nucleotide in a nucleic acid molecule is unmethylated if the nucleotide present at the 7th nucleotide in the nucleic acid molecule is cytosine (and is not 5-methylcytosine).

[0087] Methylation status can optionally be expressed or indicated by a "methylation value" (e.g., representing a frequency, rate, ratio, percent, etc. of methylation). Methylation values ​​can be generated, for example, by quantifying the amount of intact nucleic acid present after restriction digestion with a methylation-dependent restriction enzyme, or by comparing amplification profiles after a bisulfite reaction, or by comparing sequences of bisulfite-treated and untreated nucleic acid. Thus, a value, e.g., a methylation value, represents a methylation status, and thus can be used as a quantitative indicator of methylation status across multiple copies of a locus. This is of particular use when it is desirable to compare the methylation status of sequences in a sample to a threshold or reference value.

[0088] As used herein, "methylation frequency" or "percent (%) methylation" refers to the number of instances where a molecule or locus is methylated relative to the number of instances where the molecule or locus is unmethylated.

[0089] Thus, methylation state refers to the state of methylation of a nucleic acid (e.g., a genomic sequence). Moreover, methylation state refers to the characteristics of a nucleic acid segment at a particular genomic locus that are related to methylation. Such characteristics include, but are not limited to, whether any of the cytosine (C) residues in this DNA sequence are methylated, the location of the methylated C residue(s), the frequency or percentage of methylated C across any particular region of the nucleic acid, and differences in methylation between alleles, for example, due to differences in the origin of the alleles. The terms "methylation state", "methylation profile", and "methylation status" also refer to the relative concentration, absolute concentration, or pattern of methylated or unmethylated C across any particular region of a nucleic acid in a biological sample. For example, if a cytosine (C) residue(s) in a nucleic acid sequence is methylated, it can be referred to as "hypermethylated" or "increased methylation", and if a cytosine (C) residue(s) in a DNA sequence is not methylated, it can be referred to as "unmethylated", "hypomethylated" or "decreased methylation". Similarly, if a cytosine (C) residue(s) in a nucleic acid sequence is methylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual, etc.), the sequence is considered to be hypermethylated or increased in methylation compared to the other nucleic acid sequence. Alternatively, if a cytosine (C) residue(s) in a DNA sequence is unmethylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual, etc.), the sequence is considered to be hypomethylated or decreased in methylation compared to the other nucleic acid sequence. Furthermore, as used herein, the term "methylation pattern" refers to the collection of sites of methylated and unmethylated nucleotides across a region of a nucleic acid. If two nucleic acids have the same or similar number of methylated and unmethylated nucleotides across a region but different positions of methylated and unmethylated nucleotides, the methylation frequency or percentage may be the same or similar but the methylation patterns may be different.Sequences are said to be "differentially methylated" or "differentially methylated" or "differentially methylated" when they differ in the degree (e.g., one is more or less methylated than the other), frequency, or pattern of methylation. The term "differential methylation" refers to the difference in the level or pattern of nucleic acid methylation in a cancer-positive sample compared to the level or pattern of nucleic acid methylation in a cancer-negative sample. The term can also refer to the difference in the level or pattern between patients with and without cancer recurrence after surgery. Differential methylation and specific levels or patterns of DNA methylation are prognostic and predictive biomarkers, for example, after the correct cutoff or predictive features are defined.

[0090] Methylation state frequency can be used to represent a sample from a population of individuals or a single individual. For example, a nucleotide locus with a methylation state frequency of 50% has 50% of the instances methylated and 50% of the instances unmethylated. Such frequency can be used to represent, for example, the degree to which a nucleotide locus or nucleic acid region is methylated in a population of individuals or a collection of nucleic acids. Thus, if the methylation in a first population or pool of nucleic acid molecules is different from the methylation in a second population or pool of nucleic acid molecules, the methylation state frequency of the first population or pool will be different from the methylation state frequency of the second population or pool. Such frequency can also be used to represent, for example, the degree to which a nucleotide locus or nucleic acid region is methylated in a single individual. For example, such frequency can be used to represent the degree to which a group of cells from a tissue sample is methylated or unmethylated at a nucleotide locus or nucleic acid region.

[0091] As used herein, "nucleotide locus" refers to the position of a nucleotide in a nucleic acid molecule. The nucleotide locus of a methylated nucleotide refers to the position of the methylated nucleotide in a nucleic acid molecule.

[0092] Typically, methylation of human DNA occurs on dinucleotide sequences that contain adjacent guanines and cytosines, where the cytosine is located 5' to the guanine (also called CpG dinucleotide sequences). In the human genome, most cytosines within CpG dinucleotides are methylated, although some remain unmethylated in certain CpG dinucleotide-rich genomic regions known as CpG islands (see, e.g., Antequera, et al. (1990) Cell 62:503-514).

[0093] As used herein, "CpG island" refers to a G:C-rich region of genomic DNA that contains an increased number of CpG dinucleotides compared to the total genomic DNA. A CpG island can be at least 100, 200, or more base pairs in length, where the G:C content of the region is at least 50% and the ratio of observed CpG frequency to expected CpG frequency is 0.6, and in some cases, a CpG island can be at least 500 base pairs in length, where the G:C content of the region is at least 55% and the ratio of observed CpG frequency to expected CpG frequency is 0.65. The observed CpG frequency to expected CpG frequency can be calculated according to the method described in Gardiner-Garden et al (1987) J.Mol.Biol.196:261-281. For example, the observed CpG frequency to the expected CpG frequency can be calculated according to the formula R=(A×B) / (C×D), where R is the ratio of observed CpG frequency to expected CpG frequency, A is the number of CpG dinucleotides in the analyzed sequence, B is the total number of nucleotides in the analyzed sequence, C is the total number of C nucleotides in the analyzed sequence, and D is the total number of G nucleotides in the analyzed sequence. Methylation status is typically determined within CpG islands, for example in promoter regions. However, it will be recognized that other sequences in the human genome are also susceptible to DNA methylation, such as CpA and CpT (see Ramsahoye (2000) Proc. Natl. Acad. Sci. USA 97:5237-5242; Salmon and Kaye (1970) Biochim. Biophys. Acta. 204:340-351; Grafstrom (1985) Nucleic Acids Res. 13:2827-2842; Nyce (1986) Nucleic Acids Res. 14:4353-4367; Woodcock (1987) Biochem. Biophys. Res. Commun. 145:888-894).

[0094] As used herein, the terms "methylcytosine," "methyl C," "methylated cytosine," "methylated C," and "meC" are used interchangeably and include both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC).

[0095] As used herein, the term "modified cytosine" or "modified C" refers to a cytosine nucleobase in which the base moiety has a side group or other modification compared to a standard cytosine nucleotide. Modified cytosines include, but are not limited to, 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-carboxylcytosine (5caC), and 5-formylcytosine (5fC).

[0096] As used herein, a "methylation specific reagent" refers to a reagent that modifies the nucleotides of a nucleic acid molecule depending on the methylation state of the nucleic acid molecule. In certain embodiments, the term refers to a compound or composition or other agent or collection or sequence thereof that can change the nucleotide sequence of a nucleic acid molecule in a manner that reflects the methylation state of the nucleic acid molecule. Methods of treating a nucleic acid molecule with such reagents can include contacting the nucleic acid molecule with the reagent, and can be combined with additional steps as necessary to achieve the desired change in the nucleotide sequence. Such methods can be applied such that unmethylated nucleotides (e.g., each unmethylated cytosine) are modified to become a different nucleotide. For example, in some embodiments, such reagents can deaminate unmethylated cytosine nucleotides to generate deoxyuracil residues. An exemplary reagent is a bisulfite reagent.

[0097] Additionally, treatment with "methylation-specific reagents" can be applied such that methylated nucleotides are modified to different nucleotides. For example, methylated cytosines in DNA (including 5mC and 5hmC) can be oxidized by dioxygenases of the ten-eleven translocation (TET) family to borane derivatives (e.g., pyridine borane and 2-picoline borane (pic-BH)) in a process referred to herein as TAPS (TET Assisted Pyridine borane Sequencing). 3 )) can be combined with reduction by borane derivatives. See, for example, the TAPS method described in US2020 / 0370114A1, application Ser. No. 16 / 960,510, filed July 7, 2020, which combines oxidation by TET enzymes with reduction by borane derivatives. In an embodiment of the TAPS method, methylated cytosine is converted to dihydrouracil. Other methods of converting methylated C include, for example, the following: [Table 1]

[0098] (Loise Williams, et al.,Enzymatic Methyl-seq:The next generation of methylome analysis,New England Biolabs Expressions 2019.Feature Article.)

[0099] In a preferred embodiment, the methylation specific reagent modifies one of the four typically occurring nucleotides in a nucleic acid molecule (C, G, T, and A for DNA, C, G, U, and A for RNA), without modifying the other three nucleotides. The nucleotide resulting from the conversion is not limited to the four typically occurring nucleotides listed above, but may include, for example, modified or variant forms of purine or pyrimidine structures, such as nucleobase analogs discussed herein. In a preferred embodiment, the nucleotide generated by the conversion can be recognized by a DNA modifying enzyme, such as a DNA polymerase, as one of the typically occurring nucleotides listed above and serve as a template for strand replication. Conversion of a nucleotide by any of the methods described herein can be detected, for example, by determining the sequence of the resulting strand using standard sequencing methods, or by examining the position of a single or a small number of specific nucleotides to determine the identity of the nucleobase at the selected position.

[0100] As used herein, the term "converted" in reference to a nucleotide or DNA strand refers to a nucleotide or DNA strand that has been treated with a reagent(s) under conditions in which some nucleotides are converted to other nucleotides. For example, in bisulfite conversion, cytosine bases in DNA are typically deaminated to produce uracil bases at the converted locus. Although inefficient, bisulfite can cause deamination of 5-methylcytosine bases to produce thymine bases at the converted locus. "Bisulfite treated" and "bisulfite converted" are used interchangeably herein in reference to DNA or nucleotide loci that have been exposed to bisulfite reagents under conditions in which unmethylated cytosines are typically converted to uracil. In the bisulfite-free TAPS process, methylated cytosines are selectively converted to dihydrouracil (DHU), while unmethylated Cs are not converted. DHU nucleotide bases pair with A nucleotides rather than G nucleotides, allowing them to be easily distinguished from unmethylated C bases in the converted DNA strand.

[0101] As used herein, the term "poorly converted", when used in reference to conversion of nucleotides upon treatment with reagent(s) and / or conditions that convert some nucleotides to other nucleotides, refers to nucleotides that have a reduced conversion rate (preferably less than 10%, more preferably less than 1%) under a given treatment, compared to the conversion rate of nucleotides that are expected to be converted under the same treatment. For example, bisulfite-mediated deamination of cytosine is significantly slowed down by the presence of a 5-methyl group, and the rate at which 5-methylcytosine deaminates to form thymine is about two orders of magnitude less than the rate at which unmethylated cytosine deaminates to form uracil (see, for example, Hayatsu et al., Biochemistry 18:4:632-37(1979); Hayatsu, Proc. Jpn. Acad 84(8):321-330(2008), each of which is incorporated herein by reference). Thus, 5-methylcytosine is said to be underconverted compared to unmethylated cytosine under bisulfite treatment conditions typically used to convert unmethylated cytosine to uracil.

[0102] The term "bisulfite reagent" refers to a reagent comprising bisulfite, disulfite, hydrogen sulfite, or a combination thereof, useful for distinguishing between methylated and unmethylated CpG dinucleotide sequences, as disclosed herein. Methods for such treatment are known in the art (e.g., PCT / EP2004 / 011715 and WO2013 / 116375, each of which is incorporated by reference in its entirety). In some embodiments, the bisulfite treatment is carried out in the presence of a denaturing solvent, such as, but not limited to, n-alkylene glycol or diethylene glycol dimethyl ether (DME), or in the presence of dioxane or a dioxane derivative. In some embodiments, the denaturing solvent is a denaturing solvent having a concentration of 1% to 35% (v / v). In some embodiments, the bisulfite reaction is carried out in the presence of a scavenger, such as, but not limited to, a chroman derivative, such as 6-hydroxy-2,5,7,8,-tetramethylchroman 2-carboxylic acid or trihydroxybenzoic acid and its derivatives, such as gallic acid (see PCT / EP2004 / 011715, which is incorporated by reference in its entirety). In certain preferred embodiments, the bisulfite reaction involves treatment with ammonium hydrogensulfite, also known as ammonium bisulfite, as described, for example, in WO2013 / 116375.

[0103] The term "methylation assay" refers to any assay for determining the methylation status of one or more CpG dinucleotide sequences within a sequence of a nucleic acid.

[0104] As used herein, the "sensitivity" of a given marker (or set of markers used together) refers to the percentage of samples reporting DNA methylation values ​​above a threshold that distinguishes between neoplastic and non-neoplastic samples. In some embodiments, a positive is defined as a histologically confirmed tumor reporting a DNA methylation value above the threshold (e.g., in a range associated with a disease) and a false negative is defined as a histologically confirmed tumor reporting a DNA methylation value below the threshold (e.g., in a range not associated with any disease). Thus, the sensitivity value reflects the probability that a DNA methylation measurement value of a given marker from a known diseased sample falls within the range of disease-associated measurements. As defined herein, the clinical significance of a calculated sensitivity value represents an estimate of the probability that a given marker will detect the presence of a clinical condition when applied to subjects with that condition.

[0105] As used herein, the "specificity" of a given marker (or a set of markers used together) refers to the proportion of non-neoplastic samples that report DNA methylation values ​​below a threshold that distinguishes between neoplastic and non-neoplastic samples. In some embodiments, a negative is defined as a histologically confirmed non-neoplastic sample that reports a DNA methylation value below the threshold (e.g., in a range not associated with any disease) and a false positive is defined as a histologically confirmed non-neoplastic sample that reports a DNA methylation value above the threshold (e.g., in a range associated with a disease). Thus, the specificity value reflects the probability that a DNA methylation measurement of a given marker from a known non-neoplastic sample will fall within the range of non-disease-associated measurements. As defined herein, the clinical significance of a calculated specificity value represents an estimate of the probability that a given marker will detect the absence of a clinical condition when applied to patients without that condition.

[0106] As used herein, a "selected nucleotide" refers to one of the four nucleotides that typically occur within a nucleic acid molecule (C, G, T, and A in DNA and C, G, U, and A in RNA), which can include methylated derivatives of the typically occurring nucleotides (e.g., if C is a selected nucleotide, then both methylated and unmethylated C are included within the meaning of a selected nucleotide), although a methylated selected nucleotide specifically refers to a nucleotide that is typically methylated and an unmethylated selected nucleotide specifically refers to a nucleotide that typically occurs in its unmethylated form.

[0107] The term "methylation-specific restriction enzyme" refers to a restriction enzyme that selectively digests nucleic acids depending on the methylation state of its recognition site. For restriction enzymes that specifically cleave when the recognition site is unmethylated or hemimethylated, cleavage does not occur (or occurs with a much lower efficiency) if the recognition site is methylated on one or both strands. For restriction enzymes that specifically cleave only if the recognition site is methylated (methylation-dependent enzymes), cleavage does not occur (or occurs with a much lower efficiency) if the recognition site is unmethylated. Methylation-specific restriction enzymes are preferred, whose recognition sequences contain CG dinucleotides (e.g., recognition sequences such as CGCG or CCCGGG). Further preferred for some embodiments are restriction enzymes that do not cleave when the cytosine in this dinucleotide is methylated at the carbon atom C5.

[0108] The terms "selectively bind" and "specifically bind" (or selectively or specifically bound, bound, hybridized, hybridizing, annealing, annealing, etc.) when used in reference to interactions between oligonucleotides or other nucleic acids are used interchangeably herein and refer to sufficiently sequence-selective hybridization or base pairing that an oligonucleotide or nucleic acid selectively hybridizes to a particular nucleic acid (e.g., a target nucleic acid having a particular nucleotide sequence) and does not substantially bind to non-target nucleic acids (e.g., having a nucleotide sequence that differs slightly or completely from the target nucleic acid) under conditions that result in selective or specific binding.

[0109] The term "primer" refers to an oligonucleotide, whether naturally occurring, e.g., as a nucleic acid fragment obtained from a restriction digest, or produced synthetically, that can act as a point of initiation of synthesis when placed under conditions that induce synthesis of a primer extension product complementary to a nucleic acid template strand (e.g., in the presence of an inducing agent, such as DNA polymerase, and nucleotides, at a suitable temperature and pH). The primer is preferably single-stranded for maximum efficiency of amplification, but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare the extension products. Preferably, the primer is an oligodeoxyribonucleotide. In general, the primer is sufficiently long to prime the synthesis of an extension product in the presence of the inducing agent. The exact length of the primer will vary depending on many factors, such as temperature, source of primer, and method use.

[0110] The term "probe" refers to an oligonucleotide (e.g., a series of nucleotides) that can hybridize to another oligonucleotide of interest, whether naturally occurring, as in a purified restriction digest, or produced synthetically, recombinantly, or by PCR amplification. Probes can be single-stranded or double-stranded. Probes are useful for the detection, identification, and isolation of specific gene sequences (e.g., "capture probes"). It is contemplated that any probe used in the present invention can, in some embodiments, be labeled with any "reporter molecule" so as to be detectable in any detection system, including, but not limited to, enzyme systems (e.g., ELISA, and histochemical assays using enzymes), fluorescent systems, radioactive systems, and luminescent systems. It is not intended that the present invention be limited to a particular detection system or label.

[0111] The term "target" as used herein refers to a nucleic acid that is being sorted out from other nucleic acids, for example, by probe binding, amplification, isolation, capture, etc. For example, when used in the context of polymerase chain reaction, "target" refers to the region of nucleic acid bounded by the primers used in the polymerase chain reaction, whereas when used in assays that do not amplify the target DNA, for example, in some embodiments of an invasion cleavage assay, the target includes the site where the probe and the invading oligonucleotide (e.g., INVADER oligonucleotide) bind to form an invasion cleavage structure, thereby allowing the presence of the target nucleic acid to be detected. A "segment" is defined as a region of nucleic acid within a target sequence. When used in the context of double-stranded nucleic acids, the term "target" is not limited to the particular strand (e.g., coding strand) of the target that is duplexed, but can be used in the context of either one or both strands of, for example, a double-stranded gene or reference DNA.

[0112] Thus, as used herein, "non-target," when used to describe a nucleic acid such as, for example, DNA, refers to a nucleic acid that may be present in a reaction but is not the subject of detection or characterization by the reaction. In some embodiments, a non-target nucleic acid may refer to a nucleic acid present in a sample, for example, that does not contain a target sequence, while in some embodiments, a non-target may refer to an exogenous nucleic acid, i.e., a nucleic acid that does not originate from a sample that contains or is suspected of containing a target nucleic acid, and that is added to a reaction, for example, to normalize the activity of an enzyme (e.g., a polymerase) to reduce variability in the performance of that enzyme in the reaction.

[0113] Nucleic acids can be isolated by any means, such as using a commercial kit. Briefly, if the nucleic acid of interest is encapsulated by a cell membrane, the biological sample can be disrupted and dissolved by enzymatic, chemical or mechanical means. Proteins and other contaminants can then be removed from the nucleic acid solution, for example by digestion with proteinase K. The nucleic acid is then recovered from the solution. This can be done by a variety of methods, such as salting out, organic extraction, or binding of the nucleic acid to a solid support. The choice of method is influenced by several factors, such as time, cost, and the amount of nucleic acid required.

[0114] The term "isolated" when used in reference to a nucleic acid, as in "isolated oligonucleotide", refers to a nucleic acid sequence that is identified and separated from at least one contaminating nucleic acid with which it is normally associated in its natural source. An isolated nucleic acid exists in a form or context that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids, such as DNA and RNA, are found in the state in which they exist in nature. Examples of non-isolated nucleic acids include a given DNA sequence (e.g., a gene) that is found adjacent to adjacent genes on a host cell chromosome; an RNA sequence, such as a particular mRNA sequence that encodes a particular protein, that is found in a cell as a mixture with many other mRNAs that encode many proteins. However, an isolated nucleic acid that encodes a particular protein includes, by way of example, a nucleic acid in a cell that normally expresses that protein, where the nucleic acid is in a different location from that on the chromosome of the natural cell or is otherwise flanked by different nucleic acid sequences than that in which it is found in nature. An isolated nucleic acid or oligonucleotide may exist in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is to be used to express a protein, such an oligonucleotide contains at least a sense or coding strand (i.e., the oligonucleotide can be single-stranded), but may also contain both a sense strand and an antisense strand (i.e., the oligonucleotide or polynucleotide can be double-stranded).An isolated nucleic acid can be combined with other nucleic acids or molecules after being isolated from its natural or typical environment.For example, an isolated nucleic acid can be present in a host cell in which it is placed, for example, for heterologous expression.

[0115] The terms "purified" and "extracted" are used interchangeably herein to refer to molecules, either nucleic acids or polypeptides, that have been removed, isolated, or separated from their natural environment. Thus, an "isolated nucleic acid" can be a purified nucleic acid. "Substantially purified" molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated. As used herein, the terms "purified" or "purifying" also refer to the removal of contaminants from a sample.

[0116] As used herein, the terms "cell-free" and "circulating cell-free" when used with respect to nucleic acids from blood are used interchangeably and refer to nucleic acids, e.g., DNA and RNA species, that are found in blood but are not intracellular in the blood. As used herein with respect to nucleic acids extracted from blood, these terms refer to the nature and location of the nucleic acids prior to taking the sample from the subject and prior to extraction of the nucleic acids from the blood sample.

[0117] As used herein, the term "circulating tumor DNA" (or "ctDNA") is tumor-derived DNA circulating in the peripheral blood of a patient. ctDNA is of tumor origin, either directly from the tumor or from circulating tumor cells (CTCs), which are viable, intact tumor cells that are shed from the primary tumor and enter the bloodstream or lymphatic system.

[0118] As used herein, the term "marker" refers to a substance (e.g., a nucleic acid, or a region of a nucleic acid, or a protein) that can be used to distinguish between non-normal cells (e.g., cancer cells) and normal cells (non-cancerous cells), for example, based on the presence or absence, or status (e.g., methylation state) of the marker substance. As used herein, "normal" methylation of a marker refers to the degree of methylation typically found in normal cells, e.g., non-cancerous cells.

[0119] As used herein, the term "tumor" refers to any new abnormal growth of tissue, including, but not limited to, cancer. Thus, a tumor can be a pre-malignant tumor or a malignant tumor.

[0120] The term "tumor-specific marker" as used herein refers to any biological material or element that can be used to indicate the presence of a tumor. Examples of biological materials include, but are not limited to, nucleic acids, polypeptides, carbohydrates, fatty acids, cellular components (e.g., cell membranes and mitochondria), and whole cells. In some cases, the marker is a specific nucleic acid region (e.g., a gene, a region within a gene, a specific locus, etc.). A region of a nucleic acid that is a marker may be referred to, for example, as a "marker gene," a "marker region," a "marker sequence," a "marker locus," etc.

[0121] The term "sample" is used in its broadest sense. In one sense, it may refer to cells or tissues or bodily fluids of an animal. In another sense, the term refers to specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from plants or animals (including humans) and include, for example, liquids, solids, tissues, and gases. Human and animal samples include, but are not limited to, feces, tissues, sputum, mucus, blood or blood products (selected from plasma, serum, whole blood), organ wastes such as pancreatic juice, and urine. Environmental samples include environmental materials such as surface material, soil, water, and industrial samples. These examples should not be construed as limiting the types of samples applicable to the present invention. As used herein with respect to a sample, the term "sample" taken from a source or subject, e.g., from a patient, is not limited to a single physical specimen, but also encompasses samples taken in multiple aliquots, e.g., a blood "sample" may be collected into two, three, four or more different blood collection tubes or other blood collection devices (e.g., bags), or a combination of different blood collection devices.

[0122] As used herein, the terms "suspected of containing" or "suspected of containing" are used interchangeably and refer to a feature that may or may not be present in, e.g., a sample or subject, etc., which may or may not include or contain a particular feature, e.g., a marker nucleic acid, a target or combination of targets (e.g., nucleic acids), or any other material or feature.

[0123] As used herein, the term "patient" or "subject" refers to an organism that is the subject of various tests provided by the technology. The term "subject" includes animals, preferably mammals, including humans. In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the subject is a human. Furthermore, for diagnostic methods, the preferred subject is a vertebrate subject. A preferred vertebrate is a warm-blooded animal, and a preferred warm-blooded vertebrate is a mammal. A preferred mammal is most preferably a human. As used herein, the term "subject" includes both human and animal subjects. Thus, veterinary uses are provided herein. As such, the technology provides for diagnosis of mammals, such as humans, as well as mammals of endangered importance, such as the Amur tiger, economically important mammals, such as animals raised on farms for human consumption, and / or socially important animals to humans, such as animals kept as pets or kept in zoos. Examples of such animals include, but are not limited to, carnivores such as cats and dogs; swine such as pigs, hogs, and wild boars; ruminants and / or ungulates such as cows, bulls, sheep, giraffes, deer, goats, bison, and camels; pinnipeds; and horses. Thus, diagnostics and treatments of livestock are also provided, including, but not limited to, domestic pigs, ruminants, ungulates, horses (including race horses), and the like. The subject matter disclosed in the present invention further includes a system for diagnosing cancer in a subject. The system may be provided as a commercially available kit that can be used, for example, to screen for risk of cancer or to diagnose cancer in a subject from whom a biological sample was taken. Exemplary systems provided in accordance with the present technology include assessing the methylation status of the markers described herein.

[0124] The term "amplifying" or "amplification" in the context of nucleic acids refers to generating multiple copies of a polynucleotide, or a portion of such a polynucleotide, typically starting from a small amount of polynucleotide (e.g., a single polynucleotide molecule), where an amplification product or amplicon is generally detectable. Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes. The generation of multiple DNA copies from one or a few copies of a target or template DNA molecule during polymerase chain reaction (PCR) or ligase chain reaction (LCR; see, e.g., U.S. Pat. No. 5,494,810, which is incorporated by reference in its entirety) is a form of amplification. Additional types of amplification include allele-specific PCR (see, e.g., U.S. Pat. No. 5,639,611, which is incorporated by reference in its entirety), assembly PCR (see, e.g., U.S. Pat. No. 5,965,408, which is incorporated by reference in its entirety), helicase-dependent amplification (see, e.g., U.S. Pat. No. 7,662,594, which is incorporated by reference in its entirety), hot-start PCR (see, e.g., U.S. Pat. Nos. 5,773,258 and 5,338,671, each of which is incorporated by reference in its entirety), inter-sequence specific PCR, inverse PCR (see, e.g., Triglia, et al. (1988) Nucleic Acids Res., 16:8186, which is incorporated by reference in its entirety), ligation-mediated PCR (see, e.g., Guilfoyle, R. et al., Nucleic Acids Res., 16:8186, each of which is incorporated by reference in its entirety), and ligation-mediated PCR (see, e.g., Guilfoyle, R. et al., Nucleic Acids Res., 16:8186, which is incorporated by reference in its entirety). Research, 25:1854-1858 (1997); U.S. Pat. No. 5,508,169), methylation-specific PCR (see, e.g., Herman, et al., (1996) PNAS 93(13)9821-9826, which is incorporated herein by reference in its entirety), miniprimer PCR, multiplex ligation-dependent probe amplification (see, e.g., Schouten, et al., (1997) PNAS 93(13)9821-9826, which is incorporated herein by reference in its entirety), and the like., (2002) Nucleic Acids Research 30(12):e57), multiplex PCR (see, e.g., Chamberlain, et al., (1988) Nucleic Acids Research 16(23)11141-11156; Ballabio, et al., (1990) Human Genetics 84(6)571-573; Hayden, et al., (2008) BMC Genetics 9:80, each of which is incorporated by reference in its entirety), nested PCR, overlap extension PCR (see, e.g., Higuchi, et al., (1988) Nucleic Acids Research 16(15)7351-7367, each of which is incorporated by reference in its entirety), real-time PCR (see, e.g., Higuchi, et al., (1992) Biotechnology 10:413-417; Higuchi, et al., (1993) Biotechnology 11:1026-1030), reverse transcription PCR (see, e.g., Bustin, SA (2000) J. Molecular Endocrinology 25:169-193, each of which is incorporated by reference in its entirety), solid-phase PCR, thermal asymmetric interlaced PCR, and touchdown PCR (see, e.g., Don, et al., Nucleic Acids Research (1991) 19(14) 4008; Roux, K. (1994) Biotechniques 16(5) 812-814; Hecker, et al., (1996) Biotechniques 20(3) 478-485, each of which is incorporated by reference in its entirety). Amplification of polynucleotides can also be achieved using digital PCR (see, e.g., Kalinina, et al., Nucleic Acids Research. 25, 1999-2004, (1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA, each of which is incorporated herein by reference in its entirety.96, 9236-41, (1999), International Patent Publication No. WO05023091A2, U.S. Patent Application Publication No. 20070202525). In some embodiments, a portion of the target nucleic acid is copied in the amplification, and in some embodiments, non-target polynucleotides are amplified in response to the presence of the target nucleic acid, (e.g., a cleaved flap, a ligation product, a rolling circle replication product, etc.).

[0125] The term "polymerase chain reaction" ("PCR") refers to the method of K.B. Mullis, U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188, which describes a method for increasing the concentration of a segment of a target sequence in a mixture of genomic or other DNA or RNA without cloning or purification. This process for amplifying a target sequence consists of introducing a large excess of two oligonucleotide primers to a DNA mixture containing the desired target sequence, followed by a precise series of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to each of the strands of a double-stranded target sequence. To carry out the amplification, the mixture is denatured and then the primers are annealed to their complementary sequences within the target molecule. After annealing, the primers are extended with a polymerase so that a new pair of complementary strands is formed. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (e.g., denaturation, annealing, and extension constitute one "cycle" and the number of "cycles" can be many) to obtain a high concentration of the amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore this length is a controllable parameter. Due to the repetitive nature of the process, the method is called "polymerase chain reaction" ("PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified products" and are "PCR products" or "amplicons". Those skilled in the art will appreciate that the term "PCR" encompasses many variations of the originally described method, such as using real-time PCR, nested PCR, reverse transcription PCR (RT-PCR), single primer and arbitrarily primed PCR, etc.

[0126] A "polymerase" is an enzyme that generally links 5'-triphosphate nucleotides, oligomers, and their analogs to the 3'-OH. Polymerases include, but are not limited to, template-dependent DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, RNA-dependent DNA polymerases, and RNA-dependent RNA polymerases. Polymerases include T7 DNA polymerase, T3 DNA polymerase, T4 DNA polymerase, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, DNA polymerase 1, Klenow fragment, Thermophilus aquaticus DNA polymerase, Tth DNA polymerase, Vent DNA polymerase (New England Biolabs), Deep Vent DNA polymerase (New England Biolabs), Bst DNA Polymerase Large Fragment, Stoeffel Fragment, 9°N DNA Polymerase, Pfu DNA Polymerase, Tfl DNA Polymerase, RepliPHI Phi29 Polymerase, Tli DNA Polymerase, eukaryotic DNA polymerase beta, telomerase, Therminator polymerase (New England Biolabs), KOD HiFi DNA polymerase (Novagen), and KOD1. These include, but are not limited to, DNA polymerases, Q-beta replicase, terminal transferase, AMV reverse transcriptase, M-MLV reverse transcriptase, Phi6 reverse transcriptase, HIV-1 reverse transcriptase, novel polymerases discovered by bioprospecting, and polymerases cited in US2007 / 0048748, US Patent Nos. 6,329,178, 6,602,695, and 6,395,524 (incorporated by reference). These polymerases include wild-type, mutant isoforms, and genetically engineered variants.

[0127] "DNA polymerase" is a polymerase that produces DNA from deoxynucleotide monomers (dNTPs). As used herein, "eubacterial DNA polymerase" refers to Pol A-type DNA polymerases (repair polymerases) from eubacteria, including, but not limited to, DNA polymerase I from E. coli, Taq DNA polymerase from Thermus aquaticus, and DNA Pol I enzymes from other members of the Thermus genus, and from other eubacterial species, etc.

[0128] As used herein, "preamplification reaction mixture" refers to a PCR amplification mixture for amplifying a specific target sequence, in which the reaction mixture preferably does not contain reagents for directly detecting or measuring the amplification product, such as intercalating dyes or labeling probes, such as FRET probes or FRET cassettes. In a preferred embodiment, the preamplification reaction mixture does not contain a non-polymerase flap endonuclease (e.g., although a eubacterial DNA polymerase containing a 3'- or 5'-endonuclease or exonuclease domain may be present, the preamplification mixture does not contain a separate flap endonuclease, such as FEN-1 endonuclease).

[0129] As used herein, "preamplifying" a target nucleic acid refers to amplifying the target nucleic acid to obtain more copies of the target nucleic acid, e.g., the amplified target material is then used in a subsequent assay, e.g., a nucleic acid detection assay, such as PCR or PCR-flap assay, sequencing assay, etc. In a preferred embodiment, the target nucleic acid is preamplified in a preamplification reaction mixture, where the preamplification comprises thermal cycling the preamplification reaction mixture for less than 20 thermal cycles, preferably less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, or less than 5 thermal cycles. Preferably, the number of thermal cycles is selected such that the amplification is in the exponential stage of PCR at the final thermal cycle.

[0130] As used herein, the term "primer annealing" refers to conditions that allow an oligonucleotide primer to hybridize to a template nucleic acid strand, preferably sufficiently to be extended by a DNA polymerase. Conditions for primer annealing vary depending on the length and sequence of the primer and are generally determined or calculated based on the T m For example, the annealing step in an amplification method involving thermocycling may be performed by adjusting the temperature after the heat denaturation step to the T of the primer sequences for a time sufficient to allow such annealing. m This involves lowering the temperature to a temperature based on

[0131] As used herein, the term "amplifiable nucleic acid" is used in reference to a nucleic acid that can be amplified by any amplification method. "Amplifiable nucleic acid" is generally intended to include a "sample template."

[0132] As used herein, the term "sample template" refers to nucleic acid originating from a sample that is analyzed for the presence of a "target." In contrast, "background template" is used in reference to nucleic acid templates other than the sample that may or may not be present in the sample. The presence of background template is often inadvertent; it may be the result of carryover or may be due to the presence of nucleic acid contaminants that are being purified away from the sample. For example, nucleic acids from organisms other than the organism to be detected may be present as background in the test sample.

[0133] The term "next generation sequencing" or "NGS" refers to a highly parallel method of performing nucleic acid sequencing, including sequencing-by-synthesis or sequencing-by-ligation platforms (e.g., those employed by Illumina, Life Technologies, Pacific Biosciences, and Roche). Next generation sequencing methods can also include, but are not limited to, nanopore sequencing methods, such as those provided by Oxford Nanopore, or methods based on electronic detection, such as the Ion Torrent technology commercialized by Life Technologies. Suitable nucleic acid sequencing techniques for use with the present technology include sequencing-by-synthesis (see, e.g., Meyer and Kircher, "Illumina sequencing library preparation for highly multiplexed target capture and sequencing," Cold Spring Harbor Protocols 2010(6)), single molecule real-time sequencing (see, e.g., Levene et al., "Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations," Science. 299(5607):682-6(2003)), ion semiconductor sequencing (see, e.g., Rusk, "Torrents of sequence," Nat. Methods 8,44(2011)), pyrosequencing (see, e.g., Wicker et al., "454 sequencing put to the test using the complex genome of barley," BMC Genomics, 7:275,2006), and sequencing-by-ligation (see, e.g., Margulies et al., “Genome sequencing in microfabricated high-density picolitre reactors,” Nature, 437:376-80 (2005)), nanopore sequencing (see, e.g., Goodwin et al., “Oxford Nanopore sequencing, hybrid error correction, and de novo assembly of a eukaryotic genome,” Genome Res., 25(11):1750-6 (2015)), chain-termination sequencing (Sanger sequencing) (see, e.g., Sanger et al., “DNA sequencing with chain-terminating inhibitors,” Proceedings of the National Academy of Sciences of the United States of America, 74(12):5463-5467 (1977)), and mass-spectrometry sequencing (see, e.g., Edwards et al., “Mass-spectrometry DNA sequencing,” Mutation Research, 573(1-2):3-12 (2005)).

[0134] As used herein, the term "nucleic acid detection assay" refers to any method for determining the presence, absence, or amount of a nucleic acid of interest, or its nucleotide composition. Nucleic acid detection assays include DNA sequencing methods, such as next generation sequencing, nucleic acid amplification methods, probe hybridization methods, structure-specific cleavage assays (e.g., INVADER assay (Hologic, Inc.)), and are described in, for example, U.S. Patent Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543, and 6,872,816, each of which is incorporated herein by reference in its entirety for all purposes; Lyamichev et al., Nat. Biotech., 17:292 (1999); Hall et al., PNAS, USA, 97:8272 (2000), and U.S. Pat. No. 9,096,893); enzymatic mismatch cleavage methods (e.g., U.S. Pat. Nos. 6,110,684, 5,958,692, and 5,851,770 to Variagenics, which are incorporated by reference in their entireties); the polymerase chain reaction (PCR), as described above; branched hybridization methods (e.g., U.S. Pat. Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802 to Chiron, which are incorporated by reference in their entireties); rolling circle replication (e.g., U.S. Pat. Nos. 6,210, 884, 6,183,960 and 6,235,502; NASBA (e.g., U.S. Pat. No. 5,409,818, which is incorporated herein by reference in its entirety); molecular beacon technology (e.g., U.S. Pat. No. 6,150,097, which is incorporated herein by reference in its entirety); E-sensor technology (Motorola U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,170 and 6,063,573, which are incorporated herein by reference in their entirety); cycling probe methods (e.g., U.S. Pat. Nos. 5,403,711, 5,011,769 and 5,660,988, which are incorporated herein by reference in their entirety); These include, but are not limited to, the Behring signal amplification method (e.g., U.S. Pat. Nos. 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614, which are incorporated by reference in their entireties); the ligase chain reaction (e.g., Baranay Proc. Natl. Acad. Sci USA 88,189-93 (1991)); and the sandwich hybridization method (e.g., U.S. Pat. No. 5,288,609, which is incorporated by reference in its entirety).

[0135] In some embodiments, target nucleic acid is amplified (e.g., by PCR) and the amplified nucleic acid is simultaneously detected using an invasion cleavage assay. Assays configured to perform detection assays (e.g., invasion cleavage assays) in combination with amplification assays are described in U.S. Patent No. 9,096,893, which is incorporated herein by reference in its entirety for all purposes. Further configurations of amplification plus invasion cleavage detection, called QuARTS method, are described in, for example, U.S. Patent Nos. 8,361,720, 8,715,937, 8,916,344, 9,212,392, and U.S. Patent Application No. 15 / 841,006, each of which is incorporated herein by reference for all purposes. The term "invading cleavage structure" as used herein refers to a cleavage structure that includes i) a target nucleic acid, ii) an upstream nucleic acid (e.g., an invading oligonucleotide or "INVADER" oligonucleotide), and iii) a downstream nucleic acid (e.g., a probe), where the upstream and downstream nucleic acids anneal to a continuous region of the target nucleic acid, and an overlap is formed between the duplex formed between the downstream nucleic acid and the target nucleic acid and the 3' portion of the upstream nucleic acid. The overlap occurs where one or more bases from the upstream and downstream nucleic acids occupy the same position relative to a base in the target nucleic acid, whether or not the overlapping base(s) of the upstream nucleic acid are complementary to the target nucleic acid, and whether or not the bases are natural or unnatural bases. In some embodiments, the 3' portion of the upstream nucleic acid that overlaps with the downstream duplex is a non-basic chemical moiety, such as, for example, an aromatic ring structure as disclosed in U.S. Pat. No. 6,090,543, which is incorporated herein by reference in its entirety. In some embodiments, one or more of the nucleic acids may be linked to one another via a covalent bond, such as, for example, a nucleic acid stem loop, or via a non-nucleic acid chemical bond (e.g., a multi-carbon chain).As used herein, the term "flap endonuclease assay" includes the "INVADER" invasive cleavage assay and the QuARTS assay, as described above.

[0136] As used herein, the term "amplification reagents" refers to the reagents needed for amplification (deoxyribonucleoside triphosphates, buffers, etc.) excluding primers, nucleic acid template, and amplification enzymes. Typically, amplification reagents are placed and contained within a reaction vessel along with other reaction components.

[0137] A "reaction mixture" is a mixture of reagents (e.g., oligonucleotides, target nucleic acids, enzymes, etc.) in a combination and / or location that allows a reaction to occur, e.g., in a single reaction vessel, at a location in a fluidic device, at a location on a surface, etc.

[0138] As used herein, a "multiplex" reaction refers to the combination of multiple targets (two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, six, seven, eight, nine, ten, ten, twelve, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, twenty-nine, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six ... , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, etc.) (e.g., PCR, PCR-flap assay). Multiplex reactions are distinguished from reactions which operate on a single target analyte per reaction mixture. As used herein, the term "highly multiplexed" refers to a reaction that acts on at least 6, preferably at least 10, and more preferably at least 20 or more different targets (e.g., different genes, or regions of genes) in a single reaction mixture.

[0139] The terms "probe oligonucleotide," "flap probe oligonucleotide," and "flap oligonucleotide," when used in reference to a flap assay, are used interchangeably and refer to an oligonucleotide that interacts with a target nucleic acid in the presence of an invading oligonucleotide to form a cleavage structure.

[0140] As used herein, the term "label" refers to any moiety (e.g., chemical species) that can be detected or that can produce a detectable response. In some preferred embodiments, detection of the label provides quantifiable information. The label can be any known detectable moiety, such as a sequence of nucleotides (e.g., a flap sequence), a radioactive label (e.g., a radionuclide), a ligand (e.g., biotin or avidin), a chromophore (e.g., a dye or particle that imparts a detectable color), a hapten (e.g., digoxigenin), a mass label, a latex bead, a metal particle, a paramagnetic label, a luminescent compound (e.g., a bioluminescent, phosphorescent, or chemiluminescent label) or a fluorescent compound (e.g., a compound that upon excitation with radiation or light at one wavelength, emits energy, e.g., radiation or light, at a different wavelength), and the like. The label can be directly or indirectly attached to the oligonucleotide or other biomolecule. Direct labeling can be achieved through bonds or interactions that link the label to the oligonucleotide, e.g., covalent or non-covalent interactions such as hydrogen bonds, hydrophobic interactions, and ionic interactions, or through the formation of chelate or coordination complexes. Indirect labeling can be achieved through the use of a bridging moiety or "linker," such as an antibody or additional oligonucleotide(s), which is / are directly or indirectly labeled.

[0141] The term "invader oligonucleotide" refers to an oligonucleotide that hybridizes to a target nucleic acid at a position adjacent to the hybridization region between the probe and the target nucleic acid, and the 3' end of the invader oligonucleotide includes a portion (e.g., a chemical moiety, or one or more nucleotides) that overlaps with the hybridization region between the probe and the target. The 3' terminal nucleotide of the invader oligonucleotide may or may not base pair with a nucleotide in the target. In some embodiments, the invader oligonucleotide contains at its 3' end substantially the same sequence as the sequence located at the 5' end of the portion of the probe oligonucleotide that anneals to the target strand. In some embodiments, for example, in a PCR-flap assay, the primer used for amplification can also function as the invader oligonucleotide with the probe.

[0142] The term "target cleavage site," as used herein with respect to cleavage of an invading cleavage structure, refers to a preferred site (or sites) of cleavage on a nucleic acid structure (e.g., an invading cleavage structure) by a structure-specific nuclease (e.g., FEN-1 endonuclease) that recognizes the structure as a cleavage substrate. For example, as discussed by Kaiser, et al., 5' flap endonucleases such as FEN-1 endonuclease typically cleave an invading cleavage structure after the first nucleotide that is generally base-paired in the downstream nucleic acid, i.e., one nucleotide inboard from the downstream duplex.

[0143] The term "flap endonucleases" or "FENs" as used herein refers to a class of nucleolytic enzymes, typically 5' nucleases, that act as structure-specific endonucleases on a double-stranded DNA structure in which one strand is displaced by another strand of nucleic acid (e.g., to provide overlapping nucleotides at the junction between the single-stranded DNA and the double-stranded DNA) and contains a single-stranded 5' overhang or flap. FENs catalyze the hydrolytic cleavage of the phosphodiester bond at the junction between the single-stranded DNA and the double-stranded DNA to release the overhang, or flap. Flap endonucleases are reviewed by Ceska and Savers (Trends Biochem. Sci. 1998 23:331-336) and Liu, et al. (Annu. Rev. Biochem. 2004 73:589-615, the entire contents of which are incorporated herein by reference). FENs can be individual enzymes, multi-subunit enzymes, or exist as the activity of separate enzymes or protein complexes (eg, DNA polymerases).

[0144] Flap endonucleases can be thermostable. For example, FEN-1 flap endonucleases from archived thermophilic organisms are typically thermostable. As used herein, the term "FEN-1" refers to non-polymerase flap endonucleases from eukaryotic or archaeal organisms. See, for example, WO02 / 070755, and US Patent No. 7,122,364, both of which are incorporated herein by reference in their entirety for all purposes, and Kaiser MW, et al. (1999) J.Biol.Chem., 274:21387.

[0145] As used herein, the term "cleaved flap" refers to a single-stranded oligonucleotide that is the cleavage product of a flap assay.

[0146] The term "cassette," when used in reference to a flap cleavage reaction, refers to an oligonucleotide or combination of oligonucleotides configured to generate a detectable signal in response to cleavage of a flap oligonucleotide or a probe oligonucleotide, e.g., in a primary or first cleavage structure formed in a flap cleavage assay. In a preferred embodiment, the cassette hybridizes to a non-target cleavage product generated by cleavage of the flap oligonucleotide to form a second overlapping cleavage structure, which allows the cassette to be subsequently cleaved by the same enzyme, e.g., FEN-1 endonuclease.

[0147] In some embodiments, the cassette is a single oligonucleotide that contains a hairpin portion (i.e., a region where a portion of the cassette oligonucleotide hybridizes with a second portion of the same oligonucleotide under reaction conditions to form a duplex). In other embodiments, the cassette contains at least two oligonucleotides that contain complementary portions that can form a duplex under reaction conditions. In preferred embodiments, the cassette contains a label, e.g., a fluorophore. In particularly preferred embodiments, the cassette contains a label portion that produces a FRET effect.

[0148] As used herein, the term "FRET" refers to fluorescence resonance energy transfer, a process in which moieties (e.g., fluorophores) transfer energy, for example, between themselves or from a fluorophore to a non-fluorophore (e.g., a quencher molecule). In some situations, FRET involves the transfer of energy from an excited donor fluorophore to a low-energy acceptor fluorophore via short-range (e.g., about 10 nm or less) dipole-dipole interactions. In other situations, FRET involves the loss of fluorescence energy from the donor and an increase in fluorescence in the acceptor fluorophore. In yet other forms of FRET, energy can be exchanged from an excited donor fluorophore to a non-fluorescent molecule (e.g., a "dark" quenching molecule, e.g., "BHQ" quencher, Biosearch Technologies). FRET is known to those of skill in the art and has been described previously (see, e.g., Stryer et al., 1978, Ann. Rev. Biochem., 47:819; Selvin, 1995, Methods Enzymol., 246:300; Orpana, 2004 Biomol Eng 21, 45-50; Olivier, 2005 Mutant Res 573, 103-110, each of which is incorporated herein by reference in its entirety).

[0149] As used herein, the term "FRET system" refers to a pair or group of moieties that act together as donor-acceptor or donor-quencher partners for FRET-based analysis of molecules, such as probe oligonucleotides, flap oligonucleotides, or other assay reporter molecules. Meanwhile, embodiments of the present technology are exemplified with a fluorophore at one particular location and a quencher or other FRET acceptor moiety at a particular second location. For example, in some PCR probe oligonucleotides (e.g., for TAQMAN assays), the fluorophore is at or near one end of the probe oligonucleotide, and the quencher moiety is at or near the other end of the probe oligonucleotide.

[0150] Suitable fluorophores include fluorescein, rhodamine, REDMOND RED dyes, YAKIMA YELLOW dyes, hexachlorofluorescein, TAMRA dyes, ROX dyes, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7, Quasar 570 (Q570), Quasar 670 (Q670), Quasar 705 (Q705) (Quasar dyes are from LGC, Biosearch Technologies), 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5,p-methoxyphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5-styryl-4-bora-3a,4-diaza-aS-indacene-propionic acid, 6-carboxy-X-rhodamine, N,N,N',N'-tetramethyl-6-carboxyrhodamine, Texas Instruments, Inc. Red, eosin, fluorescein, 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5,p-ethoxyphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid and 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-propionic acid, 6-carboxyfluorescein (6-FAM), 2',4',1,4,-tetrachlorofluorescein (TET), 2 l,4',5l,7',l,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), fluorescein isothiocyanate (FITC), 5,6-carboxymethylfluorescein, Texas red, nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride, aminomethylcoumarin (AMCA), erythrosine, BODIPY dye, CASCADE BLUE dye, OREGON GREEN dye, pyrene, lissamine, xanthene, acridine, oxazine, phycoerythrin, QUANTUM DYE, thiazole orange-ethidium dimer, and the like, but are not limited to these.

[0151] Suitable quenchers include, but are not limited to, cyanine dyes such as Cy3, Cy3.5, Cy5, Cy5.5, and Cy7, rhodamine dyes such as tetramethyl-6-carboxyrhodamine (TAMRA) and tetrapropano-6-carboxyrhodamine (ROX), DABSYL dyes, DABCYL dyes, cyanine dyes, nitrothiazole blue (NTB), anthraquinone, malachite green, nitrothiazole, or nitroimidazole compounds, QSY7 (Molecular Probes, Eugene, OR), ECLIPSE quenchers (Nanogen, San Diego, CA), etc. Analysis of factors such as absorbance and emission spectra of various molecules in selecting pairs or groups of moieties for use in FRET configurations is well known to those of skill in the art.

[0152] In an exemplary flap detection assay, an invading oligonucleotide and a flap oligonucleotide are hybridized with a target nucleic acid to generate a first complex having an overlap as described above. An unpaired "flap" is included on the 5' end of the flap oligonucleotide. The first complex is a substrate for a flap endonuclease, such as FEN-1 endonuclease, which cleaves the flap oligonucleotide to release the 5' flap portion. In a secondary reaction, the released 5' flap product functions as an invading oligonucleotide on the FRET cassette, again creating a structure that is recognized by the flap endonuclease, thereby cleaving the FRET cassette. When the fluorophore and the quencher are separated by cleavage of the FRET cassette, a detectable fluorescent signal is generated that exceeds background fluorescence.

[0153] As used herein, the term "PCR-flap assay" refers to an assay configuration that combines PCR target amplification and detection of amplified DNA, which is performed by the formation of an overlap cleavage structure that includes the amplified target DNA, and in a preferred embodiment, the formation of a second overlap cleavage structure that includes the cleaved 5' flap from the first overlap cleavage structure and a labeled reporter oligonucleotide, such as a "FRET cassette" or a 5' hairpin FRET reporter oligonucleotide. In a preferred embodiment of the PCR-flap assay used herein, the assay reagents include a mixture containing a DNA polymerase, a FEN-1 endonuclease, and a primary probe that includes a portion complementary to the target nucleic acid. Optionally, the PCR-flap assay reagents further include a FRET cassette or a 5' hairpin FRET reporter. Typically, the target nucleic acid is amplified by PCR, and the amplified nucleic acid is detected simultaneously (i.e., detection occurs during the course of target amplification). PCR-flap assays include the QuARTS assay described in U.S. Pat. Nos. 8,361,720, 8,715,937, and 8,916,344; the flap assay described in U.S. Pat. No. 10,648,025 that uses a probe oligonucleotide with a long target-specific region (Long probe Quantitative Amplified Signal, "LQAS"); and the amplification assay of U.S. Pat. No. 9,096,893 (e.g., as illustrated in FIG. 1 of that patent), each of which is incorporated herein by reference in its entirety.

[0154] As used herein, the term "PCR-flap assay reagent" refers to one or more reagents for detecting a target sequence in a PCR-flap assay, comprising a nucleic acid molecule capable of participating in the amplification of a target nucleic acid and the formation of a flap cleavage structure in the presence of the target sequence in a mixture containing a DNA polymerase, a primer, a FEN-1 endonuclease, and a probe or flap oligonucleotide, optionally a reverse transcriptase. The PCR-flap assay reagent may further comprise a FRET cassette or a 5' hairpin FRET reporter.

[0155] As used herein, a "5' hairpin FRET reporter" or "5' hairpin FRET cassette" refers to a type of FRET probe or FRET cassette that contains a 5' hairpin-forming region upstream of a target cleavage site and contains a FRET labeling moiety that is separated upon cleavage of the reporter at the target cleavage site (see, e.g., WO2021 / 055508, which is incorporated by reference in its entirety).

[0156] As used herein, the term "amplification bias" refers to differences in amplification efficiency between and among different target nucleic acid sequences, i.e., differences in the number of copies produced from a given target nucleic acid, when treated under the same amplification reaction conditions.

[0157] The term "low bias amplification buffer" as used herein refers to an amplification buffer configured to exhibit low target-to-target variation in amplification efficiency for different targets amplified together in a multiplex amplification reaction, e.g., in a multiplex pre-amplification reaction prior to a subsequent amplification reaction, e.g., a PCR-flap assay reaction. Amplification bias between different targets may be assessed by measuring the efficiency of amplification of different targets of known concentrations. In some embodiments of the present technology, the low bias amplification buffer is a buffer useful for PCR-flap assays and containing a high concentration of magnesium (e.g., at a final concentration of at least 6 mM in the reaction mixture, preferably 6-10 mM, preferably 7-9 mM, preferably about 7.5 mM) as opposed to PCR buffers that typically contain magnesium at a final concentration of about 1-4 mM in the reaction mixture. In a preferred embodiment, the low bias amplification buffer comprises a 3-(n-morpholino)propanesulfonic acid (MOPS) buffer, and in certain preferred embodiments, the low bias amplification buffer comprises 7.5 mM MgCl 2 , 10 mM MOPS, 0.3 mM Tris-HCl (pH 8.0), 0.8 mM KCl, 0.1 μg / μL BSA, 0.0001% Tween-20, and 0.0001% IGEPAL CA-630. The terms "PCR-Flap Assay Buffer" and "Low Bias Amplification Buffer" are used interchangeably herein.

[0158] Mg used in low bias amplification buffers ++The range of amounts includes any concentration encompassed by the ranges above. For example, low bias amplification buffers can be 6 mM, 6.1 mM, 6.2 mM, 6.5 mM, 6.4 mM, 6.5 mM, 6.6 mM, 6.7 mM, 6.8 mM, 6.9 mM, 7.0 mM, 7.1 mM, 7.2 mM, 7.3 mM, 7.4 mM, 7.5 mM, 7.6 mM, 7.7 mM, 7.8 mM, 7.9 mM, 8.0 mM, 8.1 mM, 8.2 mM, 8.3 mM, 8.4 mM, 8.5 mM, 8.6 mM, 8.7 mM, 8.8 mM, 8.9 mM, 9.0 mM, 9.1 mM, 9.2 mM, 9.3 mM, 9.4 mM, 9.5 mM, 9.6 mM, 9.7 mM, 9.8 mM, 9 ... 5mM, 8.6mM, 8.7mM, 8.8mM, 8.9mM, 9.0mM, 9.1mM, 9.2mM, 9.3mM, 9.4mM, 9.5mM, 9.6mM, 9.7mM, 9.8mM, 9 .9mM, 10.0mM, 10.1mM, 10.2mM, 10.3mM, 10.4mM, 10.5mM, 10.6mM, 10.7mM, 10.8mM, 10.9mM, 11.0mM Mg ++ , or more, or any fractional value therebetween. These concentrations are listed by way of example and are not limiting.

[0159] As used herein, a "low bias amplification buffer" refers to a buffer containing greater than about 4 mM Mg ++ Although it contains high Mg ++ Reduce the helix stabilizing effect of the concentration of, for example, DMSO, (NH 4 ) 2 SO 4The low bias amplification buffer is distinct from the PCR buffer, which further comprises a reagent, sometimes referred to as an enhancer reagent, such as betaine, etc. For a discussion of such enhancer reagents, see, e.g., Ralser, et al., An efficient and economic enhancer mix for PCR, Biochemical and Biophysical Research Communications 347(2006)747-751, O. Henegariu, et al., "Multiplex PCR: Critical Parameters and Step-by-Step Protocol," BioTechniques 23:504-511 (September 1997), and Qiagen PCR Brochure "Maximizing PCR and RT-PCR Success," Third Edition (Document 1104683,10 / 2016), each of which is incorporated herein by reference in its entirety. In a preferred embodiment, the low bias amplification buffer comprises (NH 4 ) 2 SO 4 In some embodiments, the low bias amplification buffer is essentially free of ammonium ions. In some embodiments, the low bias amplification buffer is essentially free of DMSO and / or betaine.

[0160] As used herein, the term "essentially free" as used with respect to components excluded from a composition, e.g., reaction mixtures, buffers, etc., refers to a composition that is not formulated to include the excluded component, but may contain trace amounts of the component, e.g., by carryover from a previous reaction step or from a concentrated stock of a reaction component (e.g., an enzyme in a storage solution) that is added in small amounts. For example, small amounts of glycerol, or other materials may be introduced into a reaction mixture (e.g., a PCR flap assay reaction mixture) by the addition of an aliquot of an enzyme (e.g., a polymerase) that contains materials for stable storage, but such materials are typically sufficiently diluted in the reaction mixture that the trace amounts of glycerol and other materials do not alter the expected function of the reaction mixture, e.g., trace amounts of (NH 4 ) 2 SO 4 In the case of , betaine, or DMSO, it is believed that they would not alter the low-bias multiplex amplification results observed when using the low-bias amplification buffer described herein. Such trace amounts of material are not typically reported in the literature as part of the formulation of the final reaction mixture. Thus, a composition that is "essentially free" of a described component may contain the excluded component at a level that, if present, does not alter the function of the described composition compared to a pure composition, i.e., a composition that does not contain any of the excluded component.

[0161] As used herein, the terms "essentially the same amount" and "essentially the same concentration", when used with respect to mixture components present in a preamplification reaction mixture, including but not limited to primers, probes or other oligonucleotides, refer to amounts of reagents that are formulated to be at the same concentration in the mixture, but may contain slight variations in the relative amounts or relative copy numbers of the different components, for example, due to carryover of some amount of one or more of the components from previous reaction steps, or due to variations in the normal course of laboratory preparation or measurement. Thus, a composition described as containing "essentially equal amounts" of different components or containing components at "essentially the same concentrations" may contain the components in amounts that are slightly different, but such differences in absolute amounts or concentrations do not alter the function of the described composition, as compared to a composition in which the amounts or concentrations of the different components are exactly equal. The terms "essentially the same amount" and "essentially the same concentration" are used interchangeably with respect to both liquid and dry preparations. The term, when used in reference to a primer, probe or other oligonucleotide, generally refers to the molar amount of the molecule or the molar amount of a particular portion of the molecule (eg, the molar amount of the extendible 3' end of a primer).

[0162] The term "real-time" as used herein with respect to detection of nucleic acid amplification or signal amplification refers to detection or measurement of product or signal accumulation in a reaction while the reaction is proceeding, e.g., during incubation or thermal cycling. Such detection or measurement may be continuous, or may be performed at multiple discrete points during the progress of the amplification reaction, or may be combined. For example, in a polymerase chain reaction, detection (e.g., of fluorescence) may be performed continuously during all or part of the thermal cycling, or may be performed transiently at one or more points during one or more cycles. In some embodiments, PCR or PCR-flap assay reaction real-time detection is achieved by determining the fluorescence level at the same point (e.g., at a time point during a cycle, or at a temperature step during a cycle) during each of multiple cycles, or every cycle. Real-time detection of amplification may also be referred to as "during" the amplification reaction detection.

[0163] As used herein, the terms "reverse transcription" and "reverse transcribing" refer to the use of a template-dependent polymerase to generate a DNA strand complementary to an RNA template. Polymerases capable of generating a DNA strand complementary to an RNA template are commonly referred to as "reverse transcriptases," or polymerases with "reverse transcriptase activity."

[0164] As used herein, the term "nucleic acid abundance" refers to the amount of a particular target nucleic acid sequence present in a sample or aliquot. This amount is generally referred to in terms of mass (e.g., μg), mass per unit volume (e.g., μg / μL); copy number (e.g., 1000 copies, 1 attomole), or copy number per unit volume (e.g., 1000 copies per mL, 1 attomole per μL). Nucleic acid abundance can also be expressed as an amount relative to a standard amount of known concentration or copy number. Measurement of nucleic acid abundance can be based on any criterion that is understood by those skilled in the art as a suitable quantitative expression of nucleic acid abundance, such as physical density or sample, optical density, refractive properties, staining properties, or the intensity of detectable label, such as fluorescent label.

[0165] The term "amplicon" or "amplification product" refers to a segment of nucleic acid (generally DNA) generated by an amplification process such as a PCR process or another replicative process, e.g., the rolling circle amplification process or the LAMP amplification process. The term is also used in reference to RNA segments generated by amplification methods that utilize RNA polymerase, e.g., NASBA, TMA, etc.

[0166] As used herein, "rolling circle amplification" refers to in vitro rolling circle replication of a circular nucleic acid, e.g., as described in U.S. Pat. Nos. 6,210,884, 6,183,960, 6,235,502, 5,942,391, 6,316,229, 7,862,999, 11,186,863; U.S. Patent Publication No. US2015 / 0284786; and M. Ali, et al. "Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine". Chemical Society Reviews. 43-(10):3324-3341, using a strand-displacing DNA polymerase to form a DNA molecule containing tandem repeats of a sequence complementary to the circular nucleic acid.

[0167] The term "amplification curve" as used in reference to a thermal cycling amplification reaction refers to a plot of a signal indicative of amplification, such as a fluorescent signal versus cycle number. When used in reference to non-thermal cycling amplification methods, an amplification curve generally refers to a plot of signal accumulation as a function of time.

[0168] The term "baseline" as used with respect to an amplification curve refers to the detection signal obtained from an amplification reaction established prior to incubation, in the early cycles in the case of PCR, where there is little change in signal.

[0169] The terms "no template control" and "no target control" (or "NTC") as used herein with respect to control reactions refer to reactions or samples that do not contain template or target nucleic acid, which are used to check amplification quality.

[0170] As used herein, the term "quantitative amplification data set" refers to data obtained during quantitative amplification of target sample, for example, target DNA.In the case of quantitative PCR or QuARTS assay, quantitative amplification data set is the collection of fluorescence values ​​obtained during amplification, for example, during multiple thermal cycles or during all thermal cycles.Data for quantitative amplification is not limited to data collected at any particular point during reaction, and fluorescence can be measured at individual points during each cycle or continuously throughout each cycle.

[0171] As used herein with respect to real-time detection during a thermal cycled amplification reaction, "C t The term "threshold cycle" or "threshold cycle" refers to the fractional cycle number at which a detected signal (eg, fluorescence) passes a fixed threshold.

[0172] The abbreviations "Ct" and "Cp" or "threshold cycle" used herein with respect to data collected during amplification reactions, such as real-time PCR and PCR-flap assays, refer to the cycle at which a signal (e.g., a fluorescent signal) crosses a predefined threshold value indicating a positive signal. Various methods have been used to calculate the threshold value used as a determinant of signal versus concentration, and the value is generally expressed as either a "crossing threshold" (Ct) or a "crossing point" (Cp). Either the Cp value or the Ct value can be used in the embodiment of the method presented herein for the analysis of real-time signals for the determination of the percentage of variant and / or non-variant components in an assay or sample.

[0173] As used herein, the term "fish DNA" refers to large amounts of (e.g., genomic) DNA isolated from fish, for example, as described in U.S. Patent No. 9,212,392. Large amounts of purified fish DNA are commercially available, for example, in the form of cod and / or herring sperm DNA (Roche Applied Science, Mannheim, Germany) or salmon DNA (USB / Affymetrix).

[0174] As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of a reaction assay, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and / or supporting materials (e.g., buffers, written instructions for performing the assay, etc.) from one location to another. For example, a kit includes one or more enclosures (e.g., boxes) that contain the relevant reaction reagents and / or supporting materials. As used herein, the term "fragmented kit" refers to a delivery system that includes two or more separate containers, each of which contains a fragment of all the components of the kit. The containers can be delivered together or separately to the intended recipient. For example, a first container can contain an enzyme for use in the assay, and a second container contains an oligonucleotide.

[0175] The term "system" as used herein refers to a collection of articles for use for a particular purpose. In some embodiments, the articles include instructions for use, such as information provided on the article, on paper, or on a recordable medium (e.g., DVD, CD, flash drive, etc.). In some embodiments, the instructions direct the user to an online location, e.g., a website.

[0176] As used herein, the term "information" refers to any collection of facts or data. With respect to information stored or processed using a computer system(s), including but not limited to the Internet, the term refers to any data stored in any format (e.g., analog, digital, optical, etc.). As used herein, the term "information related to a subject" refers to facts or data about a subject (e.g., a human, a plant, or an animal). The term "genomic information" refers to information related to a genome, including but not limited to nucleic acid sequences, genes, methylation percentages, allele frequencies, RNA expression levels, protein expression, phenotypes correlated with genotypes, etc. "Allele frequency information" refers to facts or data related to allele frequency, including but not limited to allele identity, statistical correlation between the presence of an allele and characteristics of a subject (e.g., a human subject), the presence or absence of an allele in an individual or population, the percentage of likelihood that an allele is present in an individual with one or more particular characteristics, etc. [Brief description of the drawings]

[0177] [Figure 1] A schematic diagram of an assay is described in which different target nucleic acids (e.g., different genes and gene variants, cDNA, single nucleotide polymorphisms) are pre-amplified together in a multiplex reaction, and then the different targets are detected using the same reporter dye by performing a PCR-flap assay in separate reaction mixtures. [Diagram 2] A schematic diagram of an assay is described in which different target nucleic acids are pre-amplified together in a multiplex reaction, and then two different targets and a reference target are detected in a triplex PCR-flap assay reaction mixture using two different reporter dyes normalized to a third reference dye. [Diagram 3] A table is provided showing the configuration of the 3-dye triplex PCR-FLAP assay shown in FIG. 2, which can be used to detect 12 different targets, e.g., marker DNA pre-amplified from a DNA sample. [Figure 4]A schematic diagram of an embodiment of the technology is described in which different target nucleic acids are pre-amplified together in a multiplex reaction under conditions that minimize bias, and then the aggregate signal from multiple different targets is measured using a single dye, for example, using the same FRET cassette. In some embodiments, an additional set of markers reports to a FRET cassette with a second dye, and further sets of markers report to a third, fourth, fifth, etc. dye. [Figure 5A] A table is provided showing the configuration of two multiplex PCR-flap assays shown in FIG. 4 that can be used to detect 12 different targets, e.g., marker DNA pre-amplified from a DNA sample along with a reference gene, using one dye for all 6 markers and one dye for the reference gene. [Figure 5B] A table is provided showing the configuration of a single multiplex PCR-flap assay, shown in FIG. 4, which can be used to detect 12 different targets, e.g., marker DNA pre-amplified from a DNA sample along with a reference gene, using two dyes to detect each of two different sets of 6 markers, and a third dye for the reference gene. [Figure 5C] A table is provided showing the configuration of a single multiplex PCR-flap assay, shown in FIG. 4, which can be used to detect 24 different targets, e.g., marker DNA pre-amplified from a DNA sample along with a reference gene, using two dyes to detect each of two different sets of 12 markers, and a third dye for the reference gene. [Figure 6]The results of varying MgCl2 concentrations in multiplex preamplification in PCR-flap assay buffer are shown for strand counts measured for each of the marker DNAs shown in the subsequent LQAS PCR-flap assay reactions. These data show the variability between markers in the subsequent LQAS PCR-flap assay reactions when 100 strands or 1000 strands of marker DNA are given in the multiplex preamplification reaction. The calculated theoretical yield of amplified strands (81920 for 1000 strands of input target DNA material, or 8192 for 100 strands of input target DNA) is shown as a dashed horizontal line in each graph. [Figure 7] The results show that multiplex preamplification in low bias high Mg++ PCR-flap assay buffer (7.5 mM MgCl2) was performed with respect to the strand count measured for each of the marker DNAs shown in the subsequent LQAS PCR-flap assay reaction, and compared to preamplification of the same combination of target DNA in (NH4)2SO4 PCR buffer with 6.7 mM MgCl2 combined with an additional helix destabilizing component (e.g., dimethyl sulfoxide), varying the MgCl2 concentration in the multiplex preamplification in the PCR-flap assay buffer. These data show the variability between markers in the subsequent LQAS PCR-flap assay reaction when 100 strands or 1000 strands of marker DNA are fed in the multiplex preamplification reaction. The calculated theoretical yield of amplified strands (81920 for 1000 strands of input target DNA material, or 8192 for 100 strands of input target DNA) is shown by the dashed horizontal line in each graph. [Figure 8] A table of three-dye triplex PCR-flap assay combinations for detecting the indicated combinations of marker DNA is provided. The "Triplex Name" consists of the initial letters of each marker detected in the triplex reaction. [Figure 9A]1 shows exemplary oligonucleotide combinations (primer oligonucleotide, probe oligonucleotide and FRET cassette oligonucleotide) used in triplex PCR-flap assay in combination with dNTPs at the concentrations shown. [Figure 9B] 1 shows exemplary oligonucleotide combinations (primer oligonucleotide, probe oligonucleotide and FRET cassette oligonucleotide) used in triplex PCR-flap assay in combination with dNTPs at the concentrations shown. [Figure 9C] 1 shows exemplary oligonucleotide combinations (primer oligonucleotide, probe oligonucleotide and FRET cassette oligonucleotide) used in triplex PCR-flap assay in combination with dNTPs at the concentrations shown. [Figure 9D] 1 shows exemplary oligonucleotide combinations (primer oligonucleotide, probe oligonucleotide and FRET cassette oligonucleotide) used in triplex PCR-flap assay in combination with dNTPs at the concentrations shown. [Figure 9E] 1 shows exemplary oligonucleotide combinations (primer oligonucleotide, probe oligonucleotide and FRET cassette oligonucleotide) used in triplex PCR-flap assay in combination with dNTPs at the concentrations shown. [Figure 9F] 1 shows exemplary oligonucleotide combinations (primer oligonucleotide, probe oligonucleotide and FRET cassette oligonucleotide) used in triplex PCR-flap assay in combination with dNTPs at the concentrations shown. [Figure 9G] 1 shows exemplary oligonucleotide combinations (primer oligonucleotide, probe oligonucleotide and FRET cassette oligonucleotide) used in triplex PCR-flap assay in combination with dNTPs at the concentrations shown. [Figure 10] We describe an exemplary combination for a multiplex PCR-flap assay in which multiple markers generate signals using each of the dyes shown, and the listed markers are assayed using as few as four highly multiplex PCR-flap assays. [Figure 11A] Exemplary oligonucleotide combinations (primer oligonucleotides, probe oligonucleotides, and FRET cassette oligonucleotides) used in combination with dNTPs at the concentrations shown are shown in the four highly multiplex PCR-FLAP assays shown in the table in FIG. [Figure 11B] Exemplary oligonucleotide combinations (primer oligonucleotides, probe oligonucleotides, and FRET cassette oligonucleotides) used in combination with dNTPs at the concentrations shown are shown in the four highly multiplex PCR-FLAP assays shown in the table in FIG. [Figure 11C] Exemplary oligonucleotide combinations (primer oligonucleotides, probe oligonucleotides, and FRET cassette oligonucleotides) used in combination with dNTPs at the concentrations shown are shown in the four highly multiplex PCR-FLAP assays shown in the table in FIG. [Figure 11D] Exemplary oligonucleotide combinations (primer oligonucleotides, probe oligonucleotides, and FRET cassette oligonucleotides) used in combination with dNTPs at the concentrations shown are shown in the four highly multiplex PCR-FLAP assays shown in the table in FIG. [Figure 12] FIG. 11 shows a table of assay results for samples from individuals with cancer and healthy individuals using low-bias multiplex pre-amplification followed by four MAD-PCR-flap assay reactions with the oligonucleotide combinations shown in FIG. [Figure 13A]Low-bias multiplex pre-amplification, followed by a triplex PCR-flap assay reaction using the combination of oligonucleotides shown in FIGS. 9A-9G, shows a table of assay results of samples from individuals with cancer and healthy individuals. The percentage of methylation of each marker measured in the triplex reaction is compared with the integrated percentage of methylation measured for those markers in the corresponding MAD reaction (MAD reactions 1, 2, 3, or 4 shown). [Figure 13B] Low-bias multiplex pre-amplification, followed by a triplex PCR-flap assay reaction using the combination of oligonucleotides shown in FIGS. 9A-9G, shows a table of assay results of samples from individuals with cancer and healthy individuals. The percentage of methylation of each marker measured in the triplex reaction is compared with the integrated percentage of methylation measured for those markers in the corresponding MAD reaction (MAD reactions 1, 2, 3, or 4 shown). [Figure 13C] Low-bias multiplex pre-amplification, followed by a triplex PCR-flap assay reaction using the combination of oligonucleotides shown in FIGS. 9A-9G, shows a table of assay results of samples from individuals with cancer and healthy individuals. The percentage of methylation of each marker measured in the triplex reaction is compared with the integrated percentage of methylation measured for those markers in the corresponding MAD reaction (MAD reactions 1, 2, 3, or 4 shown). [Figure 13D] Low-bias multiplex pre-amplification, followed by a triplex PCR-flap assay reaction using the combination of oligonucleotides shown in FIGS. 9A-9G, shows a table of assay results of samples from individuals with cancer and healthy individuals. The percentage of methylation of each marker measured in the triplex reaction is compared with the integrated percentage of methylation measured for those markers in the corresponding MAD reaction (MAD reactions 1, 2, 3, or 4 shown).

DETAILED DESCRIPTION OF THE INVENTION

[0178] The present technology relates to multiplex preamplification of nucleic acids under conditions that reduce the bias between the amounts of amplification products generated from different target nucleic acid sequences co-amplified in a preamplification reaction mixture. For example, different target nucleic acids may amplify with different efficiencies, and thus generate different numbers of copies, even if each target nucleic acid is present in the same copy number before amplification. The difference in amplification efficiency between different target nucleic acid sequences and between different target nucleic acid sequences under the same reaction conditions may be referred to as "amplification bias". Provided herein is a technology related to low-bias amplification buffers and the use of low-bias amplification buffers in multiplex preamplification of nucleic acids, where the preamplified products are further assayed in a multiplexed nucleic acid detection assay, such as PCR or PCR-flap assay, nucleic acid sequencing assay, etc. Exemplary and non-limiting methods are described below.

[0179] In certain embodiments, the multiplex pre-amplified sample is further analyzed in a multiplex nucleic acid detection assay, in which two or more different target nucleic acids generate signals using the same label, e.g., the same fluorophore, in an additive manner, such that the total signal from the label comprises the signals generated by the detection of multiple different nucleic acids or nucleic acid sequences. In general, for assays configured to generate additive signals from multiple different targets, the concentrations of the amplification mixture components for each target, e.g., individual primers, are adjusted so that the amplification from different target nucleic acids in a multiplex reaction has similar amplification efficiency. See, for example, (Sint, D., et al., Methods in Ecology and Evolution 2012, 3, 898-905, and WO2006 / 050499A2). In the development of this technology, it has surprisingly been found that by using a low-bias amplification buffer during multiplex pre-amplification, it is not necessary to use different concentrations for different primers in either the multiplex pre-amplification or the subsequent multiplex nucleic acid detection assay.

[0180] Provided herein are techniques relating to methods for characterizing a sample or combination of samples from a subject, e.g., in a sample containing nucleic acids from multiple organisms or different cell types, comprising analyzing the sample(s) for the amount of different nucleic acids, e.g., different alleles, mutations, single nucleotide polymorphisms (SNPs), methylation markers, different regions of a gene or chromosome, or characteristic nucleic acids from different species or variants.

[0181] Provided herein is a technique for reducing bias in amplification-based nucleic acid detection, particularly, but not limited to, for concentrating low nucleic acid amount samples for analysis.In some embodiments, nucleic acid is pretreated, for example, DNA is pretreated with methylation-sensitive reagent or enzyme, or RNA is reverse transcribed.

[0182] Biological samples of interest may vary greatly in the amount of nucleic acids in them, and even when abundant nucleic acids are abundant, the amount of a particular nucleic acid of interest, e.g., non-normal DNA or RNA in a background of normal DNA or RNA, or human nucleic acid in a background of microbial or viral nucleic acids (or vice versa), may be very low. To compensate for low concentrations of target nucleic acids, large volumes of samples may be processed to collect sufficient nucleic acid for a particular assay. However, when it is desirable to subject a sample with a low concentration of target nucleic acid to a large number of different parallel assays, the required sample size may become prohibitively large. For example, circulating cell-free DNA (cfDNA) in plasma (e.g., of a subject) is typically very low, with a half-life of only 10-15 minutes, as it is continuously cleared from the bloodstream, primarily by the liver. Thus, typical levels of circulating DNA are very low; for example, in healthy individuals, a particular segment of DNA (e.g., from a gene of interest) may be present at about 1,500-2000 copies / mL, while a segment of DNA associated with a tumor may be present at about 5000 copies / mL in a subject with late-stage cancer. Furthermore, tumor-derived cfDNA in plasma is typically fragmented into short strands (e.g., 200 base pairs or less) (see, e.g., P. Jiang, et al., Proc. Natl Acad Sci. 112(11):E1317-E1325 (2015), which is incorporated herein by reference in its entirety). Fetal-derived cfDNA in maternal blood is not only small in size, but also represents a very small fraction of the total cfDNA circulating in the blood of pregnant subjects. Such small DNA is particularly difficult to purify, as it may be lost during typical purification steps due to inefficiencies in the purification steps, e.g., due to precipitation and / or DNA binding.

[0183] Such recovery of cfDNA from blood fraction samples may capture 75%, but often recovers much less. Similarly, viruses and / or their nucleic acids may be present in low concentrations (e.g., per mL of blood or plasma) in samples from infected individuals. Thus, depending on the sensitivity of a particular assay for these target nucleic acids, analysis of multiple nucleic acid sequences from plasma may require large amounts of plasma from the subject. Enrichment by targeted preamplification of specific target regions can increase the number of markers that can be analyzed using the same starting sample, i.e., there is no need to collect a correspondingly large amount of sample (e.g., plasma or blood) from the subject.

[0184] The present technology is also particularly suitable for multiplex analysis of any sample where the target nucleic acid of each species is a small fraction of the total preparation of nucleic acid from the sample. For example, a sample, such as an environmental sample, may contain a complex mixture of nucleic acids, such as eukaryotic cells, bacterial cells, archaeal cells, and / or nucleic acids from different bacterial, fungal, archaeal and / or viral species, or mutants or variants thereof. Multiplex pre-amplification can increase the number of genes, species, variants, etc. that can be characterized in a single sample, such as a soil or water sample, facilitating, for example, microbiome analysis or the detection of emerging variants.

[0185] Provided herein are embodiments of techniques for using low-bias multiplex pre-amplification that are particularly suited to the analysis of target nucleic acids that are low abundant and / or fragmented in the samples in which they are found.

[0186] In some embodiments, the target nucleic acid has been treated with a methylation-sensitive conversion reagent, such as a bisulfite reagent, or using the TAPS method, which combines oxidation with a TET enzyme and reduction with a borane derivative, as described herein.

[0187] Embodiments of the present technology Low bias preamplification of target regions Provided herein are techniques related to providing increased amounts of DNA for analysis in a subsequent nucleic acid detection assay, particularly a PCR assay, such as a PCR-flap assay, e.g., QuARTS or LQAS assay, as illustrated in Figure 1. In particular, embodiments of the methods and compositions disclosed herein provide for increasing the amount of multiple different nucleic acid targets of interest, e.g., from a low target sample, using a low bias multiplex preamplification step, followed by one or more detection assays, e.g., PCR-flap assays. In some embodiments, the target nucleic acid is RNA and the preamplification comprises reverse transcription.

[0188] In a preferred embodiment, the method includes using a low bias amplification buffer, e.g., a buffer containing high Mg as compared to standard PCR buffers. ++ and low KCl PCR-flap assay buffer (e.g., 6-10 mM, preferably 7.5 mM Mg ++ , and 0.0-0.8 mM KCl). For example, a typical PCR buffer (final reaction concentration, or "1x") contains 1.5 mM MgCl 2 , 20 mM Tris-HCl (pH 8), and 50 mM KCl, although an exemplary 1× PCR-Flap Assay Buffer contains 7.5 mM MgCl 2 , 10 mM MOPS, 0.3 mM Tris-HCl (pH 8.0), 0.1 μg / μL BSA, 0.0001% Tween-20, and 0.0001% IGEPAL CA-630. KCl may be included at a final concentration of, for example, 0.8 mM, 25 mM, or any other concentration that does not reduce the low bias effect of such buffers when used in multiplex amplification reactions. Preferably, the low bias amplification buffer contains (NH 4 ) 2 SO 4 In particularly preferred embodiments, the low bias amplification buffer is essentially free of DMSO, formamide and added reducing agents, such as DTT and β-mercaptoethanol.

[0189] PCR-flap assays use different buffer and salt conditions than standard PCR (e.g., PCR-flap assay buffers are typically MOPS, Tris-HCl (pH 8.0), and 7.5 mM MgCl 2 Contains little or no added KCl or other monovalent salts, and usually contains low concentrations of monovalent salts and relatively high concentrations of Mg ++ For example, "Guidelines for PCR Optimization with Taq DNA Polymerase" (https: / / www.neb.com / tools-and-resources / usage-guidelines / guidelines-for-pcr-optimization-with-taq-dna-polymerase) states that the optimal Mg concentration for Taq DNA polymerase is 1.5 mM to 2.0 mM. ++ (See, e.g., et al., "Multiplex PCR: Critical Parameters and Step-by-Step Protocol," O. Henegariu, et al., BioTechniques 23:504-511 (September 1997). During development of this technology, it was discovered that PCR-flap assay buffers reduce bias in multiplex PCR amplifications, e.g., in preamplification reactions.

[0190] In certain embodiments, the technology relates to the use of a single label, e.g., a fluorophore, to report aggregation signals from multiplex amplification of multiple different target nucleic acids amplified with target-specific (e.g., gene-specific) primer pairs in a PCR assay (e.g., TAQMAN probe cleavage assay) using the same probe. In some embodiments, aspects of the technology relate to the use of a single FRET cassette to report aggregation signals from multiplex amplification of multiple different target nucleic acids amplified with target-specific (e.g., gene-specific) primer pairs in the same PCR-flap assay. In preferred embodiments, the preamplification conditions are selected to reduce or minimize amplification bias between different target nucleic acids. In particularly preferred embodiments, conditions are selected to minimize amplification bias without the need to optimize reactions separately for each target, e.g., without the need to adjust the concentrations of different primer pairs, to make the amplification efficiencies from different targets in a multiplex reaction more similar (see, e.g., Wu, et al, Front. Immunol. v11:1631 (2020)). Not having to separately optimize primer pair concentrations is particularly advantageous in that it simplifies the process of designing primer pairs that can function together in multiplex assays, making highly complex multiplexing feasible, e.g., multiplexing of more than 20, more than 30, more than 40, more than 50 different amplification targets, in target-enhanced quantitative PCR probe assays such as PCR-flap assays.

[0191] An embodiment of the present technology is directed to combining low-bias multiplex PCR pre-amplification with detection by a multiplex PCR-flap assay, where multiple markers generate signals using individual dyes, without the need to distinguish between individual signal contributions from any of the individual targets detected in the multiplex PCR-flap assay. The process of using a single dye in a PCR-flap assay to assay multiple targets is referred to as a Multiple Analyte to one Dye PCR-flap assay or a "MAD" PCR-flap assay. If one chooses to detect different dyes in the same reaction (e.g., using additional channels in a fluorescence detector), multiple target groups reporting to different dyes can be detected in the same PCR-flap assay. The present technology provides methods and compositions for assaying multiple different target sequences in one or a few multiplex PCR-flap assays, requiring many fewer PCR-flap assay reactions than would be used if the PCR-flap assay were configured to detect each target sequence individually. This technique can also be utilized in conjunction with multiplex pre-amplification followed by other multiplex PCR FRET probe cleavage assays such as the TAQMAN assay.

[0192] During development of the embodiments of the technology provided herein, high Mg ++ It has been discovered that the use of a preamplification buffer containing 5 mM MgCl (e.g., greater than 6 mM, preferably greater than 7 mM, more preferably 7.5 mM) reduces amplification bias between targets in highly multiplex PCR compared to standard PCR assay conditions. One such buffer is MOPS, Tris-HCl (pH 8.0), and 7.5 mM MgCl. 2 and little or no added KCl or other monovalent salts. ++ Reduce the helix stabilizing effect of the concentration, e.g., DMSO, sometimes called enhancer agent, (NH 4 ) 2 SO4 It was discovered that using high Mg++ in combination with PCR buffers containing reagents such as betaine did not produce the same low bias effect.

[0193] In certain preferred embodiments, the plurality of different target nucleic acids is DNA, and the target DNA is cultured in a buffer containing different primer pairs, each of the primers at essentially equal concentrations, and at least 6 mM Mg. ++ In a particularly preferred embodiment, the preamplification reaction mixture contains essentially equal concentrations of 200-600 nM each of the primers of the different primer pairs, 7.5 mM MgCl, 10 mM dimethylformamide (DMSO) and 10 mM dNTPs. 2 , 10 mM MOPS (pH 7.5), 0.3 mM Tris-HCl (pH 8.0), 0.8 mM KCl, 0.1 μg / μL BSA, 0.0001% TWEEN-20 detergent, 0.0001% IGEPAL CA-630 detergent, 250 μM each dNTP, and 0.025 units / μL HOTSTART GOTAQ DNA polymerase. In a preferred embodiment, the preamplification reaction does not contain a labeled probe oligonucleotide, such as a TAQMAN probe or a FRET cassette. In a particularly preferred embodiment, the preamplification reaction does not contain a flap assay probe.

[0194] In certain preferred embodiments, the plurality of different target nucleic acids is RNA, and the target RNA is cultured in a culture medium containing different primer pairs, each of the primers at essentially equal concentrations, and at least 6 mM Mg. ++ The primers are preamplified in a preamplification reaction mixture containing a MOPS buffer solution containing a mixture of 200-600 nM of each of the primers of the different primer pairs at essentially equal concentrations, 0.5-1.0 units / μL of MMLV reverse transcriptase, 7.5 mM MgCl, and 1.0 mM glycerol. In a particularly preferred embodiment, the preamplification reaction mixture contains 200-600 nM of each of the primers of the different primer pairs at essentially equal concentrations, 0.5-1.0 units / μL of MMLV reverse transcriptase, 7.5 mM MgCl, and 1.0 mM glycerol. 2, 10 mM MOPS (pH 7.5), 0.3 mM Tris-HCl (pH 8.0), 0.8 mM KCl, 0.1 μg / μL BSA, 0.0001% TWEEN-20 detergent, 0.0001% IGEPAL CA-630 detergent, 250 μM each dNTP, and 0.025 units / μL HOTSTART GOTAQ DNA polymerase. In a preferred embodiment, the preamplification reaction does not contain a labeled probe oligonucleotide, such as a TAQMAN probe or a FRET cassette. In a particularly preferred embodiment, the preamplification reaction does not contain a flap assay probe.

[0195] The multiplex pre-amplification embodiments disclosed herein are used with amplification-based assays, such as PCR FRET probe assays (e.g., TAQMAN), PCR-based NGS assays, rolling circle amplification assays, and with PCR-flap assays, such as QuARTS and LQAS assays. As illustrated in FIG. 1, QuARTS and LQAS technologies combine a polymerase-based target DNA amplification process with an invading cleavage-based signal amplification process. The fluorescent signal generated by the QuARTS / LQAS reaction is monitored in a manner similar to real-time PCR. During each amplification cycle, three consecutive chemical reactions occur in each assay well, the first and second reactions on a target DNA template, and the third reaction on a synthetic DNA target labeled with a fluorophore and a quencher dye, which forms a fluorescence resonance energy transfer (FRET) donor and acceptor pair. In the first reaction, an amplified target is generated with a polymerase and an oligonucleotide primer, and in the second reaction, a highly structure-specific 5'-flap endonuclease-1 (FEN-1) enzyme reaction is used to release a 5'-flap sequence from a target-specific oligonucleotide probe, which binds to the product of the polymerase reaction to form an overlap flap substrate. In the third reaction, the cleaved flap anneals to a specifically designed oligonucleotide (FRET cassette) containing a fluorophore and a quencher that are closely linked in a FRET pair such that the fluorescence is quenched. The released probe flap hybridizes to form an overlap flap substrate, which allows the FEN-1 enzyme to cleave the 5'-flap containing the fluorophore, thereby releasing the fluorophore from the proximity of the quencher molecule. The released fluorophore emits a fluorescent signal to be detected. During the second and third reactions, the FEN-1 endonuclease can cleave multiple probes per target to generate multiple cleaved 5'-flaps per target, each of which can be involved in the cleavage of many FRET cassettes, resulting in additional fluorescent signal amplification throughout the reaction.

[0196] In using triplex format, each assay is typically designed to detect multiple targets, e.g., three genes that report to three separate fluorescent dyes. See, for example, Zou, et al., (2012) "Quantification of Methylated Markers with a Multiplex Methylation-Specific Technology", Clinical Chemistry 58:2, which is incorporated herein by reference for all purposes. In contrast, in using the highly multiplex PCR-flap assay of the present technology, multiple different targets can be assayed together, and the primary flap cleavage products (liberated probe flaps) generated from each amplified target all report to the same FRET cassette. The PCR-flap assay can be further multiplexed by having a second group of targets report to a second FRET cassette and an additional group of targets report to an additional, differently labeled FRET cassette.

[0197] Applications of this technology The technology has application to the analysis of multiple nucleic acids in any type of sample or sample mixture, such as human or animal cells, tissues, body fluids, environmental samples such as soil, water, plant cells or tissues, earth surface materials, food or food preparations, etc. Nucleic acids can be analyzed for any type of different number of sequences, such as multiple different genes or allelic or epigenetic variants in a sample from a subject, or for the amount of different nucleic acids (e.g., RNA expression products, mutant alleles, or nucleic acids from different microbial or viral strains or variants).

[0198] Methylation and mutation marker analysis The technology finds application in assaying differences between multiple nucleic acids, for example, for detection and measurement of mutations, methylation status, SNPs or other mutations, intergenic mutations, or other target nucleic acids. In some embodiments, the marker is a region of 100 bases or less, the marker is a region of 500 bases or less, the marker is a region of 1000 bases or less, the marker is a region of 5000 bases or less, or in some embodiments, the marker is a single base. In some embodiments, the marker is within a promoter with high CpG density.

[0199] The present technology is not limited by the type of sample. For example, in some embodiments, the sample is a stool sample, a tissue sample, a sputum, a blood sample (e.g., plasma, serum, whole blood), a stool sample, or a urine specimen.

[0200] Furthermore, the present technology does not limit the method used to determine methylation status. In some embodiments, the assay includes using methylation-specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation, or target capture. In some embodiments, the assay includes using methylation-specific oligonucleotides. In some embodiments, the present technology uses massively parallel sequencing (e.g., next-generation sequencing), such as sequencing by synthesis, real-time (e.g., single molecule) sequencing, bead emulsion sequencing, nanopore sequencing, etc., to determine methylation status.

[0201] In some embodiments, the design for assaying the methylation status of a marker includes analyzing background methylation at individual CpG loci within the target region of the marker to be examined by the assay technique. For example, in some embodiments, a large number of individual copies (e.g., more than 10,000, preferably more than 100,000 individual copies) of marker DNA from a sample isolated from a subject diagnosed with a disease, e.g., cancer, are examined to determine the frequency of methylation, and these data are compared to a similarly large number of individual copies of marker DNA from a sample isolated from a subject without the disease. The frequency of disease-associated methylation at individual CpG loci within the marker DNA from the sample can be compared to the frequency of background methylation, so that CpG loci with high signal-to-noise, e.g., detectable hypermethylation and / or background hypomethylation, can be selected for use in the assay design. See, e.g., U.S. Patent Nos. 9,637,792 and 10,519,510, each of which is incorporated herein by reference in its entirety. In some embodiments, a group of high signal-to-noise CpG loci (e.g., two, three, four, five or more individual CpG loci within a marker region) are interrogated simultaneously by the assay, and based on the assay results, all of the CpG loci must have a pre-defined methylation status (e.g., all must be methylated or none may be methylated) such that the marker is classified as "methylated" or "unmethylated."

[0202] When assessing methylation status, the methylation status is often expressed as the proportion or percentage of individual strands of DNA that are methylated at a particular site (e.g., a single nucleotide, a particular region or locus, a long sequence of interest, e.g., a DNA subsequence up to about 100 bp, 200 bp, 500 bp, 1000 bp or more in length) compared to the total DNA population in a sample that contains that particular site. Traditionally, the amount of unmethylated nucleic acid is determined by PCR using a calibrator. Then, a known amount of DNA is bisulfite treated, and the resulting methylation-specific sequence is determined using either real-time PCR or other exponential amplification, e.g., QuARTS assay (e.g., as described by U.S. Pat. Nos. 8,361,720, 8,715,937, 8,916,344, and 9,212,392, and U.S. Patent Application No. 15 / 841,006).

[0203] For example, in some embodiments, the method includes generating a standard curve for the unmethylated target by using an external standard. The standard curve is generated from at least two points and relates the real-time Ct value of the unmethylated DNA to a known quantitative standard. Then, a second standard curve for the methylated target is generated from at least two points and the external standard. This second standard curve relates the Ct of the methylated DNA to a known quantitative standard. Then, the Ct values ​​of the test samples are determined for the methylated and unmethylated populations, and the genome equivalent of DNA is calculated from the standard curve generated by the first two steps. The percentage of methylation at the site of interest is calculated from the amount of methylated DNA relative to the total amount of DNA in the population, e.g., (number of methylated DNA) / (number of methylated DNA+number of unmethylated DNA)×100.

[0204] Also provided herein are compositions and kits for carrying out the methods. For example, in some embodiments, reagents (e.g., primers, probes) specific to one or more markers are provided, either alone or in sets (e.g., primer pair sets for amplifying multiple markers). Additional reagents for carrying out detection assays can also be provided (e.g., enzymes, buffers, positive and negative controls for carrying out QuARTS, PCR, sequencing, bisulfite, or other assays). In some embodiments, kits are provided that contain one or more reagents that are necessary, sufficient, or useful for carrying out the methods. Also provided are reaction mixtures that contain the reagents. Further provided are master mix reagent sets that contain multiple reagents that may be added to each other and / or to the test sample to complete the reaction mixture.

[0205] Methods for isolating DNA suitable for these assay techniques are known in the art. In particular, some embodiments involve isolation of nucleic acids as described in U.S. Patent Application Serial No. 13 / 470,251 ("Isolation of Nucleic Acids"), which is incorporated herein by reference in its entirety.

[0206] Genomic DNA can be isolated by any means, such as using a commercial kit. Briefly, if the DNA of interest is encapsulated by a cell membrane, the biological sample is generally disrupted and dissolved by enzymatic, chemical, or mechanical means. The DNA solution can then be cleared of proteins and other contaminants, for example by digestion with proteinase K. The genomic DNA is then recovered from the solution. This can be done by a variety of methods, such as salting out, organic extraction, or binding of DNA to a solid support. The choice of method is influenced by several factors, such as time, cost, and the amount of DNA required. All clinical sample types, including neoplastic or pre-neoplastic material, are suitable for use in the method, such as cell lines, histological slides, biopsies, paraffin-embedded tissues, body fluids, stool, colonic effluent, urine, plasma, serum, whole blood, separated blood cells, cells separated from blood, and combinations thereof.

[0207] In the present technology, the method used for preparing sample and obtaining nucleic acid for testing is not limited.For example, in some embodiments, DNA is isolated from stool sample, or from blood, or from plasma sample, for example, by using direct capture of gene, or by related method, as described in detail in US patent application 61 / 485386.

[0208] The present technology relates to the analysis of any sample that may be associated with cancer or that may be tested to establish the absence of cancer. For example, in some embodiments, the sample comprises tissue and / or biological fluid obtained from a patient. In some embodiments, the sample comprises secretions. In some embodiments, the sample comprises sputum, blood, serum, plasma, gastric secretions, lung tissue samples, lung cells, or lung DNA recovered from stool. In some embodiments, the subject is a human. Such samples can be obtained by any number of means known in the art, as will be apparent to one of skill in the art.

[0209] Although discussed above with respect to the analysis of methylation markers, the technology is not limited to methylation analysis but is equally applicable to the analysis of mutations, allelic variations, or any type of difference between nucleic acids that may be present in a sample that contains or is suspected of containing multiple different nucleic acid targets.

[0210] Detection Assays and Kits In some embodiments, the markers described herein are utilized in a QUARTS assay performed on a stool sample. In some embodiments, methods are provided for generating DNA samples, particularly those that contain highly purified low abundance nucleic acids in small volumes (e.g., less than 100 microliters, less than 60 microliters) and that are substantially and / or virtually free of substances that interfere with the assays used to test the DNA sample (e.g., PCR, INVADER, QuARTS assay, etc.). Such DNA samples are utilized in diagnostic tests that qualitatively detect the presence or quantitatively measure the activity, expression, or amount of genes, genetic variants (e.g., alleles), or genetic modifications (e.g., methylation) present in a sample taken from a patient. For example, some cancers are correlated with the presence of certain mutant alleles or certain methylation states, and thus detection and / or quantification of such mutant alleles or methylation states is of predictive value in cancer diagnosis and treatment.

[0211] In some embodiments, the sample comprises blood, serum, plasma, or saliva. In some embodiments, the subject is a human. Such samples can be obtained by any number of means known in the art, as will be apparent to those skilled in the art. Cell-free or substantially cell-free samples can be obtained by subjecting the sample to various techniques known to those skilled in the art, including but not limited to centrifugation and filtration. Although it is generally preferred to obtain samples without using invasive techniques, it may still be preferred to obtain samples such as tissue homogenates, tissue sections, and biopsy specimens. The present technology does not limit the method used to prepare the sample and obtain nucleic acids for testing. For example, in some embodiments, DNA is isolated from stool samples, or from blood, or from plasma samples using direct gene capture, as detailed in, for example, U.S. Patent Nos. 8,808,990 and 9,169,511, and WO2012 / 155072, or by related methods.

[0212] Purpose In some embodiments, the nucleic acids present in a sample are analyzed to determine the relative amounts of nucleic acids from different cells, cell types, microorganisms, viruses that may be present in the sample, for example, in environmental samples such as soil or water (e.g., fresh water, sea water, wastewater) samples, or in other environmental samples. Such profiling of nucleic acids in samples is utilized in soil and water quality assessment, as well as to monitor the presence or spread of contamination and disease. Thus, in some embodiments, the technology is utilized in assaying samples for the presence or relative amounts of nucleic acids, for example, by multiplexing a combination of environmental target nucleic acids (e.g., bacteria, viruses, etc.) to detect the presence, absence, increase, or decrease of a particular bacterial or viral species, or genetic mutations thereof.

[0213] In some embodiments, the diagnostic test identifies the presence of a disease or condition in an individual. In some embodiments, the disease is cancer (e.g., lung, pancreatic, HCC, esophageal, gastric, ovarian, etc. cancer).

[0214] In some embodiments, the technology is applied to the treatment of patients (e.g., patients with cancer, early stage cancer, or those likely to develop cancer), such methods comprising determining the methylation status of markers in the multiplexed combinations described herein and administering a treatment to the patient based on the results of such methylation status determination. The treatment may be administering a pharmaceutical compound, administering a vaccine, performing surgery, imaging the patient, performing another test. Preferably, said uses are performed in methods of clinical screening, methods of prognostic evaluation, methods of monitoring treatment results, methods of identifying patients most likely to respond to a particular therapeutic treatment, methods of imaging patients or subjects, and methods for drug screening and development.

[0215] In some embodiments, the technology is utilized in a method for diagnosing cancer in a subject. The terms "diagnosing" and "diagnosis" as used herein refer to a method by which a person skilled in the art can estimate and even determine whether a subject suffers from a given disease or condition, or whether a subject is likely to develop a given disease or condition in the future. A person skilled in the art often performs a diagnosis based on one or more diagnostic indicators, such as a biomarker whose methylation status indicates the presence, severity, or absence of the condition.

[0216] Along with diagnosis, the clinical prognosis of cancer involves determining the aggressiveness of cancer and the likelihood of tumor recurrence in order to plan the most effective treatment. If a more accurate prognosis can be made and even the potential risk of developing cancer can be assessed, then appropriate treatment, possibly less severe treatment, can be selected for the patient. The assessment of cancer biomarkers (e.g., determining methylation status, the presence of mutations) is useful for distinguishing subjects who are likely to develop cancer or suffer from cancer recurrence, who may benefit from more intensive treatment, from subjects with a good prognosis and / or low risk of developing cancer, who may not require treatment or may only require limited treatment.

[0217] Thus, "making a diagnosis" or "diagnosing", as used herein, further includes determining the risk of developing cancer or determining a prognosis, which may provide for prediction of clinical outcome (with or without drug therapy), selection of an appropriate treatment (or whether a treatment is effective), or monitoring of current treatment and possible treatment modifications, based on the measurement of the diagnostic biomarkers disclosed herein.

[0218] Furthermore, in some embodiments of the present technology, the determination of multiple biomarkers over time can be performed to facilitate diagnosis and / or prognosis. The temporal changes in biomarkers can be used to predict clinical outcomes, monitor the progression of cancer, and / or monitor the effectiveness of appropriate therapies targeting the cancer. In such embodiments, for example, it can be predicted that during an effective therapy, a biological sample will show changes in the methylation status of one or more biomarkers disclosed herein (and possibly one or more additional biomarker(s) if monitored).

[0219] The technology is further applied to a method for determining whether to initiate or continue cancer prevention or treatment in a subject. In some embodiments, such a method includes providing a series of biological samples from a subject over a period of time; analyzing the series of biological samples to determine the methylation status of at least one biomarker disclosed herein in each of the biological samples; and comparing the measurable changes in the methylation status of the one or more biomarkers in each of the biological samples. Any changes in the methylation status of the biomarkers over the period of time can be used to predict the risk of developing cancer, predict clinical outcomes, determine whether to initiate or continue cancer prevention or treatment, and determine whether the current treatment is effectively treating the cancer. For example, a first time point can be selected before the start of treatment and a second time point can be selected sometime after the start of treatment. The methylation status can be measured in each of the collected samples from different time points, and qualitative and / or quantitative differences can be noted. The changes in the methylation status of the biomarker levels from the different samples can be correlated with the risk of developing cancer in the subject, prognosis, determining the effectiveness of treatment, and / or the progression of cancer.

[0220] In preferred embodiments, the methods and compositions of the invention are for the treatment or diagnosis of disease at an early stage, e.g., before symptoms of the disease appear, hi some embodiments, the methods and compositions of the invention are for the treatment or diagnosis of disease at a clinical stage.

[0221] As described above, in some embodiments, multiple determinations of one or more diagnostic or prognostic biomarkers can be made, and the change in the marker over time can be used to determine a diagnosis or prognosis. For example, a diagnostic marker can be determined a first time and again a second time. In such embodiments, an increase in a marker from the first time to the second time can be diagnostic of a particular type or severity of cancer, or a given prognosis. Similarly, a decrease in a marker from the first time to the second time can indicate a particular type or severity of cancer, or a given prognosis. Furthermore, the degree of change in one or more markers can be related to the severity of cancer and future adverse events. Those skilled in the art will understand that, in some embodiments, comparative measurements of the same biomarkers can be made at multiple time points, but a given biomarker can also be measured at one time point and a second biomarker at a second time point, and the comparison of these markers can provide information that leads to a diagnosis.

[0222] As used herein, the phrase "determining prognosis" refers to a method by which a person skilled in the art can predict the course or outcome of a condition of a subject. The term "prognosis" does not refer to the ability to predict the course or outcome of a condition with 100% accuracy, nor does it refer to the ability to predict the likelihood or low likelihood of a given course or outcome based on the methylation status of a biomarker. Instead, a person skilled in the art can understand that the term "prognosis" refers to a high probability of a particular course or outcome, i.e., a high likelihood of a course or outcome in a subject that exhibits a given condition, compared to an individual that does not exhibit the condition. For example, an individual that does not exhibit such a condition may have a very low probability of a given outcome (e.g., suffering from cancer).

[0223] In some embodiments, the prognostic indicator is associated with a predisposition to adverse outcomes by statistical analysis. For example, in some embodiments, a methylation status that is different from the methylation status in a normal control sample obtained from a patient without cancer, as determined by a statistical significance level, may suggest that the subject is more likely to suffer from cancer than a subject having a level more similar to the methylation status in the control sample. Furthermore, the change in methylation status from the baseline (e.g., "normal") level may reflect the subject's prognosis, and the degree of change in methylation status may be related to the severity of an adverse event. Statistical significance is often determined by comparing two or more populations to determine a confidence interval and / or p-value. See, for example, Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983, which is incorporated herein by reference in its entirety. Exemplary confidence intervals of the present subject matter are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, and exemplary p-values ​​are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.

[0224] In other embodiments, a threshold value for the degree of change in the methylation status of a prognostic or diagnostic biomarker disclosed herein can be established, and the degree of change in the methylation status of the biomarker in a biological sample is simply compared to the threshold value for the degree of change in the methylation status. Preferred threshold changes in the methylation status of the biomarkers provided herein are about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 50%, about 75%, about 100%, and about 150%. In yet another embodiment, a "nomogram" can be established, whereby the methylation status of a prognostic or diagnostic indicator (biomarker or combination of biomarkers) is directly associated with the associated predisposition to a given outcome. Those skilled in the art are familiar with the use of such nomograms to relate two values, with the understanding that the measurement is referenced to an individual sample and not a population average, and therefore the uncertainty of this measurement is the same as the uncertainty of the marker concentration.

[0225] In some embodiments, a control sample is analyzed simultaneously with the biological sample so that results obtained from the biological sample can be compared to results obtained from the control sample. It is further contemplated that a calibration curve may be provided to which the assay results of the biological sample may be compared. Such a calibration curve represents the methylation state of the biomarker as a function of assay units, e.g., as a function of fluorescent signal intensity in the case of using fluorescent labels. Using samples from multiple donors, calibration curves can be obtained for control methylation states of one or more biomarkers in normal tissues and for "risk" levels of one or more biomarkers in tissues from donors with cancer.

[0226] The assays of the present technology can be performed in a variety of physical formats. For example, the use of microtiter plates or automation can be used to facilitate the processing of multiple test samples. Alternatively, single sample formats can be created to facilitate timely and rapid treatment and diagnosis, for example, in an outpatient delivery or emergency room setting.

[0227] In some embodiments, a subject is diagnosed with cancer if there is a measurable difference in the methylation status of at least one biomarker in the sample compared to a control methylation status. Conversely, if no change in methylation status is identified in the biological sample, the subject is identified as not having cancer, not at risk of cancer, or at low risk of cancer. In this regard, subjects with or at risk of cancer can be distinguished from subjects with or at low to substantially no risk of cancer. Subjects at risk of developing cancer can be placed on a more intensive and / or more regular screening schedule. Meanwhile, subjects at low to substantially no risk may not need to undergo screening methods until future screening, e.g., screening performed according to the present technology, indicates that they are at risk of cancer.

[0228] As mentioned above, depending on the embodiment of the method of the present technology, the detection of the change in the methylation state of one or more biomarkers can be a qualitative or quantitative determination. Thus, the step of diagnosing a subject as having cancer or at risk of developing cancer indicates that a certain threshold measurement is made, e.g., that the methylation state of one or more biomarkers in a biological sample is different from a predetermined control methylation state. In some embodiments of the method, the control methylation state is any detectable methylation state of the biomarker. In other embodiments of the method, where a control sample is tested simultaneously with the biological sample, the predetermined methylation state is the methylation state of the control sample. In other embodiments of the method, the predetermined methylation state is based on and / or identified by a calibration curve. In other embodiments of the method, the predetermined methylation state is a particular state or range of states. Thus, the predetermined methylation state can be selected within acceptable limits that will be apparent to one of skill in the art, based in part on the embodiment of the method being performed and the desired specificity, etc.

[0229] Liquid Biopsy In the last few years, it has become clear that circulating epithelial cells, which represent metastatic tumor cells, can be detected in the blood of many cancer patients. Molecular profiling of rare cells is important in biological and clinical research. Applications range from characterization of circulating epithelial cells (CEpCs) in the peripheral blood of cancer patients for disease prognosis and personalized medicine (see, e.g., Cristofanilli M, et al. (2004) N Engl J Med 351:781-791; Hayes DF, et al. (2006) Clin Cancer Res 12:4218-4224; Budd GT, et al., (2006) Clin Cancer Res 12:6403-6409; Moreno JG, et al. (2005) Urology 65:713-718; Pantel et al., (2008) Nat Rev 8:329-340; and Cohen SJ, et al. (2008) J Clin Oncol 26:3213-3221). Thus, embodiments of the present disclosure provide compositions and methods for detecting the presence of metastatic cancer in a subject by determining the presence of methylation markers in plasma or whole blood. EXAMPLES

[0230] Experimental Examples The following examples are provided to illustrate the present invention, but are not intended to limit the present invention. To facilitate understanding, specific embodiments are provided to aid in interpreting technical proposals, that is, these embodiments are for illustrative purposes only, and do not limit the scope of the present invention in any way. Unless otherwise stated, embodiments that do not show specific conditions are in accordance with conventional conditions or manufacturer's recommended conditions.

[0231] Example 1 Sample preparation, preamplification, and PCR-FLAP assay Sample preparation methods Exemplary methods for isolating RNA and DNA from a sample, e.g., cells, plasma, or blood cells, and optionally processing the DNA, are detailed in WO2021 / 041726, filed August 27, 2020, which is incorporated herein by reference for all purposes. Optionally, the DNA from the sample can be treated with a methylation-specific reagent, e.g., a bisulfite reagent, or processed using the TAPS method, which combines oxidation with a TET enzyme and reduction with a borane derivative, as described above. The converted DNA is then used in a detection assay, e.g., a preamplification and / or flap endonuclease assay, as described below. For additional embodiments of bisulfite processing of nucleic acids, US10,704,081, and US Patent Application No. 63 / 058,179, filed July 29, 2020, each of which is incorporated herein by reference in its entirety for all purposes, may be applied to the techniques described herein.

[0232] In some embodiments, RNA and DNA are isolated from different blood samples from a subject. For example, blood can be collected in a first blood collection tube configured for optimal preservation and / or isolation of RNA and a second blood collection tube configured for optimal preservation and isolation of DNA, and RNA and DNA can be extracted from the portion of blood collected in this manner. In other embodiments, both RNA and DNA are extracted from a single collected blood sample, for example, using a blood collection tube configured for optimal preservation and isolation of both DNA and RNA (e.g., cf-DNA / cf-RNA Preservative Tubes (Cat. No. 63950) from NORGEN Biotek Corp. for preservation and isolation of both cell-free DNA and cell-free RNA).

[0233] In some embodiments, RNA and DNA are assayed together, e.g., in a RT-LQAS / RT-TELQAS reaction. In some embodiments, RNA and DNA are isolated separately and / or treated separately, e.g., with bisulfite, as described above, and in some embodiments, RNA and DNA are treated together, e.g., both are present during bisulfite treatment and subsequent purification, and added together to the assay reaction.

[0234] Flap endonuclease assay In QuARTS and LQAS flap assay technology, the target DNA amplification process using polymerase is combined with the signal amplification process based on invading cleavage. QuARTS technology is described, for example, in U.S. Patent Nos. 8,361,720, 8,715,937, 8,916,344, and 9,212,392, and flap assay using a probe oligonucleotide with a long target-specific region (Long probe Quantitative Amplified Signal, "LQAS") is described in U.S. Patent No. 10,648,025, each of which is incorporated herein by reference in its entirety for all purposes. The combined preamplification and LQAS assay is called "TELQAS" (for "Target Enrichment Long probe Quantitative Amplified Signal") assay.

[0235] In the QuARTS assay, the flap probe oligonucleotide has a target-specific region of 12 bases, whereas the LQAS assay uses a flap oligonucleotide with a target-specific region of at least 13 bases and employs a different thermal cycling procedure for amplification. The fluorescent signal generated by the QuARTS and LQAS reactions can be monitored in a manner similar to real-time PCR to quantify the amount of target nucleic acid in a sample.

[0236] An exemplary QuARTS reaction typically contains approximately 200-600 nmol / L (e.g., 500 nmol / L) of each primer and detection probe, approximately 100 nmol / L of invading oligonucleotide, approximately 600-700 nmol / L of each FRET cassette (FAM, e.g., as commercially available from Hologic, Inc.; HEX, e.g., as commercially available from BioSearch Technologies; and Quasar 670 ("Q670"), e.g., as commercially available from BioSearch Technologies and including a "black hole" quencher, e.g., BHQ-1, BHQ-2, or BHQ-3 from BioSearch Technologies), 6.675 ng / μL of FEN-1 endonuclease (e.g., Cleavase® 2.0, Hologic, Inc.), 1 unit of Taq DNA polymerase (e.g., GoTaq® DNA polymerase, Promega) in a 30 μL reaction volume. Corp., Madison, WI), 10 mmol / L 3-(n-morpholino)propanesulfonic acid (MOPS), 7.5 mmol / L MgCl 2 , and 250 μmol / L of each dNTP. Exemplary QuARTS cycling conditions are shown in the table below. In some applications, a quantification cycle (C q ) provides an indication of the initial number of target DNA strands (e.g., copy number) in the sample. [Table 2]

[0237] It should be noted that Amplification Stage 1 and Amplification Stage 2 are not separate amplification reactions, but rather represent the incubation of a single reaction mixture that is cycled with one thermal profile for the first 10 cycles and then with a different thermal profile for the subsequent thermal cycles (37 cycles in this example). The two amplification stages are performed sequentially on the same reaction mixture, e.g., without additions or other changes to the contents of the reaction mixture.

[0238] An exemplary LQAS reaction typically contains approximately 200-600 nmol / L of each primer, approximately 100 nmol / L of the invading oligonucleotide, and approximately 500 nmol / L of each flap oligonucleotide probe and FRET cassette. The LQAS reaction may be subjected to, for example, the following thermocycling conditions: [Table 3]

[0239] WO2021 / 041726 further describes exemplary QuARTS and LQAS / TELQAS flap assay methods that combine a polymerase-based target DNA amplification process with an invading cleavage-based signal amplification process. The fluorescent signal generated by the QuARTS and LQAS reactions can be monitored in a manner similar to real-time PCR to quantify the amount of target nucleic acid in a sample.

[0240] Also contemplated is an assay that includes multiplex reverse transcription and preamplification followed by an LQAS PCR-flap assay (reverse transcription and preamplification combined with an LQAS assay is referred to as a RT-TELQAS assay, which stands for "reverse transcription-target enrichment long probe quantitative amplified signal"). In the RT-TELQAS assay, target RNA, e.g., total RNA from a sample, is incubated with, for example, 20 U of MMLV reverse transcriptase, 1.5 U of GoTaq® DNA polymerase, 10 mM MOPS buffer (pH 7.5), 7.5 mM MgCl 2The RT-preamplification reaction is then treated with 250 μM of each dNTP, and oligonucleotide primers (e.g., for 12 targets, 12 primer pairs / 24 primers in equimolar amounts (e.g., 200 nM of each primer) or amounts modified to adjust for amplification efficiency of different target RNAs) and incubated at moderate temperature (e.g., 42° C.) for reverse transcription, followed by a limited number of thermal cycles (e.g., 10 cycles of 95° C., 63° C., 70° C.), resulting in preamplification of the target sequences corresponding to the included primer pairs. After thermal cycling, an aliquot (e.g., 10 μL) of the RT-preamplification reaction is used for the LQAS PCR-flap assay, as described below. RNA suitable for detection in the RT-TELQAS and RT-LQAS assays is not limited to any particular type of RNA target. For example, any type of RNA from tissues, cells, or circulating cell-free RNA from blood, such as protein-coding messenger RNA (mRNA), microRNA (miRNA), piRNA, tRNA, and other non-coding RNA molecules (ncRNA) (see, for example, “A comprehensive profile of circulating RNAs in human serum,” RNA Biology 15(2):242-250(2018) which is incorporated by reference in its entirety), can be assayed using the RT-TELQAS and RT-LQAS methods described below.

[0241] Multiplex preamplification of sample DNA Large amounts of isolated DNA can be used in a single multiplex amplification reaction to preamplify most or all of the target DNA from the input sample (e.g., DNA isolated from the sample or cDNA generated from isolated RNA). Preamplification can be performed using, for example, 7.5 mM MgCl 2, 10 mM MOPS, 0.3 mM Tris-HCl (pH 8.0), 0.8 mM KCl, 0.1 μg / μL BSA, 0.0001% Tween-20, 0.0001% IGEPAL CA-630, 250 μM of each dNTP, oligonucleotide primers, (e.g., for 20 targets, 20 primer pairs / 40 primers, e.g., each primer in essentially equimolar amounts, including but not limited to, in the range of 200-600 nM), a HotStart GoTaq concentration of 0.025 units / μL, and 20-50% by volume of sample DNA (e.g., 10 μL of target DNA for a 50 μL reaction mixture, or 50 μL of target DNA for a 125 μL reaction mixture).

[0242] Thermal cycling times and temperatures are selected to be appropriate for the volume of the reaction and amplification vessel. For example, the reaction can be cycled as follows: [Table 4]

[0243] After thermal cycling, an aliquot of the preamplification reaction (e.g., 10 μL) is typically diluted (e.g., to 500 μL in 10 mM Tris, 0.1 mM EDTA) with or without a large amount of endogenous DNA (e.g., fish DNA to minimize variations in polymerase activity). An aliquot of the diluted preamplification DNA (e.g., 10 μL) or of the undiluted preamplification reaction is used in a multiplex PCR-flap assay using all or a subset of the same primer pairs.

[0244] Multiplex preamplification of sample RNA Preamplification from isolated RNA or mixed RNA+DNA samples was performed using, for example, 7.5 mM MgCl 2, 10 mM MOPS, 0.3 mM Tris-HCl (pH 8.0), 0.8 mM KCl, 0.1 μg / μL BSA, 0.0001% Tween-20, 0.0001% IGEPAL CA-630, 250 μM of each dNTP, oligonucleotide primers (e.g., for 20 targets, 20 primer pairs / 40 primers, e.g., in essentially equimolar amounts, including but not limited to, in the range of 200-600 nM of each primer), 0.025 units / μL HOTSTART GOTAQ DNA polymerase, 0.67 units / μL MMLV reverse transcriptase, and 20-50% by volume of RNA sample (e.g., 10 μL of target RNA for a 50 μL reaction mixture, or 50 μL of target RNA for a 125 μL reaction mixture).

[0245] Thermal cycling times and temperatures are selected to be appropriate for the volume of the reaction and amplification vessel. For example, the reaction can be cycled as follows: [Table 5]

[0246] After thermal cycling, an aliquot of the preamplification reaction (e.g., 10 μL) is typically diluted (e.g., to 500 μL in 10 mM Tris, 0.1 mM EDTA) with or without a large amount of endogenous DNA (e.g., fish DNA to minimize variations in polymerase activity). An aliquot of the diluted preamplification DNA (e.g., 10 μL) or the undiluted preamplification reaction is used in a multiplex PCR-flap assay using all or a subset of the same primer pairs. Diluted and undiluted preamplification samples can be stored at -20°C.

[0247] PCR-FLAP assay from multiplex preamplified DNA Using the pre-amplified samples described above, QuARTS and LQAS PCR-flap assay reactions are typically set up as follows: [Table 6] * 10x oligonucleotide mix = 2 μM of each primer and 5 μM of each probe and FRET oligonucleotide ** The 20x enzyme mix contained 1 unit / μL GoTaq Hot start polymerase (Promega Corp.), 292 ng / μL Cleavase 2.0 FEN-1 flap endonuclease (Hologic, Inc.), 200 mM MOPS (pH 7.5), 150 mM MgCl2, 6.38 mM Tris-HCl (pH 8.0), 15.95 mM KCl, 2 μg / μL BSA, 0.16% Tween-20, 0.16% IGEPAL CA-630, and 25% glycerol.

[0248] As noted above, the flap oligonucleotide in the QuARTS assay has a target-specific region of 12 bases, while the LQAS assay uses a flap oligonucleotide with a target-specific region of at least 13 bases, which is subjected to different thermocycling conditions. In some embodiments, the QuARTS reaction is subjected to the following thermocycling conditions: [Table 7]

[0249] In some embodiments, the LQAS PCR-flap assay reaction is subjected to the following thermocycling conditions: [Table 8]

[0250] Example 2 Effect of magnesium concentration in PCR-flap assay buffer on amplification bias in multiplex preamplification Mg against amplification bias in multiplex reactions ++ (e.g., MgCl 2The effect of using buffers with different concentrations of Mg (provided as is) was investigated. The PCR-flap assay buffer modified for multiplexing was used with different concentrations of Mg ++ A 10-fold concentrated multiplex PCR-flap assay buffer was used as a standard for testing different concentrations of MgCl. 2 with a final concentration of 7.5 mM MgCl 2 (Standard high Mg content in PCR-flap assay buffer) ++ or a final concentration of 2.5 mM MgCl 2 (Low Mg in the range typically used for PCR assays) ++ The 10x buffers were otherwise identical and contained either 75 mM or 25 mM MgCl. 2 In addition, the solution contained 100 mM MOPS (pH 7.5), 0.08% Tween 20, 0.08% IGEPAL-CA630, and 2.5 mM of each dNTP.

[0251] Multiplex calibrators containing equal amounts of 37 different markers (plasmids containing different target sequences were digested to yield equimolar amounts of 37 individual marker DNA) were diluted in 10 mM Tris HCl, 0.1 mM EDTA to generate preparations containing 2 or 20 target DNA strands per μL, yielding either 100 or 1000 target DNA strands per 50 μL of diluted stock. Target DNA was diluted with either high or low MgCl 2 was used for pre-amplification.

[0252] Each preamplification reaction contained 25 μL of the following master mix, which was combined with 50 μL of diluted sample DNA for a final preamplification reaction mixture volume of 75.0 μL. Sample DNA aliquots contained either 1000 strands per reaction of calibrator plasmid or 100 strands per reaction of calibrator plasmid, as described above. [Table 9]

[0253] * The primer mix formulation contained 0.75 μM of forward primer (FP) and reverse primer (RP) for each of the marker DNAs listed below: [Table 10]

[0254] Each reaction was performed in duplicate.

[0255] Preamplification reactions were assembled in 96 wells and amplified using the following thermal profile: [Table 11]

[0256] A 20 μL aliquot of the preamplification reaction was diluted with 180 μL of 10 mM Tris-Cl, 0.1 mM EDTA and the undiluted and diluted preamplification products were stored at -20°C.

[0257] LQAS PCR-flap assay reaction After preamplification in the above two different buffers, the preamplification products were assayed using triplex PCR-flap assay. The B3GALT6 marker was used as an internal reference target to quantify 18 other marker DNAs in the triplex reaction described below.

[0258] 10 μL of the diluted pre-amplified sample was analyzed in each of 14 different triplex PCR flap assays, each constructed as follows: [Table 12]

[0259] Reactions were constructed to contain primer pairs for the markers in the following triplex combinations: [Table 13]

[0260] Each reaction was performed in duplicate. The results are shown in Figure 6, with the calculated theoretical yield indicated by the dashed horizontal line. The amount of deviation above or below the dashed line indicates the increase in Mg ++ The amount of signal variation seen for each marker at each concentration is shown.

[0261] These data indicate that the average dispersion coefficient of the strands was significantly higher with 2.5 mM MgCl in the preamplification reaction. 2 43% when using 7.5 mM MgCl 2 The amplification efficiency was reduced to 22% when 2.5 mM MgCl 2 The average of 70% of the total DNA was obtained by adding 7.5 mM MgCl 2 These data support the use of high Mg in multiplex PCR preamplification. ++ 1 shows that the use of PCR-flap assay buffer reduced amplification bias between different targets in subsequent LQAS PCR-flap assay reactions and increased the signal from each target.

[0262] Example 3 in low bias PCR-flap assay buffer or (NH 4 ) 2 SO 4 Comparison of amplification bias during multiplex preamplification in PCR buffer In some methods, excess Mg ++ To reduce the double-chain stabilizing effect of ions (NH 4 ) 2 SO 4 Using a PCR buffer containing Mg, for example, ++This broadens the range of magnesium that can be used without the increased background in PCR typically seen when too much is used. See, e.g., MM Blanchard, et al., PCR buffer optimization with uniform temperature regimen to facilitate automation. Genome Res. 2:234-240 (1993). (NH 4 ) 2 SO 4 PCR buffers containing Mg alone or with other helix-destabilizing additives contain Mg at concentrations above the typical 1-4 mM range commonly used for PCR. ++ One such buffer contains 16.6 mM (NH 4 ) 2 SO 4 , 67 mM Tris-Cl (pH 8.8), 6.7 mM MgCl 2 , 10 mM β-mercaptoethanol, and 0.1% DMSO. See, e.g., Sukumar, et al., US2005 / 0239101A1; Fackler et al., Cancer Research 64:4442-44452 (2004); Herman et al., Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9821-9826, September 1996). This buffer has been disclosed for use with 1.25 mM dNTPs, 2.5-5 units of Platinum Taq polymerase, and 100 ng each of forward and reverse primers in a 25 μL reaction with bisulfite-treated DNA (see, e.g., Sukumar, supra).

[0263] Multiplex preamplification in low bias PCR-flap assay buffer was performed as described above (NH 4 ) 2 SO 4Compared to multiplex preamplification in PCR buffer, all preamplification reactions were assayed using a triplex PCR-flap assay in PCR-flap assay buffer, as described above.

[0264] Two multiplex master mixes were constructed as follows: (NH 4 ) 2 SO 4 Preamplification in PCR buffer: [Table 14]

[0265] Preamplification with low bias PCR-Flap Assay Buffer: [Table 15]

[0266] For each preamplification reaction, a 3x master mix was assembled in a volume of 25 μL and combined with 50 μL of target DNA for a final reaction volume of 75 μL with the final reaction concentrations indicated above.

[0267] The reaction was amplified using the following thermal profile: [Table 16]

[0268] A 20 μL aliquot of the preamplification reaction was diluted with 180 μL of 10 mM Tris-Cl, 0.1 mM EDTA and the undiluted and diluted preamplification products were stored at -20°C.

[0269] LQAS PCR-flap assay reaction Using the triplex LQAS format described above, 18 different methylation markers and the internal reference marker B3GALT6 were quantified. 10 μL aliquots of the diluted pre-amplified samples were analyzed in each of 14 different triplex PCR flap assays, each constructed as follows: [Table 17]

[0270] Reactions were constructed to contain primer pairs for the markers in the following triplex combinations: [Table 18]

[0271] Each reaction was performed in duplicate. The results are shown in Figure 7, where the calculated theoretical yields (81,920 strands for the 1000 strand input reaction; 8190 strands for the 100 strand input reaction) are indicated by the dashed horizontal lines. The amount of deviation above or below the dashed line indicates the amount of signal variation seen for each marker in the two different preamplification buffers. These data are (NH 4 ) 2 SO 4 The PCR buffer contains 6.7 mM MgCl, which is relatively high for a PCR buffer. 2 However, the final result is 7.5 mM MgCl 2 The results are inconsistent with those achieved when a low bias PCR-flap assay buffer with (NH 4 ) 2 SO 4 Compared to results measured from the same markers pre-amplified together in PCR buffer, the signal was much more consistent across all 18 markers (i.e., reduced bias) and showed a higher overall signal.

[0272] These data show that the average coefficient of variance across the entire set of markers tested was significantly higher in the preamplification reaction (NH 4 ) 2 SO 4 The amplification efficiency was reduced from 59% using PCR buffer to 18% using low bias PCR-flap assay buffer. 4 ) 2 SO 4 The average increase was from 59% when PCR buffer was used to 117% when low bias PCR-flap assay buffer was used in the preamplification reaction. These data support the use of low bias (high Mg) in multiplex PCR preamplification. ++ ) PCR-flap assay buffer was used to preamplify (NH 4 ) 2 SO 4 This shows that amplification bias between different targets in the subsequent LQAS PCR-flap assay reaction was reduced and the signal from each target was increased compared to the results observed when PCR buffer was used.

[0273] Example 4 Low bias multiplex preamplification using multiplex PCR-flap assay: Multiple Analytes Reported to One Dye (MAD) The above data indicate that high Mg preamplification is ++ We show that the use of a MOPS buffer containing allows for more uniform coamplification of different targets without the need to adjust primer or primer pair concentrations to reduce bias between markers. Development of this technology shows that the use of these low-bias preamplification conditions allows for highly multiplexed analysis of markers without the need to resolve signals from each individual target.

[0274] In the above examples, each pre-amplified target DNA is then measured in a triplex PCR-FLAP assay, where each triplexed marker reports to an individual FRET cassette, such that the signal from each individual target nucleic acid can be distinguished from the other two targets in each triplex reaction.

[0275] Low bias preamplification reactions were tested in a configuration where 3-5 different markers in a reaction use the same FRET cassette reporter (Multiple Analytes per Dye, or "MAD" multiplex reactions) such that the individual signals from any one marker cannot be distinguished from one another. Such an assay configuration is shown diagrammatically in Figure 4 and Figures 5A-5C. The sensitivity and specificity for detecting these markers in cancer samples (DNA isolated from plasma from subjects known to have cancer) was compared to results achieved using a triplex configuration, where the results from the different markers are resolved from one another but mathematically integrated to achieve a higher sensitivity than achievable using any single marker.

[0276] In the MAD multiplexing step, each reaction contained a second FRET cassette for integrated signal detection from a second set of 3-5 markers and a third FRET cassette for detection of the reference marker B3GALT6. DNA from healthy subjects or from subjects with cancer was preamplified under the low bias multiplex conditions described above using the PCR-flap assay buffer described above. The 35 markers + B3GALT6 reference DNA in the preamplified reaction mixture was then measured using either 14 triplex PCR-flap assay reactions using the oligonucleotide combinations shown in Figures 9A-9G or 4 MAD multiplex PCR-flap assays using the oligonucleotide combinations shown in Figures 11A-11D.

[0277] Figure 12 shows the results of the assay sensitivity for four MAD PCR-flap assays targeting 35 different methylated marker DNA, and the results for triplex PCR-flap assays using the same markers are shown in the tables of Figures 13A-13D. These data show that the individual markers measured in the triplex LQAS assay showed a maximum methylation% ranging from 0 to 1.2% in healthy subjects, and the maximum methylation% of markers measured in the MAD group in healthy subjects ranged from 0 to 1.7%. The average sensitivity for detecting cancer samples for individual markers measured using the triplex configuration was 60%, whereas the average sensitivity for detecting cancer samples using the MAD group of markers was 90%.

[0278] Example 5 Multiplexed analysis of target nucleic acids from environmental samples Environmental samples such as soil and wastewater typically contain diverse populations of viruses and microorganisms, and the present technique is utilized in characterizing the microbial and viral populations of such samples.

[0279] Sample preparation Nucleic acids are prepared from environmental samples by standard methods, for example, using commercially available kits suitable for the extraction of DNA and / or RNA from a particular type of sample. Examples of suitable kits include, but are not limited to, ThermoFisher Scientific's MAGMAX Wastewater Ultra Nucleic Acid Isolation Kit (MagMAX™ Wastewater Ultra Nucleic Acid Isolation Kit User Guide, 03 / 2022), Qiagen's RNeasy PowerSoil Total RNA Kit (RNeasy PowerSoil Total RNA Kit Handbook 06 / 2017), and Qiagen's DNeasy PowerSoil Pro Kit (DNeasy PowerSoil Pro Kit Handbook 03 / 2021), which handbooks are incorporated herein by reference in their entirety for all purposes.

[0280] Multiplex preamplification of sample DNA For preamplification of 30 different target and reference DNAs, 50 μL of prepared DNA sample was combined with 31 different primer pairs (62 primers) at 200–600 nM each (different primers were at essentially equal concentrations) in 75 μL of preamplification reaction mixture, which was further supplemented with 7.5 mM MgCl 2 , 10 mM MOPS (pH 7.5), 0.3 mM Tris-HCl (pH 8.0), 0.8 mM KCl, 0.1 μg / μL BSA, 0.0001% TWEEN-20 detergent, 0.0001% IGEPAL CA-630 detergent, 250 μM of each dNTP, and 0.025 units / μL HOTSTART GOTAQ DNA polymerase. The reaction mixture is subjected to thermal cycling as follows: [Table 19]

[0281] Multiplex preamplification of sample RNA For preamplification of 30 different target and reference RNAs, 50 μL of isolated RNA or mixed RNA+DNA sample was combined with 200–600 nM of 31 different primer pairs (62 primers) (primers at essentially equal concentrations) in a 75 μL preamplification reaction mixture, which further contained 0.67 units / μL MMLV reverse transcriptase, 7.5 mM MgCl 2 , 10 mM MOPS (pH 7.5), 0.3 mM Tris-HCl (pH 8.0), 0.8 mM KCl, 0.1 μg / μL BSA, 0.0001% TWEEN-20 detergent, 0.0001% IGEPAL CA-630 detergent, 250 μM of each dNTP, and 0.025 units / μL HOTSTART GOTAQ DNA polymerase. The RT-preamplification reaction mixture is subjected to thermal cycling as follows: [Table 20]

[0282] After thermal cycling, the preamplification reaction vessel is centrifuged to recover the concentrate. A 10 μL aliquot of the preamplification reaction is diluted to 500 μL with 10 mM Tris, 0.1 mM EDTA, optionally containing a large amount of fish DNA. The diluted and undiluted preamplification samples can be stored at -20°C.

[0283] PCR-flap assay from preamplification products Multiple different targets reporting to each color Three different PCR-flap assay reactions are performed. Each PCR flap assay reaction mixture contains primer pairs for 10 different pre-amplified targets + 1 reference amplicon or control amplicon. For 30 different targets + reference, three 11-plex flap assay reactions are performed with primers, probes, and FRET cassettes combined as follows: [Table 21-1] [Table 21-2]

[0284] Similarly, in reaction 2, the flap probes for targets 11-15 report to the arm 5 FAM FRET cassette, the flap probes for targets 16-20 report to the arm 1 HEX FRET cassette and the reference target flap probe reports to the arm 3 Q670 FRET cassette, and in reaction 3, the flap probes for targets 21-25 report to the arm 5 FAM FRET cassette, the flap probes for targets 26-30 report to the arm 1 HEX FRET cassette and the reference target flap probe reports to the arm 3 Q670 FRET cassette.

[0285] Make a separate PCR-Flap Assay Master Mix for each of reactions 1, 2, and 3 by combining the following: [Table 22]

[0286] For each of PCR-flap assay reactions 1, 2, and 3, 10 μL of the diluted preamplification reaction product is mixed with 20 μL of PCR-flap assay master mix and the reaction mixture is sealed in a reaction vessel (e.g., a microcentrifuge tube or the well of an assay plate).

[0287] The PCR-Flap assay reaction mixture is subjected to thermal cycling as follows, and fluorescent signals of FAM, HEX, and Q670 are acquired at the indicated points of the amplification cycle: [Table 23]

[0288] In the configuration of reaction 1 above, the FAM signal measured during PCR-flap assay thermal cycling is the total signal from preamplified targets 1-5, the HEX signal is the total signal from preamplified targets 6-10, and the Q670 signal is from the preamplified reference target nucleic acid. Similarly, for reaction 2, the FAM signal measured during PCR-flap assay thermal cycling is the total signal from preamplified targets 11-15, and the HEX signal is the total signal from preamplified targets 16-20, and for reaction 3, the FAM signal measured during PCR-flap assay thermal cycling is the total signal from preamplified targets 21-25, and the HEX signal is the total signal from preamplified targets 26-30.

[0289] Each target reports a different color The signal specific for each of the 30 individual preamplified target DNAs (preamplified from DNA or RNA sample material) is measured in a triplex reaction configured as follows; [Table 24]

[0290] In a similar format, targets 3-30 are measured in pairs (e.g., 3 and 4, 5 and 6, 7 and 8, etc.) in 14 additional triplex reactions, with a reference target for each pair. The results measured for HEX and FAM signals can be normalized to the Q670 signal measured from the reference target, allowing the relative amounts of all markers to be compared across the 15 triplex reactions.

[0291] All literature and similar materials cited in this application, including but not limited to patents, patent applications, papers, books, articles, and Internet web pages, are expressly incorporated by reference in their entirety for any purpose. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the various embodiments described herein belong. If the definition of a term in an incorporated reference differs from the definition set forth in the present teachings, the definition set forth in the present teachings shall control.

[0292] Although certain embodiments of the present invention have been described, these embodiments are presented by way of example only and are not intended to limit the scope of the present disclosure. Indeed, the novel methods and systems described herein may be embodied in other various forms. Furthermore, various modifications, omissions, substitutions, and variations of the compositions, methods, systems, and uses of the present technology that may be described will be apparent to those skilled in the art without departing from the scope and spirit of the present technology as described. Although the present technology has been described in connection with specific exemplary embodiments, it should be understood that the invention described in the claims should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods for carrying out the invention that are apparent to those skilled in the art of molecular biology, biochemistry, medicine, or related fields are intended to be within the scope of the following claims. The appended claims and their equivalents are intended to cover any form or modification that would fall within the scope and spirit of the present disclosure. Accordingly, the scope of the present invention is defined solely by reference to the appended claims.

[0293] The scope of the present disclosure is not intended to be limited by the specific disclosure of preferred embodiments in this section or elsewhere herein, but may be defined by the claims presented in this section or elsewhere herein or presented in the future. The claim language shall be interpreted broadly based on the language used in the claims, and not limited to the examples described in this specification or during the prosecution of the application, which examples should be interpreted as non-exclusive.

[0294] It should be understood that a feature, material, feature, or group described in conjunction with a particular aspect, embodiment, or example is applicable to any other aspect, embodiment, or example described in this section or elsewhere herein, unless incompatible with such other aspect, embodiment, or example. All of the features disclosed in this specification (including any accompanying claims, abstract, and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. This protection is not limited to the details of any of the foregoing embodiments. Protection extends to any novel, or any novel combination, of features disclosed in this specification (including any accompanying claims, abstract, and drawings), or to any novel, or any novel combination of steps of any method or process so disclosed.

[0295] Moreover, certain features described in this disclosure in the context of separate embodiments can be combined and implemented in a single embodiment. Conversely, various features described in the context of a single embodiment can be implemented in multiple embodiments separately or in any suitable subcombination. Furthermore, although features may be described above as working in a particular combination, one or more features from the claimed combination can, in some cases, be deleted from the combination, and the combination can be claimed as a subcombination or a variation of the subcombination.

[0296] Furthermore, although operations may be shown in the figures or described herein in a particular order, such operations need not be performed in the particular order shown, or sequentially, to achieve desirable results, nor need all operations be performed. Other operations not shown or described can be incorporated into the example methods and processes. For example, one or more additional operations can be performed before, after, simultaneously with, or between any of the operations. Furthermore, operations can be rearranged or reordered to form other implementations. Those skilled in the art will appreciate that in some embodiments, the actual steps taken in the illustrated and / or disclosed processes may differ from those steps shown. Depending on the embodiment, some of the steps described above may be omitted, or other steps may be added. Furthermore, features and attributes of the specific embodiments disclosed above can be combined in different ways to form additional embodiments, all of which are within the scope of the present disclosure.

[0297] For purposes of this disclosure, certain aspects, advantages, and novel features are described herein. Not necessarily all such advantages may be achieved in accordance with any particular embodiment. Thus, for example, one skilled in the art will recognize that the present disclosure can be embodied or practiced in a manner that achieves one advantage or a group of advantages taught herein, but not necessarily other advantages that may be taught or suggested herein.

Claims

1. A method for analyzing a mixture suspected to contain multiple target nucleic acids, a) A sample suspected to contain multiple different target nucleic acids is treated in a pre-amplification reaction mixture containing a low-bias amplification buffer to produce a multiplex pre-amplified mixture, The pre-amplification reaction mixture comprises at least four different target-specific primer pairs for generating amplification regions from at least four different target nucleic acids (if present in the sample), and at least one reference primer pair for generating amplification regions from a reference nucleic acid. Each of the primers in the pair of at least four different target-specific primers and the pair of at least one reference primers is at essentially the same concentration, produced by the process. b) Adding a portion of the pre-amplified multiplex mixture to the multiplex PCR assay reaction mixture, wherein the multiplex PCR assay reaction mixture is i) An additional amount of each of the at least four different target-specific primer pairs and the reference primer pair, wherein the primer in the additional amount of the at least four different target-specific primer pairs and the at least one reference primer pair is added in essentially the same amount, ii) at least four different target-specific probe oligonucleotides, each of which hybridizes specifically to one of the at least four different target nucleic acids (step a), and each of the at least four different target-specific probe oligonucleotides, comprising a first label, and iii) A reference probe flap oligonucleotide that specifically hybridizes to an amplification region from the reference nucleic acid, comprising a second label, The addition of the at least four different target-specific probe oligonucleotides and the reference probe flap oligonucleotide is present in the multiplex PCR assay reaction mixture at essentially the same concentration. Furthermore c) Performing a PCR assay using the multiplex PCR assay reaction mixture, wherein each of the reference nucleic acid region and the at least four different target regions (if amplified in step a) is amplified in the PCR assay reaction mixture, the target-specific probe oligonucleotide and the reference probe oligonucleotide specifically hybridize to the target region and the reference nucleic acid region amplified in the multiplex PCR assay reaction mixture, are cleaved to release the first and second labels, and the released first and second labels are measured. The method, including the method described above.

2. The method according to claim 1, wherein the multiplex PCR assay reaction mixture comprises a low-bias amplification buffer.

3. The method according to claim 2, wherein the multiplex PCR assay reaction mixture comprises the same low-bias amplification buffer used in the pre-amplification reaction mixture.

4. The following: (A) The first label comprises a first 5' flap sequence, and the first 5' flap sequence is not substantially complementary to any of the amplification regions from the at least four different target nucleic acids; (B) The second label comprises a second 5' flap sequence, the second 5' flap sequence being different from the first 5' flap sequence and not substantially complementary to the amplification region from the reference nucleic acid; (C) The PCR assay reaction mixture further comprises a first FRET cassette labeled with a first fluorophore and / or a second FRET cassette labeled with a second fluorophore, wherein the first FRET cassette comprises a sequence complementary to the first 5' flap sequence and the second FRET cassette comprises a sequence complementary to the second 5' flap sequence; (D) The PCR assay reaction mixture further comprises a flap endonuclease, preferably a FEN-1 endonuclease, preferably an archaeal FEN-1 endonuclease; (E) The first label comprises a first FRET system containing a first fluorophore, and the second label comprises a second FRET system containing a second fluorophore, and the fluorescence from the first and second fluorophore is measured during the PCR assay; (F) The at least four different target-specific primer pairs are at least five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, Includes 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different target-specific primer pairs; (G) The at least four different target-specific probe oligonucleotides are at least five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, Includes 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different target-specific probe oligonucleotides; (H) The concentrations of the at least four different target-specific primer pairs and the reference primer pair in the PCR assay reaction mixture are essentially the same as the concentrations of the at least four different target-specific primer pairs and the reference primer pair in the pre-amplification reaction mixture; (I) The low-bias amplification buffer is 3-(n-morpholino)propanesulfonic acid (MOPS) buffer and at least about 6 mM, preferably 6.1 mM, 6.2 mM, 6.5 mM, 6.4 mM, 6.5 mM, 6.6 mM, 6.7 mM, 6.8 mM, 6.9 mM, 7.0 mM, 7.1 mM, 7.2 mM, 7.3 mM, 7.4 mM, 7.5 mM, 7.6 mM, 7.7 mM, 7.8 mM, 7.9 mM, 8.0 mM, 8.1 mM, 8.2 mM, 8.3 mM, 8.4 mM, 8.5 mM, 8.6 mM, 8.7 mM , including Mg++ of 8.8 mM, 8.9 mM, 9.0 mM, 9.1 mM, 9.2 mM, 9.3 mM, 9.4 mM, 9.5 mM, 9.6 mM, 9.7 mM, 9.8 mM, 9.9 mM, 10.0 mM, 10.1 mM, 10.2 mM, 10.3 mM, 10.4 mM, 10.5 mM, 10.6 mM, 10.7 mM, 10.8 mM, 10.9 mM, or 11.0 mM; and (J) The low-bias amplification buffer contains approximately 7.5 mM Mg++, The method according to claim 1, further defined by at least one of the following.

5. The pre-amplification reaction mixture comprises at least one additional target-specific primer pair for generating an amplified region from an additional target nucleic acid (if present in the sample) that is different from both the at least four different target nucleic acids and the reference nucleic acid, and the multiplex PCR assay reaction mixture i) an additional amount of at least one additional target-specific primer pair, which is essentially the same amount as the additional amount of the at least four different target-specific primer pairs, and ii) at least one additional target-specific probe oligonucleotide that specifically hybridizes to the amplification region (step a) from the at least one additional target nucleic acid, the at least one additional target-specific probe oligonucleotide having a third label different from the first label and the second label, It further includes, The third label comprises a third 5' flap sequence, the third 5' flap sequence being different from the first 5' flap sequence and the second 5' flap sequence, and not substantially complementary to the amplification region from the additional target nucleic acid. The PCR assay reaction mixture further comprises a third FRET cassette labeled with a third fluorophore, the third FRET cassette comprising a sequence complementary to the third 5' flap sequence. The method according to claim 4.

6. The aforementioned at least one additional target-specific primer pair is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different additional target nucleic acids (present in the sample) 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 5 Includes 2, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different additional target-specific primer pairs, and The multiplex PCR assay reaction mixture contains two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, ten, fifteenth The additional target-specific probe oligonucleotides further comprise 3, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100, and the additional target-specific probe oligonucleotides The b-nucleotides are the aforementioned 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, The method according to claim 5, which specifically hybridizes to an amplified region (in step a) from 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different additional target nucleic acids.

7. A method for analyzing a sample for at least 10 different target nucleic acids using a single PCR-flap assay reaction, a) Processing nucleic acids containing multiple different target nucleic acids in a pre-amplification reaction mixture containing PCR-flap assay buffer to produce a multiplex pre-amplified mixture, The pre-amplification reaction mixture comprises at least 10 different target-specific primer pairs for generating amplification regions from at least 10 different target nucleic acids in the sample and at least 1 reference primer pair for generating amplification regions from a reference nucleic acid, wherein each of the primers in the at least 10 different target-specific primer pairs and the at least 1 reference primer pair is at essentially the same concentration in the pre-amplification reaction mixture, the process to be produced b) Adding a portion of the pre-amplified multiplex mixture to the multiplex PCR-flap assay reaction mixture, wherein the multiplex PCR-flap assay reaction mixture is i) An additional amount of each of the at least 10 different target-specific primer pairs and the reference primer pair, wherein the primers in the additional amount of the at least 10 different target-specific primer pairs and the at least one reference primer pair are at essentially the same concentration in the multiplex PCR-flap assay reaction mixture. ii) At least 10 different target-specific flap oligonucleotides, each of which specifically hybridizes to one of the amplification regions of the at least 10 different target nucleic acids and is present in essentially the same concentration in the multiplex PCR-flap assay reaction mixture, The flap oligonucleotides are divided into a first group and a second group, wherein each of the flap oligonucleotides in the first group contains a first 5' flap sequence, and each of the flap oligonucleotides in the second group contains a second 5' flap sequence, and the at least 10 different target-specific flap oligonucleotides are divided into a first group and a second group, iii) A reference flap oligonucleotide that specifically hybridizes to the amplification region from the reference nucleic acid, having a third 5' flap sequence, wherein the third 5' flap sequence is different from the first 5' flap sequence and the second 5' flap sequence, iv) A first FRET cassette labeled with a first fluorophore, comprising an array complementary to the first 5' flap array, v) A second FRET cassette labeled with a second fluorophore, comprising an arrangement complementary to the second 5' flap arrangement, vi) A third FRET cassette labeled with a third fluorophore, comprising an arrangement complementary to the third 5' flap arrangement, and vi) PCR-flap assay buffer, including the above addition, Furthermore c) Performing a PCR-flap assay using the multiplex PCR-flap assay reaction mixture, wherein each of the reference nucleic acid and the at least 10 different target regions is amplified in the PCR-flap assay reaction mixture, and fluorescence from the first fluorophore, the second fluorophore, and the third fluorophore is measured. The method, including the method described above.

8. A composition, a) A group of oligonucleotides, i) A first set of at least four different target-specific primer pairs for generating amplification regions from a first group of at least four different target nucleic acids, ii) At least one reference primer pair for generating an amplification region from a reference nucleic acid, Each of the primers in the at least four different target-specific primer pairs and the at least one reference primer pair is at essentially the same concentration as the at least one reference primer pair iii) A first set of at least four different target-specific flap oligonucleotides, each of which specifically hybridizes to one different amplification region from the group of at least four different target nucleic acids, A first set of at least four different target-specific flap oligonucleotides, each of which comprises a first 5' flap sequence. iv) A reference flap oligonucleotide that specifically hybridizes to the amplification region from the reference nucleic acid, having a second 5' flap sequence, The second 5' flap sequence is different from the first 5' flap sequence, the reference flap oligonucleotide, v) A first FRET cassette labeled with a first fluorophore, comprising a sequence complementary to the first 5' flap sequence and not substantially complementary to the second 5' flap sequence, and vi) A second FRET cassette labeled with a second fluorophore, comprising an array complementary to the second 5' flap array and substantially not complementary to the first 5' flap array. A group of oligonucleotides including the above, b) dNTP, and c) Low-bias amplification buffer The composition comprising the mixture.

9. x) DNA polymerase, preferably a heat-stable DNA polymerase, xi) Flap endonuclease, preferably FEN-1 endonuclease, preferably archaeal FEN-1 endonuclease, and xi) A portion of a multiplex pre-amplified mixture amplified in a low-bias amplification buffer using at least four different target-specific primer pairs and at least one reference primer pair at essentially the same concentration, the portion of the multiplex pre-amplified mixture comprising the amplification regions of a first group of at least four different target nucleic acids and the amplification region of the reference nucleic acid. The composition according to claim 8, further comprising one or more of the following.

10. The following: (A) The first set of at least four different target-specific flap oligonucleotides is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , including 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different flap oligonucleotides; (B) The first set of at least four different target-specific primer pairs is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, Includes 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different primer pairs; and (C) The composition is vii) A second set of different target-specific primer pairs for generating amplification regions from a second group of different target nucleic acids, The second set of different target-specific primer pairs, wherein each of the primers in the second set is in essentially the same amount or concentration as the primer in the first set of target-specific primer pairs. viiii) A second set of different target-specific flap oligonucleotides, each of which specifically hybridizes to one of the different amplification regions from a second group of different target nucleic acids, The second set of different target-specific flap oligonucleotides, wherein each of the flap oligonucleotides in the second set of target-specific flap oligonucleotides contains a third 5' flap sequence, and ix) A third FRET cassette labeled with a third fluorophore, comprising an array complementary to the third 5' flap array and substantially not complementary to either the first or second 5' flap array. Further including, The composition according to claim 8, further defined by at least one of the following.

11. The following: (I) The second set of the different target-specific flap oligonucleotides is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , including 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different flap oligonucleotides; (II) The second set of the different target-specific primer pairs is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 4 Including 9, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different primer pairs, The composition according to claim 8, further defined by at least one of the following.

12. a) i) A first set of at least four different target-specific primer pairs for generating amplification regions from a first group of at least four different target nucleic acids in the sample, ii) At least one pair of reference primers for generating an amplification region from a reference nucleic acid in the sample, Each of the primers in the at least four different target-specific primer pairs and the at least one reference primer pair is in essentially the same amount or concentration as the at least one reference primer pair iii) A first set of at least four different target-specific flap oligonucleotides, each of which specifically hybridizes to one different amplification region from the group of at least four different target nucleic acids, A first set of at least four different target-specific flap oligonucleotides, each of which comprises a first 5' flap sequence. iv) A reference flap oligonucleotide that specifically hybridizes to the amplification region from the reference nucleic acid, having a second 5' flap sequence, The second 5' flap sequence is a reference flap oligonucleotide that is different from the first 5' flap sequence, The four different target-specific flap oligonucleotides and the reference flap oligonucleotide are present in essentially the same amount or concentration as the reference flap oligonucleotide. v) A first FRET cassette labeled with a first fluorophore, comprising a sequence complementary to the first 5' flap sequence and not substantially complementary to the second 5' flap sequence, and vi) A second FRET cassette labeled with a second fluorophore, comprising an array complementary to the second 5' flap array and substantially not complementary to the first 5' flap array, A kit comprising a mixture containing a group of oligonucleotides.

13. b) DNA polymerase, preferably a heat-stable DNA polymerase, c) Flap endonuclease, preferably FEN-1 endonuclease, preferably archaeal FEN-1 endonuclease, d) Low-bias amplification buffer, and e) dNTP The kit according to claim 12, further comprising one or more of the following.

14. The following: (A) The first set of at least four different target-specific flap oligonucleotides is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , including 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different flap oligonucleotides; (B) The first set of at least four different target-specific primer pairs is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , including 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different primer pairs; (C) the mixture is vii) A second set of different target-specific primer pairs for generating amplification regions from a second group of different target nucleic acids in a sample, The second set of different target-specific primer pairs, wherein each of the primers in the second set is in essentially the same amount or concentration as the primer in the first set of target-specific primer pairs. viiii) A second set of different target-specific flap oligonucleotides, each of which specifically hybridizes to one of the different amplification regions from a second group of different target nucleic acids, The second set of different target-specific flap oligonucleotides, wherein each of the flap oligonucleotides in the second set of target-specific flap oligonucleotides contains a third 5' flap sequence, and ix) A third FRET cassette labeled with a third fluorophore, comprising a sequence complementary to the third 5' flap sequence and substantially not complementary to either the first or second 5' flap sequence, further comprising: The kit according to claim 12, further defined by at least one of the following.

15. (I) A second set of the different target-specific flap oligonucleotides is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, Includes 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different flap oligonucleotides; and (II) The second set of the different target-specific primer pairs is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 4 Including 9, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 different primer pairs, The kit according to claim 14.

16. f) A portion of the oligonucleotide group in the second mixture, i) A first set of at least four different target-specific primer pairs for generating amplification regions from a first group of at least four different target nucleic acids in a sample, ii) comprising the at least one reference primer pair for generating an amplification region from the reference nucleic acid in the sample, Each of the primers in the pair of at least four different target-specific primers and the pair of at least one reference primers contains essentially the same amount or concentration of the oligonucleotide group. The kit according to claim 12, further comprising:

17. The kit according to claim 13, wherein the mixture in a) is in the form of a dry or a solution.

18. The kit according to claim 16, wherein the mixture of (f) is in the form of a dry or a solution.

19. The low-bias amplification buffer is 3-(n-morpholino)propanesulfonic acid (MOPS) buffer and Mg in the PCR reaction mixture. ++ The final concentration should be at least about 6 mM, preferably 6.1 mM, 6.2 mM, 6.5 mM, 6.4 mM, 6.5 mM, 6.6 mM, 6.7 mM, 6.8 mM, 6.9 mM, 7.0 mM, 7.1 mM, 7.2 mM, 7.3 mM, 7.4 mM, 7.5 mM, 7.6 mM, 7.7 mM, 7.8 mM, 7.9 mM, 8.0 mM, 8.1 mM, 8.2 mM, 8.3 mM, 8.4 mM, 8.5 mM, 8.6 mM, 8.7 mM , 8.8 mM, 8.9 mM, 9.0 mM, 9.1 mM, 9.2 mM, 9.3 mM, 9.4 mM, 9.5 mM, 9.6 mM, 9.7 mM, 9.8 mM, 9.9 mM, 10.0 mM, 10.1 mM, 10.2 mM, 10.3 mM, 10.4 mM, 10.5 mM, 10.6 mM, 10.7 mM, 10.8 mM, 10.9 mM, or 11.0 mM of Mg ++ Mg at this concentration ++ The kit according to claim 13, including the following:

20. The low-bias amplification buffer is Mg in the PCR reaction mixture. ++ The final concentration of Mg is approximately 7.5 mM. ++ Mg at this concentration ++ The kit according to claim 19, comprising:

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