Multiplex amplification detection assay for plasma DNA and isolation and detection

Abstract

The technology provided herein relates to amplification-based detection of bisulfite treated DNA, and in particular, but not exclusively, to methods and compositions for multiplex amplification of low levels of sample DNA prior to further characterization of the sample DNA. The technology also provides methods for isolating DNA from a blood or blood product sample, such as a plasma sample.

Description

[0001] This application is a divisional application of a patent application filed on October 26, 2016, with the earliest priority date of October 30, 2015, application number 2016800616311, entitled "Multiple Amplification Detection and Separation of Plasma DNA".

[0002] Cross-references to related applications.

[0003] This application claims priority to U.S. Provisional Application Serial No. 62 / 249,097, filed on October 30, 2015, which is incorporated herein by reference. Invention Field

[0004] The techniques provided herein relate to amplification-based detection of nucleic acids, specifically (but not only) methods and compositions for multiplex amplification of low-level sample DNA prior to further characterization of sample DNA. The techniques also provide methods for isolating DNA from blood or blood product samples, such as plasma samples. background

[0005] Quantitative nucleic acid methods are important in many areas of molecular biology, particularly for molecular diagnostics. At the DNA level, such methods are used, for example, to determine 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 at specific loci between or within genes. Additionally, quantitative nucleic acid methods are used to measure the amount of mRNA as a measure of gene expression.

[0006] Among the many different analytical methods for detecting and quantifying nucleic acids or nucleic acid sequences, variants of polymerase chain reaction (PCR) have become the most powerful and widely used technique, the principles of which have been disclosed in U.S. Patents 4,683,195, 4,683,202, and 4,965,188.

[0007] Detecting nucleic acids present at low levels in samples (e.g., DNA from disease loci, such as tumor DNA, collected from samples far from disease loci, such as DNA found in feces, sputum, urine, plasma, etc., "remote DNA samples") can be challenging, partly because many of the DNAs found in such samples are not only few in number but are also often fragmented. See, for example, WO 2006 / 113770 of Ballhause and U.S. Patent Publication US201110009277 A1 of Davos, each of which is incorporated herein by reference in its entirety. Cell-free DNA (cfDNA) found in plasma, for example, can be highly fragmented, and many DNAs of potential interest, such as tumor-derived DNA, can be very small, for example, 200 nucleotides or less in length. Nucleic acids of this size can be lost during routine purification processes due to, for example, poor binding to purification columns or ineffective alcohol precipitation.

[0008] Analyzing nucleic acids from samples is particularly challenging if multiple targets or loci in the nucleic acid need to be detected. For example, collected samples with a small number of copies of the target of interest often cannot be divided into a sufficient number of aliquots to allow testing of all targets without the risk of compromising the accuracy of individual target testing (e.g., through false negative results).

[0009] DNA in a sample can be enriched by pre-amplification of target nucleic acids (e.g., genomic DNA, cDNA, etc.) in a low-target sample, and then the sample can be aliquoted for further target-specific analysis. For example, whole-genome amplification using simple primers (e.g., random hexamers) has been used to increase the amount of virtually all DNA in a sample in a non-specific manner to any particular target. (Sigma-Aldrich's GenomePlex systems, Arneson et al., Cold Spring Harb. Protoc.; 2008; doi:10.1101 / pdb.prot4920).

[0010] Another approach is to amplify one or more regions of particular interest in a semi-targeted manner to produce a mixture of amplified fragments (amplifiers) containing different mutations or loci that will be further analyzed. Amplification using consecutive rounds with the same primers is prone to producing high background nonspecific amplification, as well as artifacts such as artificial recombinant molecules, highly nonspecific background, and biased amplification of different intended targets. Therefore, such preamplification PCR is often performed under specific conditions, such as a limited number of cycles, and / or using low primer concentrations (e.g., 1 / 20 to 1 / 10 of those in standard PCR) to avoid increased nonspecific background amplification, as it has been shown that using concentrations of more than approximately 160 nM of each primer in multiplex preamplification increases the amplification background in negative control reactions (see, for example, Andersson et al., Expert Rev. Mol. Diagn. Earlyonline, 1–16 (2015)).

[0011] After the first round of amplification in multiplex PCR, the pre-amplified DNA is typically diluted and aliquoted into new amplification reactions for quantitative or qualitative PCR analysis, which uses standard PCR conditions such as higher reagent concentrations and a greater number of cycles, and typically uses different primer pairs (e.g., "nested" primers that anneal to sites within the pre-amplified fragment rather than to the original primer sites at the ends of the amplicons) for a second amplification.

[0012] When examining DNA methylation, the analysis is further complicated by the fact that common methods used to prepare samples for methylation detection often result in significant loss of sample DNA. For example, bisulfite treatment is commonly used to convert unmethylated cytosine residues to uracil residues, but this method typically yields only about 30% recovery of the input DNA. Furthermore, amplification of DNA after bisulfite treatment is particularly challenging. For instance, the conversion of unmethylated cytosine reduces the complexity of the DNA sequence, and the treatment itself is known to cause significant DNA damage, such as strand breaks, both of which can contribute to background enhancement in amplification reactions, especially in multiplex amplification.

[0013] Overview

[0014] In the development of the method described herein, it has been determined that bisulfite-treated DNA from low-target samples can be pre-amplified and amplified for real-time detection without whole-genome pre-amplification or the use of nested or semi-nested primers. Surprisingly, targeted pre-amplification can be multiplexed, for example, in the Quantitative Allele-Specific Real-Time Target and Signal Amplification (QuARTS) assay (see, for example, U.S. Patents 8,361,720, 8,715,937, and 8,916,344), using a combination of the same primer pairs used for a second round of amplification of a single target locus, which combines PCR targeted amplification with FEN-1-mediated flap cleavage for signal amplification.

[0015] In some implementations, this technology provides a method for analyzing samples that allows for the individual detection of multiple distinct targets present at low copy numbers, reducing the risk of false negative results due to sample aliquoting. For example, in some implementations, this technology provides a method for analyzing multiple target nucleic acids in a sample, including:

[0016] a) Provide a sample of volume x, the sample containing bisulfite-treated DNA suspected of containing one or more of a plurality of n distinct target regions, wherein at least one of the target regions is a low-copy target, and if present in the sample, it is present in the sample at a copy number such that:

[0017] i) Each of the n portions of the sample has a volume of x / n, and one or more of the n portions do not contain the low-copy target, or

[0018] ii) Each of the n portions of the sample has a volume of x / n, and the low-copy target in one or more of the n portions is below the sensitivity level for the detection of the low-copy target;

[0019] b) Under conditions in which the n different target regions (if present in the sample) are amplified to form a pre-amplified mixture having a volume y, the volume x of the sample is treated to the amplification reaction;

[0020] c) Dispense the pre-amplified mixture into multiple different assay reaction mixtures, wherein each assay reaction mixture contains a portion of the pre-amplified mixture, the pre-amplified mixture having a volume of y / n or less, and wherein the low-copy target, if present in the sample in step a), is present in each of the assay reaction mixtures; and

[0021] d) Perform multiple detections using the detection reaction mixture, wherein the different target regions, if present in the sample in step a), are detected in the detection reaction mixture.

[0022] In some embodiments, the bisulfite-treated DNA is derived from a human subject. In some preferred embodiments, the sample is prepared from the subject's bodily fluids (preferably containing plasma). In some embodiments, the bisulfite-treated DNA is circulating cell-free DNA (cfDNA) isolated from plasma, such as cell-free DNA less than 200 base pairs in length. In a particularly preferred embodiment, cell-free DNA is isolated from plasma by a method comprising combining a plasma sample with a protease (e.g., streptokinase, proteinase K) and a first lysis reagent comprising guanidine isothiocyanate and a nonionic detergent to form a mixture (where proteins are digested by the protease), and then, under conditions in which DNA is bound to silica particles, adding silica particles and a second lysis reagent comprising a mixture of guanidine thiocyanate, a nonionic detergent, and isopropanol. In some embodiments, the nonionic detergents in the first lysis reagent and the second lysis reagent may be the same or different, and are selected from, for example, polyethylene glycol sorbitan monolaurate (Tween-20), octylphenoxy polyethoxyethanol (Nonidet P-40), and branched octylphenoxy poly(ethoxy)ethanol (IGEPAL CA-630).

[0023] The method further includes separating silica particles with bound DNA from the mixture, washing the separated silica particles with bound DNA with a first wash solution containing guanidine hydrochloride or guanidine thiocyanate and ethanol, separating the silica particles with bound DNA from the first wash solution, and washing the silica particles with bound DNA with a second wash solution containing a buffer (e.g., Tris pH 8.0 and ethanol). In a preferred embodiment, the silica particles with bound DNA are washed multiple times with the second wash buffer, for example, 2-6 times. In a particularly preferred embodiment, a smaller volume of the second wash buffer is used for each wash than in previous washes with the same buffer. In some embodiments, the washed silica particles are separated from the final wash buffer treatment, and DNA is eluted from the silica particles, for example, with an elution buffer such as 10 mM Tris-HCl pH 8.0 and 0.1 mM EDTA. In a preferred embodiment, the silica particles with bound DNA are dried, for example, by heating to about 70°C, before eluting the DNA.

[0024] This technique is not limited to any particular sample size, but it is particularly suitable for samples with low copy numbers of the target in large samples. For example, in some embodiments, bisulfite-treated DNA is prepared from a bodily fluid, such as plasma, sample with an initial volume of at least 1 mL, preferably at least 5 mL, more preferably at least 10 mL, and / or the volume x of said bisulfite-treated DNA sample is at least 10 μl, preferably at least 25 μl, more preferably at least 50 μl, more preferably at least 100 μl. In a preferred embodiment, the volume of the treated DNA sample present in the pre-amplification reaction is at least 5%, preferably at least 10%-60%, preferably 15%-55%, more preferably about 20%-50% of the total volume of the amplification reaction.

[0025] The present invention is not limited to the specific number of fractions into which the sample is divided. In some embodiments, n (number of fractions) is at least 3, preferably at least 5, more preferably at least 10, more preferably at least 20, and even more preferably at least 100.

[0026] In some implementations, this technology provides a method for analyzing multiple target nucleic acids in a sample using PCR pre-amplification and PCR-valve assay, the method comprising:

[0027] a) A first reaction mixture containing PCR amplification reagents is provided with bisulfite-treated DNA (in a preferred embodiment, human DNA) comprising a plurality of different target regions, wherein the PCR amplification reagents comprise:

[0028] i) Multiple different primer pairs for amplifying the multiple different target regions (if present in the sample) from the bisulfite-treated DNA;

[0029] ii) Thermostable DNA polymerase;

[0030] iii) dNTPs; and

[0031] iv) Contains Mg ++ buffer solution

[0032] b) Exposing the first reaction mixture to thermal cycling conditions, wherein multiple different target regions (if present in the sample) are amplified to produce a pre-amplified mixture, and wherein the thermal cycling conditions are limited to multiple thermal cycles that maintain the amplification in an exponential range, preferably fewer than 20, more preferably fewer than 15, and even more preferably 10 or fewer.

[0033] c) Dispensing the pre-amplified mixture into multiple PCR-lobe assay reaction mixtures, wherein each PCR-lobe assay reaction mixture contains:

[0034] i) An additional amount of primer pairs selected from the plurality of different primer pairs described in step a)i);

[0035] ii) Thermostable DNA polymerase;

[0036] iii) dNTPs;

[0037] iv) Contains Mg ++ The buffer solution

[0038] v) Valvular endonuclease, preferably FEN-1 endonuclease;

[0039] vi) Valeroid oligonucleotides, and

[0040] vi) A hairpin oligonucleotide comprising a region complementary to a portion of the valve oligonucleotide, preferably a FRET box oligonucleotide;

[0041] and

[0042] d) Detect the amplification of one or more different target regions from the bisulfite-treated DNA during the PCR-valve assay by detecting the cleavage of the hairpin oligonucleotide by the valve endonuclease.

[0043] In a preferred embodiment, the FEN-1 endonuclease is thermostable FEN-1, preferably derived from archaea, such as Afu FEN-1.

[0044] In some embodiments, the pre-amplified mixture described above is diluted with a diluent before being dispensed into the PCR-valve assay reaction mixture, while in other embodiments, the pre-amplified mixture is added directly to the undiluted PCR-valve assay reaction mixture.

[0045] In some embodiments, the primers used in the PCR-valve assay reaction are used at essentially the same concentration as those used when the specific primers were used in the first reaction mixture, excluding any primers left over from the first reaction. For example, in some embodiments, the additional amount of primers in the primer pair added to the PCR-valve assay reaction mixture is added to a concentration such that the concentration of the primers added in the PCR-valve assay (i.e., primers not counted from the pre-amplified mixture) is substantially the same as the concentration of the primers in that primer pair in the PCR amplification reagent. In other embodiments, the additional amount of primers in the primer pair added to the PCR-valve assay reaction mixture is added to a concentration such that the concentration of the primers added in the PCR-valve assay is lower or higher than the concentration of the primers in that primer pair in the first reaction mixture.

[0046] Although this method is not limited to the Mg in the first reaction mixture and the buffer used in the PCR-valve assay reaction mixture. ++ The specific concentration, but in a preferred embodiment, the buffer contains at least 3 mM Mg ++ Preferably, Mg > 4 mM ++ More preferably, greater than 5 mM Mg ++ More preferably, greater than 6 mM Mg ++ More preferably about 7 mM to 7.5 mM Mg ++ In some embodiments, the buffer contains less than about 1 mM of KCl. In a preferred embodiment, the buffer comprises 10 mM 3-(n-morpholino)propanesulfonic acid (MOPS) buffer and 7.5 mM MgCl2.

[0047] In some embodiments, the first reaction mixture and / or the plurality of PCR-valve assay reaction mixtures contain exogenous non-target DNA, preferably native fish DNA.

[0048] In some embodiments, the thermostable DNA polymerase is a bacterial DNA polymerase, preferably from the genus *Thermus*, more preferably from *Thermus aquaticus*. In some embodiments, the DNA polymerase is modified for hot-start PCR, for example, by using reagents (e.g., antibodies, chemical adducts, etc.) to activate the DNA polymerase upon heating.

[0049] In some embodiments, the bisulfite-treated DNA contains human DNA, and multiple distinct target regions comprise target regions selected from the group consisting of SFMBT2, VAV3, BMP3, and NDRG4. In some embodiments, multiple distinct primer pairs target at least two, preferably at least three, and more preferably all four target regions.

[0050] In some embodiments, multiple different target regions include a reference target region, and in some preferred embodiments, the reference target region includes β-actin and / or ZDHHC1 and / or B3GALT6.

[0051] In some embodiments, at least one of a plurality of different primer pairs is selected to generate an amplicon less than about 100 base pairs in length, preferably less than about 85 base pairs, from the target region. In some preferred embodiments, all different primer pairs are selected to generate an amplicon less than about 100 base pairs in length from the target region.

[0052] In some embodiments, the methods provided herein involve amplifying substantially all of the bisulfite-treated DNA generated during the processing of a sample, such as a bodily fluid sample. In some embodiments, the preparation of the bisulfite-treated DNA constitutes a substantial part of the first reaction mixture; for example, in some embodiments, the volume of the sample containing the bisulfite-treated DNA in the first reaction mixture constitutes at least 20-50% of the total volume of the first reaction mixture. For example, in some embodiments, the volume of the bisulfite-treated DNA in the first reaction mixture is at least 5% of the total volume of the first reaction mixture, preferably at least 10%-60%, preferably 15%-55%, more preferably about 20%-50%.

[0053] In some embodiments, the method of this technology provides a method for analyzing multiple target nucleic acids in human plasma samples using PCR pre-amplification and PCR-valve assay, the method comprising:

[0054] a) Provided in a first reaction mixture containing PCR amplification reagents, bisulfite-treated DNA prepared from at least 1 mL of plasma, the bisulfite-treated DNA containing multiple distinct target regions, wherein the PCR amplification reagents comprise:

[0055] i) Multiple different primer pairs for amplifying the multiple different target regions selected from SFMBT2, VAV3, BMP3 and NDRG4, if present in the sample, from the bisulfite-treated DNA, wherein each of the multiple different primer pairs is selected to generate an amplicon of less than about 100 base pairs in length from the target region.

[0056] ii) DNA polymerase from aquatic thermophilic bacteria;

[0057] iii) dNTPs; and

[0058] iv) Contains 7.5 mM Mg ++ buffer solution

[0059] b) Exposing the first reaction mixture to thermal cycling conditions, wherein multiple different target regions (if present in the sample) are amplified to produce a pre-amplified mixture, and wherein the thermal cycling conditions are limited to multiple thermal cycles that maintain the amplification in an exponential range, preferably fewer than 20, more preferably fewer than 15, and even more preferably 10 or fewer.

[0060] c) Dispensing the pre-amplified mixture into multiple PCR-lobe assay reaction mixtures, wherein each PCR-lobe assay reaction mixture contains:

[0061] i) An additional amount of primer pairs selected from the plurality of different primer pairs described in step a)i);

[0062] ii) DNA polymerase from aquatic thermophilic bacteria;

[0063] iii) dNTPs;

[0064] iv) Contains 7.5 mM Mg ++ buffer solution

[0065] v) Thermally stable FEN-1 valve endonuclease;

[0066] vi) Valeroid oligonucleotides, and

[0067] vi) FRET box oligonucleotides containing a region complementary to a portion of the valve oligonucleotide;

[0068] and

[0069] d) Detect amplification of one or more different target regions selected from SMBT2, VAV3, BMP3 and NDRG4 during PCR-valve assay reaction.

[0070] In a preferred embodiment, multiple distinct target regions include a reference target region, preferably comprising β-actin and / or ZDHHC1. In some embodiments, from Figures 5A-5F Choose one or more target regions and / or primer pairs from the target regions and primer pairs depicted in the diagram.

[0071] This document also provides improved methods for isolating DNA (e.g., cell-free DNA) from blood or blood fractions (e.g., plasma or serum). For example, embodiments provide a method for processing plasma samples, the method comprising combining the plasma sample with a protease and a first lysis reagent comprising guanidine thiocyanate and a nonionic detergent to form a mixture in which proteins are digested by the protease, and then, under conditions in which DNA is bound to silica particles, adding miscible silica particles and a second lysis reagent comprising a mixture of guanidine thiocyanate, a nonionic detergent, and isopropanol. In some embodiments, the nonionic detergents in the first and second lysis reagents may be the same or different and are selected, for example, from polyethylene glycol sorbitan monolaurate (Tween-20), octylphenoxypolyethoxyethanol (Nonidet P-40), and branched octylphenoxypoly(ethyleneoxy)ethanol (IGEPAL CA-630). In some preferred embodiments, the silica particles are magnetic particles.

[0072] The method further includes separating silica particles with bound DNA from the mixture, washing the separated silica particles with bound DNA with a first wash solution containing guanidine hydrochloride or guanidine thiocyanate and ethanol, separating the silica particles with bound DNA from the first wash solution, and washing the silica particles with bound DNA with a second wash solution containing a buffer (e.g., Tris pH 8.0) and ethanol. In a preferred embodiment, the silica particles with bound DNA are washed multiple times with the second wash buffer, for example, 2-6 times. In a particularly preferred embodiment, a smaller volume of the second wash buffer is used for each wash than the volume used in previous washes with the buffer. In some embodiments, the washed silica particles are separated from the final wash buffer treatment, and the DNA is eluted from the silica particles, for example, with water or with an elution buffer such as 10 mM Tris-HCl pH 8.0, 0.1 mM EDTA. In a preferred embodiment, for example, the silica particles with bound DNA are dried after a final washing step by heating (to, for example, 37°C to 75°C, preferably about 45°C to 70°C, more preferably about 70°C), and then the DNA is eluted.

[0073] During the development of this technology, it was discovered that using two different lysis reagents added at different times during the process improved the yield of DNA from plasma. In a preferred embodiment, an aliquot of a mixture containing a first lysis reagent and a protease, incubated at room temperature to 55°C for, for example, about 5 to 60 minutes, preferably 30 to 60 minutes, is added after incubation. In a preferred embodiment, the mixture is incubated at room temperature. In some embodiments, the first lysis reagent comprises guanidine thiocyanate and a nonionic detergent, and the second lysis reagent comprises guanidine thiocyanate, a nonionic detergent, and an alcohol. In a preferred embodiment, the first lysis reagent comprises about 4.3 M guanidine thiocyanate and 10% w:v IGEPAL CA-630, and in some embodiments, the second lysis reagent comprises 4.3 M guanidine thiocyanate and 10% w:v IGEPAL CA-630 in combination with isopropanol.

[0074] During the development of this technology, it was discovered that using two different wash solutions at different steps of the process improved the yield of DNA from plasma. In some embodiments, the first wash solution used as described above comprises guanidine hydrochloride or guanidine thiocyanate and ethanol, and the second wash solution comprises a buffer and ethanol. In a particularly preferred embodiment, the first wash solution comprises about 3 M guanidine hydrochloride or guanidine thiocyanate and about 57% ethanol, and the second wash solution used as described above comprises about 80% ethanol and about 20% 10 mM Tris pH 8.0 buffer.

[0075] In a particularly preferred embodiment, all pyrolysis and washing steps are performed at room temperature.

[0076] In some embodiments, the plasma sample is mixed with a DNA treatment control (e.g., DNA that is not cross-reactive with an assay configured to detect DNA found in the plasma sample). For example, in some embodiments, the plasma is human plasma, and the DNA treatment control contains a zebrafish RASSF1 sequence. In a preferred embodiment, the DNA treatment control is synthetic DNA, such as a synthetic DNA fragment containing a zebrafish RASSF1 sequence. In a particularly preferred embodiment, the DNA treatment control is double-stranded. In a preferred embodiment, the processing / treatment control is added to the plasma sample prior to DNA extraction from the sample, for example, along with a first or second lysis reagent.

[0077] In some embodiments, exogenous DNA (e.g., DNA that does not cross-react with assays configured to detect DNA found in plasma samples) is added to the plasma sample. For example, in a preferred embodiment, the plasma is human plasma, and exogenous fish DNA, such as genomic DNA from salmon, is added to the sample.

[0078] The embodiments of the methods provided herein are particularly suitable for processing relatively large plasma samples (e.g., greater than 1 mL). In preferred embodiments, the plasma sample has a volume of at least 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or at least 10 mL, or any fractional volume therebetween. In some embodiments, the volume of the plasma sample is greater than 10 mL.

[0079] In some embodiments, the method further includes analyzing specific target nucleic acids in the isolated DNA sample. In a preferred embodiment, the method includes analyzing multiple methylated target nucleic acids in the isolated DNA, the method comprising treating the isolated DNA sample with bisulfite to produce a bisulfite-treated DNA sample, and treating the bisulfite-treated DNA sample under conditions in which multiple different target regions (e.g., 2, 3, 4, 5, etc. target regions) (if present in the sample) are amplified to form an amplified mixture.

[0080] In some preferred embodiments, the method further includes dispensing the amplified mixture into a plurality of different detection reaction mixtures and performing a plurality of different detection assays with the detection reaction mixtures, wherein a plurality of different target regions, if present in the sample, are detected in one or more of the plurality of different detection reaction mixtures. In a preferred embodiment, the plurality of different target regions includes at least five different target regions.

[0081] This document provides kits and systems for performing the methods described herein. In some embodiments, the technology provides a kit for isolating DNA from plasma, the kit comprising, for example:

[0082] a) A first pyrolysis reagent containing guanidine thiocyanate and a nonionic detergent or a component used to prepare the first pyrolysis reagent;

[0083] b) A second cleavage reagent comprising guanidine thiocyanate, a nonionic detergent, and isopropanol, or a component used to prepare the second cleavage reagent;

[0084] c) A first washing solution containing guanidine hydrochloride or guanidine thiocyanate and ethanol or components used to prepare the first washing solution;

[0085] d) A second wash solution containing Tris buffer and ethanol or components used to prepare the second wash solution; and

[0086] e) Silica particles.

[0087] In a preferred embodiment, the nonionic detergent comprises IGEPEAL CA-630. In some embodiments, the silica particles are magnetic particles, and in a particularly preferred embodiment, the kit comprises a magnet, for example, for separating particles during the steps of the method. In some embodiments, the kit also comprises an elution buffer or components for preparing the elution buffer.

[0088] In some embodiments, the kit also includes a DNA treatment control, such as a DNA treatment control containing the zebrafish RASSF1 sequence. In some embodiments, the kit also includes a preparation of the native fish DNA, and in a particularly preferred embodiment, the DNA treatment control is used for the preparation of the native fish DNA.

[0089] In some implementations, the technology provides a system for processing plasma samples, the system comprising:

[0090] a) A first pyrolysis reagent containing guanidine thiocyanate and a nonionic detergent or a component used to prepare the first pyrolysis reagent;

[0091] b) A second cleavage reagent comprising guanidine thiocyanate, a nonionic detergent, and isopropanol, or a component used to prepare the second cleavage reagent;

[0092] c) A first washing solution containing guanidine hydrochloride or guanidine thiocyanate and ethanol or components used to prepare the first washing solution;

[0093] d) A second wash solution containing Tris buffer and ethanol or components used to prepare the second wash solution; and

[0094] e) Silica particles.

[0095] In a preferred embodiment, the nonionic detergent comprises IGEPEAL CA-630.

[0096] In some implementations, the system also includes an elution buffer or components for preparing the elution buffer.

[0097] In some embodiments, the system also includes a DNA treatment control, such as a DNA treatment control containing the zebrafish RASSF1 sequence. In some embodiments, the system also includes a preparation of the native fish DNA; in a particularly preferred embodiment, the DNA treatment control is used for the preparation of the native fish DNA.

[0098] In some embodiments, the system further includes one or more of the following: a magnet, a container for processing plasma, and / or a container or plate for receiving purified DNA. In some embodiments, the system includes means for performing all or part of the steps, such as means such as the STARlet automation platform.

[0099] In some embodiments, the system also includes reagents for analyzing DNA separated from plasma. For example, in some embodiments, the system includes reagents for treating DNA with bisulfite to produce bisulfite-treated DNA, such as bisulfite reagents, desulfonate reagents, and materials for purifying the bisulfite-treated DNA (e.g., silica beads, binding buffers, solutions containing bovine serum albumin and / or casein, such as those described in U.S. Patent No. 9,315,853 (incorporated herein by reference)).

[0100] In a preferred embodiment, the system further includes DNA analysis reagents, such as PCR amplification reagents and / or flap assay reagents. In a particularly preferred embodiment, the system includes PCR amplification reagents comprising:

[0101] i) Multiple different primer pairs for amplifying multiple different target regions (if present in the plasma);

[0102] ii) Thermostable DNA polymerase;

[0103] iii) dNTPs; and

[0104] iv) Contains Mg ++ buffer solution

[0105] In some embodiments, the system further includes a PCR-valve assay reagent. In some preferred embodiments, the PCR-valve assay reagent comprises:

[0106] i) Multiple different primer pairs for amplifying multiple different target regions (if present in the plasma);

[0107] ii) Thermostable DNA polymerase;

[0108] iii) dNTPs;

[0109] iv) Contains Mg ++ buffer solution

[0110] v) Valvular endonuclease;

[0111] vi) Valeroid oligonucleotides, and

[0112] vi) A hairpin oligonucleotide comprising a region complementary to a portion of the valve oligonucleotide.

[0113] In another embodiment, the system includes a thermal cycler for performing PCR amplification and / or PCR flap assay reactions. In a preferred embodiment, the thermal cycler is configured to detect signals, such as fluorescence, during the amplification reaction process using assay reagents.

[0114] Based on the teachings contained herein, alternative implementation methods will be apparent to those skilled in the art. Brief description of the attached diagram

[0115] These and other features, aspects, and advantages of this technology will become better understood with reference to the following figures:

[0116] Figure 1 A schematic diagram of a combined PCR invasive cutting assay (“PCR-valve assay”) such as the Quarts assay is provided.

[0117] Figure 2 This diagram illustrates nested PCR in conjunction with a PCR-valve assay, showing a first amplification (or pre-amplification) using outer primers, followed by a PCR-valve assay using a second pair of primers with binding sites within the outer primer sites. The smaller amplicons produced in the second amplification are shown at the bottom. The FRET cassette portion of the reaction is not shown.

[0118] Figure 3 A schematic diagram is provided showing PCR pre-amplification followed by PCR-lobe assay, where the pre-amplification and PCR-lobe assay use the same primer pairs and produce copies of the same amplicons. The FRET cassette portion of the reaction is not shown.

[0119] Figure 4A schematic diagram of multiplex pre-amplification is provided, in which multiple different target regions in a sample are amplified in a single multiplex PCR reaction containing primer pairs for each target region, followed by a single PCR-lobe assay reaction, wherein each PCR-lobe assay uses only primer pairs that are specific to the target loci to be detected in the final PCR-lobe assay reaction.

[0120] Figures 5A-5F This diagram displays the combined analytical assay of methylated nucleic acid sequences used for detection using bisulfite transformation, pre-amplification, and PCR-valve assay. Each figure shows one strand of the DNA target region before bisulfite treatment and the expected sequence of that region upon transformation with bisulfite reagent; unmethylated C residues after transformation are shown as 'T'. Primer binding sites for the outer primer and the inner primer for the PCR-valve assay (this can be used for nested assay designs) are boxed. Each figure also shows the alignment of the PCR-valve assay primers with the valve probes on fragments of the transformed sequence. Figures 5A-5F The target regions of the markers SFMBT2, VAV3, BMP3, NDRG4, and the reference DNA β-actin and ZDHHC1 are shown. The 'arms' on the valve oligonucleotides used for PCR-valve assay are as follows: arm 1 is 5'-CGCCGAGG-3'; arm 3 is 5'-GACGCGGAG-3'; arm 5 is 5'-CCACGGACG-3'.

[0121] Figure 6 The table shows a comparison of the detection of specified bisulfite-treated target DNA using different numbers of pre-amplification cycles with outer primers, followed by PCR-valve assay amplification and detection using nested (inner) primers. Comparison assays were performed directly on bisulfite-treated DNA using the Quarts PCR-valve assay without pre-amplification.

[0122] Figure 7 Comparison of usage, such as Figures 5A-5F The results of nested or non-nested amplification primer configurations are shown, and different primer concentrations and different buffers in the PCR pre-amplification step are compared, as described in Example 3.

[0123] Figures 8A-8C The results show the effects of using different numbers of cycles during the pre-amplification phase of the assay. Figure 8A This displays the expected number of strands for each target type in normal plasma samples or plasma samples incorporating known amounts of target DNA, without pre-amplification or using 5, 10, 20, or 25 cycles of amplification. Figure 8B The detection of the number of chains in each reaction was compared under the displayed conditions, as described in Example 4.

[0124] Figure 9The results of using non-nested multiplex pre-amplification on DNA isolated from feces are shown, as described in Example 5.

[0125] Figure 10A-10I The results show the effects of using non-nested multiplex pre-amplification on DNA isolated from plasma, as described in Example 6.

[0126] Figure 11A-11C The graph shows a comparison of the yields of β-actin DNA (untreated and bisulfite-converted after extraction) and the B3GALT6 gene (bisulfite-converted after extraction) under different plasma separation conditions, as described in Example 8.

[0127] Figure 12A-1 2-C shows a graph comparing the yields of β-actin DNA (untreated and bisulfite-converted after extraction) and the B3GALT6 gene (bisulfite-converted after extraction) under different plasma separation conditions, as described in Example 10.

[0128] Figure 13 shows a table of nucleic acid sequences associated with the implementation scheme described herein.

[0129] It should be understood that the accompanying drawings are not necessarily drawn to scale, nor are the objects in the drawings necessarily drawn to scale relative to each other. The drawings are descriptions intended to make clear and understandable various embodiments of the instruments, systems, and methods disclosed herein. Wherever possible, the same reference numerals will be used throughout the drawings to refer to the same or similar parts. Furthermore, it should be understood that the drawings are not intended to limit the scope of this teaching in any way.

[0130] The present invention includes the following embodiments:

[0131] 1. A method for analyzing a sample containing multiple target nucleic acids, the method comprising:

[0132] a) Provide a sample of volume x, the sample containing bisulfite-treated DNA suspected of containing one or more of a plurality of n distinct target regions, wherein at least one of the target regions is a low-copy target, and if present in the sample, it is present in the sample at a copy number such that:

[0133] i) Each of the n portions of the sample has a volume of x / n, and one or more of the n portions do not contain the low-copy target, or

[0134] ii) Each of the n portions of the sample has a volume of x / n, and the low-copy target in one or more of the n portions is below the sensitivity level for the detection of the low-copy target;

[0135] b) wherein, under the condition that the target regions are amplified to form a pre-amplified mixture having a volume y when the n different target regions are present in the sample, the volume x of the sample is treated to the amplification reaction;

[0136] c) Dispense the pre-amplified mixture into a plurality of different detection reaction mixtures, wherein each detection reaction mixture contains a portion of the pre-amplified mixture having a volume of y / n or less, and wherein the low-copy target, if present in the sample in step a), is present in each of the detection reaction mixtures;

[0137] d) Perform multiple detections using the detection reaction mixture, wherein the different target regions, if present in the sample in step a), are detected in the detection reaction mixture.

[0138] 2. The method according to embodiment 1, wherein the bisulfite-treated DNA is derived from a human subject.

[0139] 3. The method according to embodiment 1 or 2, wherein the sample is prepared from body fluid.

[0140] 4. The method according to embodiment 3, wherein the body fluid includes plasma.

[0141] 5. The method according to embodiment 4, wherein the sample is prepared from cell-free DNA isolated from plasma.

[0142] 6. The method according to embodiment 5, wherein the length of the cell-free DNA is less than 200 base pairs.

[0143] 7. The method according to embodiment 5, wherein the cell-free DNA is isolated from the plasma by a method comprising:

[0144] a) Combine the plasma sample with the following reagents:

[0145] i) Proteases; and

[0146] ii) A first lysis reagent, the first lysis reagent comprising

[0147] - Guanidine thiocyanate; and

[0148] - Nonionic detergents;

[0149] To form a mixture in which the proteins are digested by the protease;

[0150] b) Adding to the mixture of step a) under conditions in which DNA is bound to the silica particles.

[0151] iii) Silica particles, and

[0152] iv) A second lysis reagent, comprising:

[0153] - Guanidine thiocyanate;

[0154] - Nonionic detergents; and

[0155] - Isopropanol;

[0156] c) Separate silica particles with bound DNA from the mixture in b);

[0157] d) Add the first washing solution to the isolated silica particles containing bound DNA, the first washing solution comprising: i) guanidine hydrochloride or guanidine thiocyanate, and ii) ethanol;

[0158] e) Separate silica particles with bound DNA from the first washing solution;

[0159] f) Add the second washing solution, which contains a buffer and ethanol, to the isolated silica particles containing bound DNA;

[0160] g) Separate the washed silica particles containing bound DNA from the second washing solution; and

[0161] h) Elute DNA from the washed silica particles containing bound DNA.

[0162] 8. The method according to embodiment 7, wherein the protease is a proteinase K protease.

[0163] 9. The method according to any one of embodiments 3-8, wherein the sample is prepared from at least 1 mL, preferably at least 5 mL, more preferably at least 10 mL of body fluid, and / or wherein the volume x of the sample is at least 10 μl, preferably at least 25 μl, more preferably at least 50 μl, more preferably at least 100 μl, and wherein the volume of the sample in the amplification reaction of step b is at least 20 to 50% of the total volume of the amplification reaction.

[0164] 10. The method according to any one of embodiments 1-9, wherein n is at least 3, preferably at least 5, more preferably at least 10, and even more preferably at least 20.

[0165] 11. A method for analyzing multiple target nucleic acids in a sample using PCR pre-amplification and PCR-valve assay, the method comprising:

[0166] a) Bisulfite-treated DNA comprising multiple different target regions is provided in a first reaction mixture containing PCR amplification reagents, wherein the PCR amplification reagents comprise:

[0167] i) Multiple different primer pairs for amplifying the multiple different target regions from the bisulfite-treated DNA when the target regions are present in the sample;

[0168] ii) Thermostable DNA polymerase;

[0169] iii) dNTPs; and

[0170] iv) Contains Mg ++ buffer solution

[0171] b) Exposing the first reaction mixture to thermal cycling conditions in which multiple different target regions, if present in the sample, are amplified to produce a pre-amplified mixture, and wherein the thermal cycling conditions are limited to multiple thermal cycles that maintain the amplification in an exponential range, preferably fewer than 20, more preferably fewer than 15, and even more preferably 10 or fewer.

[0172] c) Dispensing the pre-amplified mixture into multiple PCR-lobe assay reaction mixtures, wherein each PCR-lobe assay reaction mixture contains:

[0173] i) An additional amount of primer pairs selected from the plurality of different primer pairs described in step a)i);

[0174] ii) Thermostable DNA polymerase;

[0175] iii) dNTPs;

[0176] iv) The one containing Mg ++ The buffer solution

[0177] v) Valvular endonuclease;

[0178] vi) Valeroid oligonucleotides, and

[0179] vi) A hairpin oligonucleotide comprising a region complementary to a portion of the valve oligonucleotide;

[0180] and

[0181] d) Detect amplification of one or more different target regions from the bisulfite-treated DNA during the PCR-valve assay reaction.

[0182] 12. The method according to any one of embodiments 1-11, wherein the pre-amplified mixture is diluted with a diluent prior to the dispensing.

[0183] 13. The method according to embodiment 11 or embodiment 12, wherein the primers in the additional amount of primer pair added to the PCR-valve assay reaction mixture are added to a concentration such that the concentration of the primers added in the PCR-valve assay is substantially the same as the primer concentration of the primer pair in the PCR amplification reagent.

[0184] 14. The method according to any one of embodiments 11-13, wherein the hairpin oligonucleotide comprises a fluorophore portion.

[0185] 15. The method according to embodiment 14, wherein the hairpin oligonucleotide is a FRET box oligonucleotide.

[0186] 16. The method according to any one of embodiments 11-15, wherein the Mg-containing ++ The buffer solution contains approximately 6 to 10 mM Mg ++ .

[0187] 17. The method according to embodiment 16, wherein the method comprises Mg ++ The buffer solution contains approximately 7.5 mM Mg. ++ .

[0188] 18. The method according to any one of embodiments 11-17, wherein the Mg-containing ++ The buffer solution contains less than 1 mM of KCl.

[0189] 19. The method according to any one of embodiments 11 to 18, wherein the Mg-containing ++ The buffer contains 10 mM 3-(n-morpholino)propanesulfonic acid (MOPS) buffer and 7.5 mM MgCl2.

[0190] 20. The method according to any one of embodiments 11-19, wherein the first reaction mixture and / or the plurality of PCR-valve assay reaction mixtures contain exogenous non-target DNA.

[0191] 21. The method according to embodiment 20, wherein the exogenous non-target DNA is native fish DNA.

[0192] 22. The method according to any one of embodiments 11-21, wherein the thermostable DNA polymerase is derived from aquatic thermophilic bacteria.

[0193] 23. The method according to any one of embodiments 11-22, wherein the thermostable DNA polymerase is modified for hot-start PCR.

[0194] 24. The method according to any one of embodiments 11-23, wherein the valve endonuclease is a FEN-1 endonuclease.

[0195] 25. The method according to embodiment 24, wherein the FEN-1 endonuclease is derived from an archaea.

[0196] 26. The method according to any one of embodiments 1-25, wherein the bisulfite-treated DNA comprises human DNA.

[0197] 27. The method according to embodiment 26, wherein the plurality of different target regions comprise target regions selected from the group consisting of SFMBT2, VAV3, BMP3 and NDRG4.

[0198] 28. The method according to embodiment 26, wherein the plurality of different target regions includes a reference target region.

[0199] 29. The method according to embodiment 28, wherein the reference target region comprises β-actin and / or ZDHHC1 and / or B3GALT6.

[0200] 30. The method according to any one of embodiments 11-29, wherein at least one of the plurality of different primer pairs is selected to generate an amplicon from a target region of less than about 100 base pairs in length.

[0201] 31. The method according to embodiment 30, wherein each of the plurality of different primer pairs is selected to generate an amplicon from a target region of less than about 100 base pairs, preferably less than about 85 base pairs.

[0202] 32. The method according to any one of embodiments 1-31, wherein the volume of the sample containing bisulfite-treated DNA in the pre-amplified mixture or the first reaction mixture is 20-50% of the total volume of the pre-amplified mixture or the first reaction mixture.

[0203] 33. A method for processing a plasma sample, the method comprising:

[0204] a) Combine the plasma sample with the following reagents:

[0205] i) Protease;

[0206] ii) A first lysis reagent, the first lysis reagent comprising

[0207] - Guanidine thiocyanate,

[0208] - Nonionic detergents;

[0209] To form a mixture in which the proteins are digested by the protease;

[0210] b) Adding to the mixture of step a) under conditions in which DNA is bound to the silica particles.

[0211] iii) Silica particles, and

[0212] iv) A second lysis reagent, comprising:

[0213] - Guanidine thiocyanate;

[0214] - Nonionic detergents; and

[0215] - Isopropanol;

[0216] c) Separate silica particles with bound DNA from the mixture in b);

[0217] d) Add a first washing solution to the separated silica particles containing bound DNA, the first washing solution comprising: i) guanidine hydrochloride or guanidine thiocyanate, and ii) ethanol;

[0218] e) Separate the silica particles containing bound DNA from the first washing solution;

[0219] f) Add a second washing solution containing a buffer and ethanol to the isolated silica particles containing bound DNA;

[0220] g) Separate the washed silica particles containing bound DNA from the second washing solution; and

[0221] h) Elute the DNA from the washed silica particles containing bound DNA to produce a separated DNA sample.

[0222] 34. The method according to implementation scheme 1, wherein steps f)-g)1 to 3 are repeated before step h).

[0223] 35. The method according to embodiment 33 or embodiment 34, wherein the nonionic detergent in the first lysis reagent and the second lysis reagent is the same or different, and is selected from the group consisting of: polyethylene glycol sorbitan monolaurate (Tween-20), octylphenoxy polyethoxyethanol (Nonidet P-40), and branched octylphenoxy poly(ethoxy)ethanol (IGEPAL CA-630).

[0224] 36. The method according to any one of embodiments 33-35, wherein the protease is a proteinase K protease.

[0225] 37. The method according to any one of embodiments 33-36, the method further comprising the step of drying the washed silica particles with bound DNA after step g) and before step h).

[0226] 38. The method according to any one of embodiments 33-37, wherein the mixture of step a) further comprises a DNA treatment control.

[0227] 39. The method according to embodiment 38, wherein the DNA treatment control comprises a zebrafish RASSF1 sequence.

[0228] 40. The method according to embodiment 8 or 39, wherein the DNA treatment control is synthetic DNA.

[0229] 41. The method according to any one of embodiments 33-40, wherein the mixture in step a) further comprises the host fish DNA.

[0230] 42. The method according to any one of embodiments 33-41, wherein the silica particles are magnetic particles.

[0231] 43. The method according to any one of embodiments 33-42, wherein the plasma sample has a volume of at least 2 mL.

[0232] 44. The method according to any one of embodiments 33-43, wherein the first lysis reagent comprises about 4.3M guanidine thiocyanate and 10% w / v IGEPAL CA-630.

[0233] 45. The method according to any one of embodiments 33-44, wherein the second cleavage reagent comprises 4.3 M guanidine thiocyanate and 10% w:v IGEPAAL CA-630 in combination with isopropanol.

[0234] 46. ​​The method according to any one of embodiments 33-45, wherein the first washing solution comprises i) about 3M guanidine hydrochloride or 3M guanidine thiocyanate, and ii) about 57% ethanol.

[0235] 47. The method according to any one of embodiments 33-46, wherein the second washing solution comprises about 80% ethanol and about 20% 10 mM Tris pH 8.0 buffer.

[0236] 48. The method according to any one of embodiments 33-47, wherein the elution comprises eluting the DNA in an elution buffer containing 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA.

[0237] 49. The method according to any one of embodiments 33-48, wherein the conditions under which the protein is digested by the protease include incubating the mixture at room temperature for 30 to 60 minutes.

[0238] 50. The method according to any one of embodiments 33-49, wherein steps a)-g) are carried out at room temperature.

[0239] 51. The method according to any one of embodiments 33-50, further comprising analyzing multiple target nucleic acids of the isolated DNA sample, the method comprising:

[0240] a) Treat the isolated DNA sample with bisulfite to produce a bisulfite-treated DNA sample;

[0241] b) The bisulfite-treated DNA sample is treated to an amplification reaction under conditions in which at least five different target regions are amplified to form a pre-amplified mixture when the target region is present in the sample;

[0242] c) Dispense the pre-amplified mixture into multiple different detection and assay reaction mixtures; and

[0243] d) Perform multiple detection assays using the detection assay reaction mixture, wherein at least five different target regions, if present in the sample in step a), are detected in one or more of the multiple different detection assay reaction mixtures.

[0244] 52. A kit for isolating DNA from plasma, the kit comprising:

[0245] a) A first pyrolysis reagent comprising guanidine thiocyanate and a nonionic detergent or a component used to prepare the first pyrolysis reagent;

[0246] b) A second cleavage reagent comprising guanidine thiocyanate, a nonionic detergent, and isopropanol, or components used to prepare the second cleavage reagent;

[0247] c) A first washing solution comprising i) guanidine hydrochloride or guanidine thiocyanate and ii) ethanol or components used to prepare the first washing solution;

[0248] d) A second wash solution containing Tris buffer and ethanol or the components used to prepare the second wash solution; and

[0249] e) Silica particles.

[0250] 53. The kit according to embodiment 52, wherein the nonionic detergent in the first lysis reagent and the second lysis reagent is the same or different, and is selected from the group consisting of: polyethylene glycol sorbitan monolaurate (Tween-20), octylphenoxy polyethoxyethanol (Nonidet P-40), and branched octylphenoxy poly(ethoxy)ethanol (IGEPAL CA-630).

[0251] 54. The kit according to embodiment 52 or embodiment 53, wherein the silica particles are magnetic particles.

[0252] 55. The kit according to embodiment 54, wherein the kit further comprises a magnet.

[0253] 56. The kit according to any one of embodiments 52-55, wherein the kit further comprises an elution buffer or a component for preparing the elution buffer.

[0254] 57. The kit according to any one of embodiments 52-55, wherein the kit further comprises a DNA treatment control, preferably a synthetic DNA treatment control.

[0255] 58. The kit according to embodiment 57, wherein the DNA treatment control comprises a zebrafish RASSF1 sequence.

[0256] 59. The kit according to any one of embodiments 52-58, wherein the kit further comprises a preparation of the fish DNA.

[0257] 60. The kit according to embodiment 57 or 58, wherein the DNA treatment control is present in the preparation of the native fish DNA.

[0258] 61. A system for processing plasma samples, the system comprising:

[0259] a) A first pyrolysis reagent comprising guanidine thiocyanate and a nonionic detergent or a component used to prepare the first pyrolysis reagent;

[0260] b) A second cleavage reagent comprising guanidine thiocyanate, a nonionic detergent, and isopropanol, or components used to prepare the second cleavage reagent;

[0261] c) A first washing solution comprising i) guanidine hydrochloride or guanidine thiocyanate and ii) ethanol or components used to prepare the first washing solution;

[0262] d) A second wash solution containing Tris buffer and ethanol or the components used to prepare the second wash solution; and

[0263] e) Silica particles.

[0264] 62. The system according to embodiment 61, wherein the nonionic detergent in the first lysis reagent and the second lysis reagent is the same or different, and is selected from the group consisting of: polyethylene glycol sorbitan monolaurate (Tween-20), octylphenoxy polyethoxyethanol (Nonidet P-40), and branched octylphenoxy poly(ethoxy)ethanol (IGEPAL CA-630).

[0265] 63. The system according to embodiment 61 or 62, wherein the system further comprises one or more of the following:

[0266] i) Protease;

[0267] ii) Elution buffer or components used to prepare said elution buffer;

[0268] iii) DNA treatment control;

[0269] iv) Preparation of the fish's DNA;

[0270] v) A reagent used to process DNA to produce bisulfite-treated DNA;

[0271] vi) Containers used for handling plasma; and / or

[0272] vii) Magnet.

[0273] 64. The system according to embodiment 63, wherein the DNA treatment control is synthetic DNA containing a zebrafish RASSF1 sequence.

[0274] 65. The system according to embodiment 63 or embodiment 64, wherein the DNA treatment control is present in a solution containing the DNA of the host fish.

[0275] 66. The system according to any one of embodiments 61-65, the system further comprising a reagent for treating DNA with bisulfite to produce bisulfite-treated DNA.

[0276] 67. The system according to any one of embodiments 61-66, wherein the system further comprises PCR amplification reagents.

[0277] 68. The system according to any one of embodiments 61-66, further comprising PCR amplification reagents and / or PCR flap assay reagents, wherein the PCR amplification reagents comprise:

[0278] i) Used to amplify multiple different primer pairs of multiple different target regions when the target region is present in the plasma;

[0279] ii) Thermostable DNA polymerase;

[0280] iii) dNTPs; and

[0281] iv) Contains Mg ++ buffer solution;

[0282] And the PCR valve assay reagent contains:

[0283] i) Used to amplify multiple different primer pairs of multiple different target regions when the target region is present in the plasma;

[0284] ii) Thermostable DNA polymerase;

[0285] iii) dNTPs;

[0286] iv) Contains Mg ++ buffer solution

[0287] v) Valvular endonuclease;

[0288] vi) Valeroid oligonucleotides, and

[0289] vi) A hairpin oligonucleotide comprising a region complementary to a portion of the valve oligonucleotide.

[0290] definition

[0291] To facilitate understanding of this technology, numerous terms and phrases are defined below. Further definitions are provided throughout the detailed description.

[0292] Throughout the specification and claims, unless the context clearly specifies otherwise, the following terms shall have the meaning explicitly associated herein. However, the phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, although it may refer to the same embodiment. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to different embodiments, although it may refer to different embodiments. Therefore, as described below, various embodiments of the present technology can be readily combined without departing from the scope or spirit of the present technology.

[0293] Furthermore, as used herein, unless the context explicitly states otherwise, the term "or" is an inclusive "or" operator and is equivalent to the term "and / or". Unless the context explicitly states otherwise, the term "based on" is not exclusive and allows for basing on additional factors not described. Additionally, throughout the specification, "a", "an", and "the" include the plural referent. "In" includes both "in" and "on".

[0294] As used in the claims of this application, the transitional phrase “consistently of…” limits the scope of the claims to the specified materials or steps “and those materials or steps that do not materially affect the essential and novel features of the claimed invention,” as discussed in In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976). For example, a composition “consistently of the exemplified elements” may contain unlisted contaminants at a certain level such that the contaminants, though present, do not alter the function of the exemplified composition compared to the pure composition (i.e., a composition “consisting of the exemplified components”).

[0295] As used herein with respect to non-target DNA, the term "exogenous" refers to non-target DNA isolated and purified from a source other than the source or sample containing the target DNA. For example, purified fish DNA is exogenous DNA relative to a sample containing human target DNA, as described, for example, in U.S. Patent No. 9,212,392 (which is incorporated herein by reference). The exogenous DNA does not need to originate from an organism different from the target DNA. For example, if commercially available purified fish DNA is added to a reaction configured to detect target nucleic acids in a sample from a particular fish, the purified fish DNA is exogenous. In a preferred embodiment, the exogenous DNA is selected to be undetectable by assays configured to detect and / or quantify the target nucleic acids in a reaction in which the exogenous DNA has been added.

[0296] As used in this article, "DNA fragment" or "small DNA" or "short DNA" refers to DNA consisting of no more than about 200 base pairs or nucleotides in length.

[0297] The term "primer" refers to an oligonucleotide that can act as a synthetic initiation site when placed under conditions that initiate primer extension. Oligonucleotide "primers" can be naturally occurring, such as in purified restriction digests, or can be synthesized. In some embodiments, oligonucleotide primers are used in conjunction with template nucleic acids, and primer extension is template-dependent, resulting in the formation of the complementary strand of the template.

[0298] In the context of nucleic acids, the term "amplifying" or "amplification" refers to the generation of multiple copies of a polynucleotide or a portion of a polynucleotide, typically starting from a small number of polynucleotides (e.g., a single polynucleotide molecule), where the amplification product or amplicon is usually detectable. Polynucleotide amplification involves a variety of chemical and enzymatic processes. In polymerase chain reaction (PCR) or ligase chain reaction (LCR; see, for example, U.S. Patent No. 5,494,810; incorporated herein by reference in its entirety), the generation of multiple copies of DNA from one or more copies of a target or template DNA molecule is a form of amplification. Other types of amplification include, but are not limited to, allele-specific PCR (see, for example, U.S. Patent No. 5,639,611; incorporated herein by reference in its entirety), assembly PCR (see, for example, U.S. Patent No. 5,965,408; incorporated herein by reference in its entirety), helicase-dependent amplification (see, for example, U.S. Patent No. 7,662,594; incorporated herein by reference in its entirety), hot-start PCR (see, for example, U.S. Patent Nos. 5,773,258 and 5,338,671; each incorporated herein by reference in its entirety), sequence-specific PCR, inverse PCR (see, for example, Triglia et al., (1988) NucleicAcids Res., 16:8186; incorporated herein by reference in its entirety), and ligation-mediated PCR (see, for example, Guilfoyle, R.).Nucleic Acids Research, 25:1854-1858 (1997); U.S. Patent No. 5,508,169; each of which is incorporated herein by reference in its entirety), methylation-specific PCR (see, for example, Herman et al., (1996) PNAS 93(13) 9821-9826; incorporated herein by reference in its entirety), small primer PCR, multiplex ligation-dependent probe amplification (see, for example, Schouten et al., (2002) Nucleic Acids Research 30(12): e57; incorporated herein by reference in its entirety), multiplex PCR (see, for example, 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 these articles is incorporated into this paper in its entirety by reference), nested PCR, overlapping extension PCR (see, for example, Higuchi et al., (1988) Nucleic Acids Research 16(15) 7351-7367; incorporated into this paper in its entirety by reference), real-time PCR (see, for example, Higuchi et al., (1992) Biotechnology 10:413-417; Higuchi et al., (1993) Biotechnology 11:1026-1030; each of these articles is incorporated into this paper in its entirety by reference), reverse transcription PCR (see, for example, Bustin, SA (2000) J. Molecular Endocrinology 25:169-193; incorporated into this paper in its entirety by reference), solid-phase PCR, thermally asymmetric interleaved PCR and falling PCR (see, for example, 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 these articles is incorporated herein by reference in its entirety. Polynucleotide amplification can also be performed using digital PCR (see, for example, Kalinina et al., Nucleic Acids Research).25; 1999-2004, (1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA. 96; 9236-41, (1999); International Patent Publication No. WO05023091A2; U.S. Patent Application Publication No. 20070202525; each of which is incorporated herein by reference in its entirety.

[0299] The term "polymerase chain reaction" ("PCR") refers to the methods described in U.S. Patents 4,683,195, 4,683,202, and 4,965,188 by KB Mullis, which describe methods for increasing the concentration of a target sequence fragment in a mixture of genomic or other DNA or RNA without cloning or purification. This method for amplifying the target sequence comprises introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired target sequence, followed by precise thermal cycling in the presence of DNA polymerase. The two primers are complementary to the respective strands of the double-stranded target sequence. For 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 by polymerase to form a new pair of complementary strands. The denaturation, primer annealing, and polymerase extension steps can be repeated multiple times (i.e., denaturation, annealing, and extension constitute a "cycle"; there can be many "cycles") to obtain a high concentration of amplified fragments of the desired target sequence. The length of the amplified fragment of the desired target sequence is determined by the relative positions of the primers to each other, and therefore this length is a controllable parameter. Due to the reproducible aspect of this method, it is called a “polymerase chain reaction” (“PCR”). Because the desired amplified fragment of the target sequence becomes the dominant sequence in the mixture (in terms of concentration), they are called “PCR-amplified” and are “PCR products” or “amplifiers.” Those skilled in the art will understand that the term “PCR” includes many variations of the methods originally described, such as real-time PCR, nested PCR, reverse transcription PCR (RT-PCR), single-primer PCR, and arbitrary-priming PCR.

[0300] As used in this article, the term "nucleic acid detection assay" refers to any method for determining the nucleotide composition of a target nucleic acid.Nucleic acid detection assays include, but are not limited to, DNA sequencing methods, probe hybridization methods, structure-specific cleavage assays (e.g., as described in U.S. Patent Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543, and 6,872,816; Lyamichev et al., Nat. Biotech., 17:292 (1999); Hall et al., PNAS, USA, 97:8272 (2000), and in combination PCR / invasive cleavage assays (Hologic, Inc., such as the “INVADER” flap assay or invasive cutting assay described in U.S. Patent Publications 2006 / 0147955 and 2009 / 0253142 (each of which is incorporated herein by reference in its entirety for all purposes); enzyme mismatch cutting methods (e.g., Variagenics, U.S. Patents 6,110,684, 5,958,692, and 5,851,770, which... (All of which are incorporated herein by reference); polymerase chain reaction (PCR) as described above; branch hybridization (e.g., U.S. Patent Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802 of Chiron, which are incorporated herein by reference in their entirety); rolling circle replication (e.g., U.S. Patent Nos. 6,210,884, 6,183,960, and 6,235,502, which are incorporated herein by reference in their entirety); NASBA (e.g., U.S. Patent No. 5,409,818, which is incorporated herein by reference in its entirety); molecular beacon technology (e.g., U.S. Patent No. 6,150,097, which is incorporated herein by reference in its entirety); electronic sensor technology (U.S. Patent Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573, which are incorporated herein by reference in their entirety); cyclic probe technology (e.g., U.S. Patent Nos. 5,403,711, 5,011,769, and 5,660,988, which are incorporated herein by reference in their entirety); Dade Behring signal amplification methods (e.g., U.S. Patents 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614, which are incorporated herein by reference in their entirety); ligase chain reaction (e.g., Barany Proc. Natl. Acad. Sci. USA 88,189-93 (1991)); and sandwich hybridization methods (e.g., U.S. Patent 5,288,609, which is incorporated herein by reference in its entirety).

[0301] In some embodiments, the target nucleic acid is amplified (e.g., by PCR) and the amplified nucleic acid is simultaneously detected using an invasive cleavage assay. Assays configured to perform detection assays (e.g., invasive cleavage assays) in combination with amplification assays are described in U.S. Patent Publication No. 20090253142A1 (Application Serial No. 12 / 404,240) (incorporated herein by reference in its entirety for all purposes), and as follows Figure 1 The diagram illustrates this. Because multiple copies of the FRET cassette are cut for each copy of the resulting target amplicon, this assay is considered to produce “signal amplification” in addition to target amplification. An additional amplification-enhancing invasive cut detection configuration known as the Quarts method is described in U.S. Patents 8,361,720, 8,715,937, and 8,916,344 (incorporated herein by reference in its entirety for all purposes).

[0302] As used herein, the term “PCR-lobe assay” and the term “PCR-invasive cut assay” are used interchangeably and refer to an assay configuration that combines PCR amplification and detection of amplified DNA by forming a first overlapping cut structure containing amplified target DNA and a second overlapping cut structure containing a 5' lobe of a cut from the first overlapping cut structure and a hairpin detection oligonucleotide called a “FRET box”. In the PCR-lobe assay as used herein, the assay reagent contains a mixture of DNA polymerase, FEN-1 endonuclease, a primary probe containing a portion complementary to the target nucleic acid, and a hairpin FRET box, and amplifies the target nucleic acid by PCR and simultaneously detects (i.e., detects during the target amplification process) the amplified nucleic acid. PCR-lobe assays include the Quarts assay described in U.S. Patent Nos. 8,361,720, 8,715,937, and 8,916,344 and the amplification assay in U.S. Patent No. 9,096,893 (e.g., as described in that patent). Figure 1 (As illustrated in the diagram), each of the patents is incorporated herein by reference in its entirety.

[0303] As used herein, the term "PCR-valve assay reagent" refers to one or more reagents used to detect a target sequence in a PCR-valve assay, the reagents comprising nucleic acid molecules capable of participating in the amplification of the target nucleic acid and forming a valve-cutting structure in the presence of the target sequence in a mixture containing DNA polymerase, FEN-1 endonuclease and FRET cassette.

[0304] As used herein, the terms "valve assay reagent" or "invasive cleavage assay reagent" refer to all reagents required for performing a valve assay or invasive cleavage assay on a substrate. As is known in the art, a valve assay generally includes the invasive oligonucleotide, valve oligonucleotide, valve endonuclease, and optionally a FRET cassette as described above. Valve assay reagents may optionally contain the invasive oligonucleotide and the target bound to the valve oligonucleotide.

[0305] As used herein, the term "valve oligonucleotide" refers to an oligonucleotide that can be cleaved by a valve endonuclease in detection assays (such as invasive cleavage assays). In a preferred embodiment, the valve oligonucleotide forms an invasive cleavage structure with other nucleic acids, such as target nucleic acids and invasive oligonucleotides.

[0306] As used herein, the term "FRET box" refers to a hairpin oligonucleotide containing a fluorophore portion and a neighboring quencher portion of the fluorophore. A cleaved lobe (e.g., a cleavage from a target-specific probe in a PCR-lobe assay) hybridizes with the FRET box to produce a second substrate of a lobe endonuclease, such as FEN-1. Once this substrate is formed, a base containing the 5' fluorophore is cleaved from the box, generating a fluorescent signal. In a preferred embodiment, the FRET box contains an unpaired 3' portion to which the cleavage product (e.g., a portion of the cleaved lobe oligonucleotide) hybridizes to form an aggressively cleaved structure that can be cleaved by the FEN-1 endonuclease.

[0307] As used in this article, a nucleic acid “hairpin” refers to a region of a single-stranded nucleic acid that contains a double-stranded stem and loop (i.e., base pairing), which is formed when the nucleic acid contains two parts that are sufficiently complementary to each other to form multiple consecutive base pairs.

[0308] As used herein, the term "FRET" refers to fluorescence resonance energy transfer, a method in which a portion (e.g., a fluorophore) transfers energy, for example, between itself or from a fluorophore to a non-fluorophore (e.g., a quencher molecule). In some cases, FRET involves the transfer of energy to an excited donor fluorophore via short-range (e.g., about 10 nm or less) dipole-dipole interactions. In other cases, FRET involves a loss of fluorescence energy from the donor and an increase in fluorescence from the acceptor fluorophore. In other forms of FRET, energy may be exchanged from the excited donor fluorophore for a non-fluorescent molecule (e.g., a quencher molecule). FRET is known to those skilled in the art and has been described (see, for example, Streyer et al., 1978, Ann. Rev. Biochem., 47:819; Selvin, 1995, MethodsEnzymol., 246:300; Orpana, 2004 Biomol Eng 21, 45-50; Olivier, 2005 MutantRes 573, 103-110, each of which is incorporated herein by reference in its entirety).

[0309] As used herein, the term “FEN-1” for enzymes refers to a non-polymerase valve endonuclease derived from eukaryotes or archaea, such as the one encoded by the FEN-1 gene. See, for example, WO 02 / 070755 and Kaiser MW et al., (1999) J. Biol. Chem., 274:21387, which are incorporated herein by reference in their entirety for all purposes.

[0310] As used in this article, the term “FEN-1 activity” refers to any enzymatic activity of the FEN-1 enzyme.

[0311] As used herein, the term "primer annealing" refers to the conditions that allow an oligonucleotide primer to hybridize with the template nucleic acid strand. Primer annealing conditions vary with primer length and sequence and are typically based on T0 values ​​determined or calculated for the primer. m For example, the annealing step in amplification methods involving thermal cycling involves lowering the temperature to a T value based on the primer sequence after a thermal denaturation step. m The temperature is maintained at a sufficient time to allow for such annealing.

[0312] As used herein, the term "amplifiable nucleic acid" refers to nucleic acid that can be amplified by any amplification method. It is expected that "amplifiable nucleic acid" will typically include a "sample template".

[0313] As used herein, the term "real-time" in relation to the detection of nucleic acid amplification or signal amplification refers to the detection or measurement of the accumulation of products or signals in a reaction during its execution (e.g., during incubation or thermal cycling). Such detection or measurement may occur continuously, or may occur at multiple discrete points during the amplification reaction, or may be a combination thereof. For example, in polymerase chain reaction (PCR), detection (e.g., fluorescence detection) may occur continuously during all or part of the thermal cycling, or it may occur instantaneously at one or more points during one or more cycles. In some embodiments, real-time detection of PCR is achieved by measuring the fluorescence level at the same point (e.g., a time point or temperature step within a cycle) in each of multiple cycles. Real-time detection of amplification may also be referred to as detection "during the amplification reaction."

[0314] As used herein, the term "nucleic acid abundance" refers to the amount of a specific target nucleic acid sequence present in a sample or aliquot. This amount is typically expressed as mass (e.g., μg), mass per unit volume (e.g., μg / μL), copy number (e.g., 1000 copies, 1 Åmolar), or copy number per unit volume (e.g., 1000 copies / mL, 1 Åmolar / μL). Nucleic acid abundance can also be expressed as a quantity relative to a standard amount of known concentration or copy number. A measure of nucleic acid abundance can be understood by those skilled in the art on any basis as a suitable quantitative representation of nucleic acid abundance, including the physical density, optical density, refractive index, staining properties of the sample, or based on the intensity of a detectable marker such as a fluorescent marker.

[0315] The terms "amplifier" or "amplification product" refer to a fragment of nucleic acid (usually DNA) generated by amplification methods such as PCR. The term is also used to refer to RNA fragments generated by amplification methods using RNA polymerases (such as NASBA, TMA, etc.).

[0316] When used for thermal cycling amplification reactions, the term "amplification curve" refers to a graph indicating the amplified signal (e.g., fluorescence signal) versus the cycle number. When used for non-thermal cycling amplification methods, the amplification curve typically refers to a curve showing the accumulation of the signal as a function of time.

[0317] When used in relation to amplification curves, the term "baseline" refers to the signal detected from the assembled amplification reaction before incubation, or in the case of PCR during the initial cycle (where the signal changes little).

[0318] As used in this article for real-time detection during amplification reactions undergoing thermal cycling, the term "C" t "Threshold cycling" or "threshold cycling" refers to the fractional number of cycles a detected signal (e.g., fluorescence) passes through a fixed threshold.

[0319] As used in this article for control reactions, the terms "template-free control" and "target-free control" (or "NTC") refer to reactions or samples that do not contain template or target nucleic acids. They are used to verify amplification quality.

[0320] As used herein, the term "sample template" refers to nucleic acids derived from the sample in which the "target" for analysis is present. Conversely, "background template" is used to refer to nucleic acids other than the sample template that may or may not be present in the sample. The presence of a background template is usually unintentional. This can be a residual result or attributable to the presence of nucleic acid contaminants that are being attempted to be purified from the sample. For example, nucleic acids from organisms other than those being tested can be present as background in the test sample.

[0321] Samples “suspected of containing” nucleic acid may or may not contain the target nucleic acid molecule.

[0322] As used herein, the term "sample" is used in its broadest sense. For example, in some embodiments, it is intended to include a sample or culture (e.g., a microbial culture), while in other embodiments, it is intended to include biological and environmental samples (e.g., samples suspected of containing a target sequence, gene, or template). In some embodiments, the sample may include a synthetically derived sample. The sample may be unpurified, or may be partially or fully purified, or otherwise processed.

[0323] This technology is not limited to the type of biological sample used or analyzed. It can be used with a wide variety of biological samples, including but not limited to tissues (e.g., organs (e.g., heart, liver, brain, lungs, stomach, intestines, spleen, kidneys, pancreas, and reproductive organs), glands, skin, and muscle), cells (e.g., blood cells (e.g., lymphocytes or erythrocytes), muscle cells, tumor cells, and skin cells), gases, body fluids (e.g., blood or portions thereof, serum, plasma, urine, semen, saliva, etc.), or solid (e.g., feces) samples obtained from humans (e.g., adults, infants, or embryos) or animals (e.g., cattle, poultry, mice, rats, dogs, pigs, cats, horses, etc.). In some embodiments, the biological sample may be solid food and / or feed products and / or ingredients, such as dairy products, vegetables, meat and meat by-products, and waste. Biological samples can be obtained from all different families of livestock and undomesticated or wild animals, including but not limited to animals such as ungulates, bears, fish, rabbits, rodents, pinnipeds, etc.

[0324] Biological samples also include biopsy tissue samples and tissue sections (e.g., biopsy tissue samples or sections or paraffin-embedded sections of tumors, growths, rashes, infections), medical or hospital samples (e.g., including but not limited to blood samples, saliva, oral swabs, cerebrospinal fluid, pleural fluid, milk, colostrum, lymph, sputum, vomit, bile, semen, oocytes, cervical cells, amniotic fluid, urine, feces, hair, and sweat), laboratory samples (e.g., subcellular fractions), forensic samples (e.g., blood or tissue (e.g., droplets or residues), hair and skin cells containing nucleic acids), and archaeological samples (e.g., fossil organisms, tissues, or cells).

[0325] Environmental samples include, but are not limited to, environmental materials such as surface materials, soil, water (e.g., fresh or seawater), algae, lichens, geological samples, air containing materials containing nucleic acids, crystals and industrial samples, as well as samples obtained from food and dairy processing equipment, instruments, apparatus, appliances, disposable and non-disposable items.

[0326] Samples can be prepared by any desired or suitable method. In some embodiments, nucleic acids are analyzed directly from bodily fluids, feces, or other samples using the method described in U.S. Patent No. 9,000,146 (which is incorporated herein by reference in its entirety for all purposes).

[0327] However, the above examples are not to be construed as limiting samples to which this technology is applicable (e.g., samples suspected of containing a target sequence, gene, or template (e.g., whose presence or absence can be determined using the compositions and methods of this technology)).

[0328] As used in this article, the terms “nucleic acid sequence” and “nucleic acid molecule” refer to oligonucleotides, nucleotides or polynucleotides and their fragments or portions. This term encompasses sequences of analogues including DNA and RNA nucleotides, including those listed above, and also including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxy-methyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy-amino-methyl-2-thiouracil. Uracil, beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentene adenine, methyl uracil-5-hydroxyacetate, uracil-5-hydroxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-hydroxyacetate, uracil-5-hydroxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 2,6-diaminopurine and pyrazolo[3,4-d]pyrimidines such as guanine analogues 6amino1H-pyrazolo[3,4d]pyrimidine 4(5H)1 (ppG or PPG, also known as Super G) and the adenine analog 4-amino1H-pyrazolo[3,4d]pyrimidine (ppA or PPA). The xanthine analog 1H-pyrazolo[5,4d]pyrimidine-4(5H)-6(7H)-dione (ppX) can also be used. These base analogs, when present in oligonucleotides, can enhance hybridization and improve mismatch identification. The oligonucleotide conjugates of this technique may include all tautomer forms of naturally occurring bases, modified bases, and base analogs. Other modified bases that can be used in this technology include 6-amino-3-prop-1-ynyl-5-hydropyrazolo[3,4-d]pyrimidin-4-one, PPPG; 6-amino-3-(3-hydroxyprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidin-4-one, HOPPPG; 6-amino-3-(3-aminoprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidin-4-one, NH2PPPG; 4-amino-3-(prop-1-ynyl)pyrazolo[3,4-d]pyrimidin, PPPA;4-Amino-3-(3-hydroxypropyl-1-ynyl)pyrazolo[3,4-d]pyrimidine, HOPPPA; 4-Amino-3-(3-aminopropyl-1-ynyl)pyrazolo[3,4-d]pyrimidine, NH2PPPA; 3-propyl-1-ynylpyrazolo[3,4-d]pyrimidine-4,6-diamino, (NH2)2PPPA; 2-(4,6-diaminopyrazolo[3,4-d]pyrimidine-3-yl)ethyn-1-ol, (NH2)2PPPAOH; 3-(2-aminoethynyl)pyrazolo[3,4-d]pyrimidine-4,6-diamine, (NH2)2 PPPANH2; 5-prop-1-ynyl-1,3-dihydropyrimidin-2,4-dione, PU; 5-(3-hydroxyprop-1-ynyl)-1,3-dihydropyrimidin-2,4-dione, HOPU; 6-amino-5-prop-1-ynyl-3-dihydropyrimidin-2-one, PC; 6-amino-5-(3-hydroxyprop-1-ynyl)-1,3-dihydropyrimidin-2-one, HOPC; and 6-amino-5-(3-aminoprop-1-ynyl)-1,3-dihydropyrimidin-2-one, NH2PC; 5-[4-amino-3-(3-methoxyprop-1-ynyl)pyrazol[3,4-d]pyrimidinyl]-2-(hydroxymethyl)oxazolylamine-3-ol, CH3 OPPPA; 6-amino-1-[4-hydroxy-5-(hydroxymethyl)oxadiazon-2-yl]-3-(3-methoxypropyl-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidin-4-one, CH3 OPPPG; 4,(4,6-diamino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-but-3-yn-1-ol, Super A; 6-amino-3-(4-hydroxy-but-1-ynyl)-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one; 5-(4-hydroxy-but-1-ynyl)-1H-pyrimidin-2,4-dione, Super T; 3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine ((NH2)2PPAI); 3-bromo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine ((NH2)2PPABr); 3-chloro-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine ((NH2)2PPACl); 3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4-ylamine (PPAI); 3-bromo-1H-pyrazolo[3,4-d]pyrimidine-4-ylamine (PPABr); and 3-chloro-1H-pyrazolo[3,4-d]pyrimidine-4-ylamine (PPACl).

[0329] Nucleic acid sequences or molecules can be genomic or synthetically derived DNA or RNA, and can be single-stranded or double-stranded, representing sense or antisense strands. Therefore, nucleic acid sequences can be dsDNA, ssDNA, mixed ssDNA, mixed dsDNA, ssDNA prepared as ssDNA (e.g., through unwinding, denaturation, helicase, etc.), A-, B-, or Z-DNA, triple-stranded DNA, RNA, ssRNA, dsRNA, mixed ss and dsRNA, dsRNA prepared as ssRNA (e.g., through unwinding, denaturation, helicase, etc.), messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), catalytic RNA, snRNA, microRNA, or protein nucleic acid (PNA).

[0330] This technology is not limited by the type or source of the nucleic acid (e.g., sequence or molecule (e.g., target sequence and / or oligonucleotide)) utilized. For example, the nucleic acid sequence can be amplified or produced (e.g., by synthesis (e.g., polymerization (e.g., primer extension (e.g., RNA-DNA hybridization primer technology)) and reverse transcription (e.g., RNA to DNA)) and / or amplified (e.g., polymerase chain reaction (PCR), rolling circle amplification (RCA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), ligase chain reaction (LCR), cycling probe technology, Q-β replicase, strand displacement amplification (SDA), branched DNA signal amplification (bDNA), hybridization capture, and helicase-dependent amplification).

[0331] Unless otherwise stated herein, the terms “nucleotide” and “base” are used interchangeably when referring to nucleic acid sequences. A “nucleobase” is a heterocyclic base such as adenine, guanine, cytosine, thymine, uracil, inosine, xanthine, hypoxanthine, or their heterocyclic derivatives, analogs, or tautomers. Nucleobases can be naturally occurring or synthetic. Non-limiting examples of nucleobases are adenine, guanine, thymine, cytosine, uracil, xanthine, hypoxanthine, 8-azapurine, purines substituted with a methyl or bromine at the 8-position, 9-oxo-N6-methyladenine, 2-aminoadenine, 7-deazoxanthine, 7-deazoguanine, 7-deazo-adenine, N4-ethanol cytosine, 2,6-diaminopurine, N6-ethanol-2,6-diaminopurine, 5-methylcytosine, 5 -(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, thiouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, 7,8-dimethylpyrazine, 6-dihydrothymidine, 5,6-dihydrouracil, 4-methyl-indole, vinylidene adenine, and in U.S. Patent Nos. 5,432,272 and 6,150,510 and PCT Application WO Non-naturally occurring nucleobases described in WO 92 / 002258, WO 93 / 10820, WO 94 / 22892 and WO 94 / 24144, and in Fasman (“Practical Handbook of Biochemistry and Molecular Biology”, pp. 385-394, 1989, CRC Press, Boca Raton, LO) (all of which are incorporated herein by reference in their entirety).

[0332] As used herein, the term "oligonucleotide" is defined as a molecule containing two or more nucleotides (e.g., deoxyribonucleotides or ribonucleotides), preferably at least five nucleotides, more preferably at least about 10-15 nucleotides, and even more preferably at least about 15 to 30 nucleotides or longer (e.g., oligonucleotides are typically less than 200 residues in length (e.g., between 15 and 100 nucleotides), however, as used herein, the term is also intended to cover longer polynucleotide chains). The exact size will depend on many factors, which in turn depend on the final function or use of the oligonucleotide. Oligonucleotides are generally expressed by their length. For example, a 24-residue oligonucleotide is referred to as a "24-mer". Oligonucleotides can form secondary and tertiary structures by self-hybridization or by hybridization with other polynucleotides. Such structures can include, but are not limited to, duplexes, hairpins, crosses, bends, and triplexes. Oligonucleotides can be produced in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or combinations thereof. In some embodiments, oligonucleotides forming invasive cleavage structures are generated in a reaction (e.g., by extending primers in an enzymatic extension reaction).

[0333] Since mononucleotides react to produce oligonucleotides by having the 5' phosphate of a mononucleotide's pentose ring attached in one direction to the 3' oxygen of its adjacent pentose ring via a phosphodiester bond, an oligonucleotide is referred to as having a "5' end" if the 5' phosphate at the end of the oligonucleotide is not attached to the 3' oxygen of the mononucleotide's pentose ring, and a "3' end" if the 3' oxygen at the end of the oligonucleotide is not attached to the 5' phosphate of the subsequent mononucleotide's pentose ring. As used herein, even within larger oligonucleotides, nucleic acid sequences can be said to have both 5' and 3' ends. If, when moving along the nucleic acid chain in a 5' to 3' direction, the 3' end of a first region precedes the 5' end of a second region, then the first region along the nucleic acid chain is considered to be upstream of the other region.

[0334] As used herein, the terms “complementary” or “complementarity” are used to refer to polynucleotides (e.g., sequences of two or more nucleotides, such as oligonucleotides or target nucleic acids) that are associated according to the base pairing rules. For example, the sequence “5'-AGT-3'” is complementary to the sequence “3'-TCA-5'”. Complementarity can be “partial,” where only some nucleic acid bases pair according to the base pairing rules. Alternatively, “complete” or “full” complementarity can exist between nucleic acid bases. The degree of complementarity between nucleic acid chains has a significant impact on the efficiency and strength of hybridization between nucleic acid chains. This is particularly important in amplification reactions and detection methods that depend on the association of two or more nucleic acid chains. Either term can also be used to refer to a single nucleotide, particularly in the case of polynucleotides. For example, a particular nucleotide within an oligonucleotide may be noted for its complementarity with or lack thereof in a nucleotide within another nucleic acid sequence (e.g., a target sequence), in contrast to or in comparison to the complementarity between the rest of the oligonucleotide and the nucleic acid sequence.

[0335] As used herein, a complementary sequence of a nucleic acid sequence refers to an oligonucleotide that is in "antiparallel association" when aligned with a nucleic acid sequence such that the 5' end of one sequence pairs with the 3' end of another sequence. As described above, nucleotide analogs can be included in the nucleic acids of this technology, and include them. Complementarity need not be perfect; stable duplexes can contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can empirically determine duplex stability based on a number of variables, including, for example, the length of the oligonucleotide, the base composition and sequence of the oligonucleotide, ionic strength, and the incidence of mismatched base pairs.

[0336] As used herein, the term "marker" refers to any part (e.g., a type of chemical substance) that is detectable or can cause a detectable reaction. In some preferred embodiments, the detection of a marker provides quantifiable information. A marker can be any known detectable part, such as, for example, a radiolabel (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 marker, latex beads, metal particles, paramagnetic markers, luminescent compounds (e.g., bioluminescent, phosphorescent, or chemiluminescent markers), or fluorescent compounds.

[0337] Labels can be directly or indirectly linked to oligonucleotides or other biomolecules. Direct labeling can occur through bonds or interactions that link the label to the oligonucleotide (including covalent bonds or non-covalent interactions such as hydrogen bonds, hydrophobic and ionic interactions), or through the formation of chelates or coordination complexes. Indirect labeling can be performed using bridging portions or "connectors," such as antibodies or other oligonucleotides that are directly or indirectly labeled.

[0338] The marker can be used alone or in combination with portions of the emission spectrum of the marker (e.g., fluorescence resonance energy transfer (FRET)) that can suppress (e.g., quench), excite, or shift (e.g., deflect) the marker (e.g., luminescent marker).

[0339] "Polymerase" is an enzyme commonly used to link 3'-OH 5'-triphosphate nucleotides, oligomers, and their analogues. 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, but are not limited to, 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 β, telomere transferase, Therminator polymerase (New England Biolabs), KOD HiFi DNA polymerase (Novagen), and KOD1. DNA polymerases, Q-β replicase, terminal transferases, AMV reverse transcriptase, M-MLV reverse transcriptase, Phi6 reverse transcriptase, HIV-1 reverse transcriptase, novel polymerases discovered through biosurveying, and polymerases exemplified in US 2007 / 0048748, US Patent Nos. 6,329,178, 6,602,695, and 6,395,524 (incorporated by reference). These polymerases include wild-type, mutant subtypes, and genetically engineered variants.

[0340] "DNA polymerase" is a polymerase that produces DNA from deoxyribonucleotide monomers (dNTPs). As used herein, "bacterial DNA polymerase" refers to Pol A type DNA polymerase (repair polymerase) 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 genus Thermus and other eubacterial species.

[0341] As used in this article, the term “target” refers to a nucleic acid species or nucleic acid sequence or structure to be detected or characterized.

[0342] Therefore, as used herein, "non-target," for example, as it is used to describe nucleic acids (e.g., DNA), refers to nucleic acids that may be present in a reaction but are not detected or characterized by the reaction. In some embodiments, non-target nucleic acids may refer to nucleic acids present in a sample that do not contain, for example, target nucleic acids, while in other embodiments, non-target may refer to exogenous nucleic acids, i.e., nucleic acids not derived from samples containing or suspected of containing target nucleic acids, and added to a reaction, for example, to normalize the activity of an enzyme (e.g., polymerase) to reduce variability in the enzyme's performance during the reaction.

[0343] As used herein, the term "amplification reagents" refers to those reagents (such as deoxyribonucleoside triphosphates, buffers, etc.) required for amplification, excluding primers, nucleic acid templates, and amplification enzymes. Typically, amplification reagents are placed and contained together with other reaction components in a reaction vessel.

[0344] As used herein, when referring to nucleic acid detection or analysis, the term "control" refers to a nucleic acid with known characteristics (e.g., known sequence, known copy number per cell) used for comparison with an experimental target (e.g., nucleic acid at an unknown concentration). A control can be an endogenous, preferably invariant gene that can be used against the test nucleic acid or target nucleic acid in its standardized assay. Such standardized controls are used to address inter-sample variations that can occur, such as in sample processing, assay efficiency, etc., and allow for accurate inter-sample data comparisons. Genes that can be used to standardize nucleic acid assays for human samples include, for example, β-actin, ZDHHC1, and B3GALT6 (see, for example, U.S. Patent Application Sequences 14 / 966,617 and 62 / 364,082, each incorporated herein by reference).

[0345] Controls can also be external. For example, in quantitative assays such as qPCR, Quarts, etc., a “calibrator” or “calibration control” is a nucleic acid having a known sequence (e.g., a sequence identical to a portion of the experimental target nucleic acid) and a known concentration or serial concentration (e.g., a serially diluted control target used for calibration generated in quantitative PCR). Typically, the calibration control is analyzed using the same reagents and reaction conditions used for the experimental DNA. In some embodiments, the measurement of the calibrator is performed simultaneously with the experimental assay, for example, in the same thermal cycler. In a preferred embodiment, multiple calibrators may be contained in a single plasmid, making it easy to provide different calibrator sequences in equimolar amounts. In a particularly preferred embodiment, the plasmid calibrator is digested, for example, with one or more restriction endonucleases to release the calibrator portion from the plasmid vector. See, for example, WO 2015 / 066695, which is incorporated herein by reference.

[0346] As used in this article, “ZDHHC1” refers to the gene encoding a DHHC type zinc finger containing a 1-type protein located in human DNA at Chr 16 (16q22.1) and belonging to the DHHC palmitoyltransferase family.

[0347] As used herein, the term "treatment control" refers to a foreign molecule, such as a foreign nucleic acid added to a sample prior to the extraction of target DNA, which can be measured post-extraction to assess the efficiency of the treatment and determine the success or failure mode. The nature of the treatment control nucleic acid used typically depends on the type of assay and the analyte. For example, if the assay used is for the detection and / or quantification of double-stranded DNA or mutations therein, a double-stranded DNA treatment control is typically incorporated into a pre-extracted sample. Similarly, for assays monitoring mRNA or microRNA, the treatment control used is typically an RNA transcript or synthetic RNA. See, for example, U.S. Patent Application Serial No. 62 / 364,049, filed July 19, 2016, which is incorporated herein by reference and describes the use of zebrafish DNA as a treatment control for human samples.

[0348] As used herein, the term "zebrafish DNA" is distinct from the intrinsic "fish DNA" (e.g., purified salmon DNA) and refers to DNA isolated from zebrafish (Danio rerio) or DNA generated in vitro (e.g., enzymatically, synthetically) to have nucleotides found in zebrafish DNA. In a preferred embodiment, the zebrafish DNA is a methylated DNA added as a detectable control DNA, for example, a treatment control used to verify DNA recovery through sample processing steps. Specifically, zebrafish DNA containing at least a portion of the RASSF1 gene can be used as a treatment control, for example, for human samples, as described in U.S. Patent Application Serial No. 62 / 364,049.

[0349] As used herein, the term "fish DNA" is distinct from zebrafish DNA and refers to in vivo (e.g., genomic) DNA isolated from a fish, such as that described in U.S. Patent No. 9,212,392. In vivo purified fish DNA is commercially available, for example, in the form of cod and / or herring sperm DNA (Roche Applied Science, Mannheim, Germany) or salmon DNA (USB / Affymetrix).

[0350] As used herein, the terms “particle” and “bead” are used interchangeably, and the terms “magnetic particle” and “magnetic bead” are used interchangeably, and refer to particles or beads that respond to a magnetic field. Typically, magnetic particles comprise materials that do not possess a magnetic field but form magnetic dipoles when exposed to a magnetic field, such as materials that can be magnetized in the presence of a magnetic field but are not inherently magnetic in its absence. As used in this context, the term “magnetic” includes materials that are paramagnetic or superparamagnetic. As used herein, the term “magnetic” also encompasses temporarily magnetic materials, such as ferromagnetic or ferrimagnetic materials with low Curie temperatures, provided that such temporarily magnetic materials are paramagnetic within the temperature range in which they are used to separate biomaterials using silica magnetic particles containing such materials according to this method.

[0351] As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of nucleic acid purification systems and reaction assays, such delivery systems include systems that allow the storage, transport, or delivery of reagents and devices (e.g., dissociative salts, particles, buffers, denaturants, oligonucleotides, filters, etc. in appropriate containers) and / or supporting materials (e.g., sample processing or storage containers, written instructions for performing procedures, etc.) from one location to another. For example, a kit may include one or more shells (e.g., boxes) containing the relevant reaction reagents and / or supporting materials. As used herein, the term “fragmented kit” refers to a delivery system comprising two or more separate containers, each containing a sub-part of all kit components. These containers may be delivered together or separately to the intended recipient. For example, the first container may contain materials and buffers for sample collection, while the second container contains capture oligonucleotides and denaturants. The term “fragmented kit” is intended to cover kits containing, but not limited to, analyte-specific reagents (ASRs) as defined in, section 520(e) of the Federal Food, Drug, and Cosmetic Act. In fact, any delivery system comprising two or more separate containers (each containing a sub-part of the total kit components) is included in the term "fragmented kit". Conversely, a "combination kit" refers to a delivery system that contains all components of the reaction assay in a single container (e.g., in a single box containing each desired component). The term "kit" includes both fragmented kits and combination kits.

[0352] As used herein, the term "system" refers to a collection of items for a specific purpose. In some embodiments, the items include instructions for use, provided as information, for example, on the item itself, on paper, or on a recordable medium (e.g., floppy disk, CD, flash drive, etc.). In some embodiments, the instructions direct the user to an online location, such as a website for viewing, listening to, and / or downloading instructions. In some embodiments, the instructions or other information are provided as an application ("app") on a mobile device. Invention Details

[0353] This article provides techniques related to amplification-based detection of nucleic acids, and specifically, but not exclusively, relates to methods for enriching low-DNA, bisulfite-converted samples for analysis.

[0354] Target biological samples can contain vastly different amounts of DNA, and even those abundant in the host DNA may contain very low levels of target DNA, such as abnormal DNA in a normal DNA background, or human DNA in a microbial DNA background (or vice versa). To compensate for low concentrations of target DNA, large samples can sometimes be processed to collect enough DNA for a specific assay. However, when multiple different assays with low concentrations of target DNA need to be performed in parallel, the required sample volume can become excessive. For example, circulating cell-free DNA in a subject's plasma is typically very low because it is primarily cleared from the bloodstream via the liver and has a half-life of only 10 to 15 minutes. Therefore, common levels of circulating DNA are very low; for example, a specific fragment of DNA, such as that from a target gene, may be present at approximately 1,500–2,000 copies / mL in a healthy individual, while a tumor-associated DNA fragment may be present at approximately 5,000 copies / mL in a subject with advanced cancer. In addition, tumor-derived cfDNA in plasma is often fragmented into short chains, for example, having 200 or fewer base pairs (see, for example, P. Jiang et al., Proc. Natl Acad Sci. 112(11): E1317-E1325 (2015), which is incorporated herein by reference in its entirety). Such small DNAs are particularly difficult to purify because they can be lost during typical purification steps, such as through precipitation and / or inefficiencies in DNA binding purification steps.

[0355] DNA can be recovered from such blood fractions up to 75%, but often much less is recovered. Therefore, depending on the sensitivity of the specific assay targeting these targets, analysis of multiple DNA biomarkers from plasma may require large amounts of plasma from the subject. Enrichment through targeted pre-amplification of specific target regions can increase the number of biomarkers that can be analyzed using the same starting sample, eliminating the need to collect correspondingly large samples (e.g., plasma or blood) from the subject.

[0356] This document provides embodiments of a technique for extracting DNA (e.g., cell-free circulating DNA) from plasma samples. In a preferred embodiment, the method provided herein does not include the use of organic extraction (e.g., phenol-chloroform extraction), alcohol precipitation, or columns, making the method easily scalable and automated. In a particularly preferred embodiment, essentially the entire separation procedure—from the plasma sample to the purified DNA bound to beads for elution—is performed at room temperature.

[0357] This document provides embodiments of a multiplex preamplification technique particularly suitable for the analysis of target DNAs present in low abundance and / or fragmented in samples where they are found and treated with bisulfite reagents, such as those described in Leontiou et al., PLoS ONE 10(8): e0135058. doi:10.1371 / journal.pone.0135058 (2015). In some preferred embodiments, the bisulfite treatment comprises the use of ammonium bisulfite, preferably for desulfonation of the vector-bound DNA, as described in U.S. Patent No. 9,315,853.

[0358] Implementation plan of this technology

[0359] 1. Isolation of non-circulating cell DNA from plasma

[0360] This document provides techniques related to the separation of fragmented DNA from samples (e.g., blood or plasma samples). Specifically, this document provides techniques related to the extraction of low-copy small DNA (e.g., less than about 200 base pairs in length) from plasma samples using miscible particles, such as silica particles, that bind DNA. This document provides methods using two different lysis reagents added at different times during the lysis treatment of the plasma sample, and using a combination of two different wash buffers in the treatment of the DNA bound to the particles. In a preferred embodiment, the techniques provided herein include adding native exogenous non-target DNA (e.g., native fish DNA) to the DNA to be separated for further analysis, said exogenous non-target DNA preferably being added to the plasma before or during the first particle binding step.

[0361] 2. Pre-amplification of target regions for PCR-lobe assay analysis

[0362] This article provides information on providing increased amounts of DNA for use in PCR-valve assays (e.g., as...). Figure 1This describes a technique for analysis using the Quartz assay (illustrated in the diagram). Specifically, embodiments of the methods and compositions disclosed herein provide a method for using multiple pre-amplification steps, such as increasing the amount of target DNA target from a low-target sample, followed by target-specific detection to further amplify and detect the target locus.

[0363] It is known that reamplified DNA fragments previously amplified in a targeted manner (e.g., amplification of aliquots or dilutions of target-specific PCR amplicon products) are prone to producing undesirable artifacts, such as high background noise from unwanted DNA products. Therefore, analysis of target nucleic acids using sequential rounds of target-specific PCR is typically performed under specific conditions, such as using different primer pairs in consecutive reactions. For example, in “nested PCR,” a first round of amplification is performed to produce a first amplicon, and a second round of amplification is performed using a primer pair, wherein one or both of the primers anneal to a site within the region defined by the initial primer pair; i.e., the second primer pair is considered “nested” within 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 undesirable effects include using very low concentrations of primers in the first amplification.

[0364] Multiplex amplification of several different specific target sequences is typically performed using a relatively standard PCR reagent mixture. For example, for Amplitaq DNA polymerase, a mixture containing approximately 50 mM KCl, 1.5 to 2.5 mM MgCl2, and Tris-HCl buffer at approximately pH 8.5 is used. As mentioned above, if a second amplification is to be performed, primers are usually present in limited quantities (Andersson, ibid.). For subsequent assays, the amplified DNA is diluted or purified, and then small aliquots are added to the detection assay (e.g., PCR-lobe assay), which uses buffer and salt conditions different from standard PCR (e.g., a buffer containing MOPS, Tris-HCl pH 8.0, and 7.5 mM MgCl2, and little or no added KCl or other monovalent salts, and due to the low monovalent salts and relatively high MgCl2 concentration). ++ Conditions generally considered unfavorable for PCR (see, 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, which discloses optimal Mg2+ concentrations of 1.5 mM to 2.0 mM for Taq DNA polymerase) are also unfavorable.++ The range was optimized by supplementing magnesium concentration to 4 mM in increments of 0.5. See also “Multiplex PCR: Critical Parameters and Step-by-Step Protocol”, O. Henegariu et al., BioTechniques 23:504-511 (September 1997). Variations in reaction conditions between the first and second amplifications (or other assays) are typically achieved by purifying the DNA from the first amplification reaction or by using sufficient dilution (so that the amount of reaction components remaining in subsequent reactions is negligible).

[0365] The implementation of this technology involves combined bisulfite modification, multiplex PCR amplification, and PCR-lobe assay for detecting low copy number DNA. During the development of the implementation of the technology provided herein, it was found that using KCl with very low concentrations and containing elevated Mg... ++ PCR-valve assay buffer (e.g., >6 mM, preferably >7 mM, more preferably 7.5 mM) produces significantly better signals for both multiplex pre-amplification and subsequent PCR-valve assays in the absence of valve assay reagents (e.g., in the absence of hairpin oligonucleotides and FEN-1 endonuclease). Furthermore, it was unexpectedly determined that using the same primer pairs to amplify the target region in both the pre-amplification and subsequent PCR-valve assay reactions yielded better results than using nested primers. Using PCR-valve assay primer pairs in both initial amplification and PCR-valve detection has the advantage of generating signals from very small target DNA fragments, as can be expected in remote DNA samples. For example, in the examples below, amplicons of approximately 50 to 85 base pairs were generated and detected.

[0366] In some embodiments, one or both of the pre-amplification and PCR-valve assays contain exogenous non-target DNA in the reaction mixture, as described, for example, in U.S. Patent Application Serial No. 14 / 036,649, filed September 25, 2013 (which is incorporated herein by reference in its entirety). In some preferred embodiments, the exogenous non-target DNA comprises fish DNA. While not limiting the invention to any particular mechanism of action, the presence of hairpin oligonucleotides (e.g., hairpin FRET cassettes used in some embodiments of invasive cut assays) has been observed to have an inhibitory effect on DNA polymerases present in the same container, as assessed by sample and signal amplification. See, for example, U.S. Patent Publication 2006 / 0147955, belonging to Allawi, which is incorporated herein by reference for all purposes. Allawi et al. observed that hairpin FRET oligonucleotides affect polymerase performance when PCR and invasive cut assay components are combined, and that the use of purified exogenous non-target DNA, particularly genomic DNA, improves the consistency of signals generated in such assays. Therefore, in a preferred embodiment, purified exogenous non-target DNA is added to the sample before and / or simultaneously with contacting the sample with an enzyme (such as a polymerase). When an enzyme of about 0.01 to 1.0 U / μL, such as a 0.05 U / μL enzyme (e.g., a polymerase such as Taq polymerase), is used in the assay, the non-target DNA is typically added to the sample or reaction mixture at a concentration of about 2 to 20 ng / μL, preferably about 6 to about 7 ng / μL.

[0367] The multiplex pre-amplification implementation schemes disclosed herein can be used with PCR-valve assays such as the Quarts assay. Figure 1As shown, the Quarts technology combines a polymerase-based target DNA amplification method with an invasive cleavage-based signal amplification method. The fluorescence signal generated by the Quarts reaction is monitored in a manner similar to real-time PCR. During each amplification cycle, three consecutive chemical reactions are performed in each well: the first and second reactions occur on the target DNA template, and the third reaction occurs on a synthetic DNA target labeled with a fluorophore and a quencher dye, thereby forming a fluorescence resonance energy transfer (FRET) donor-acceptor pair. The first reaction utilizes polymerase and oligonucleotide primers to generate the amplified target. The second reaction uses a highly structure-specific 5'-lobe endonuclease-1 (FEN-1) reaction to release a 5'-lobe sequence from the target-specific oligonucleotide probe, which binds to the product of the polymerase reaction to form an overlapping lobe substrate. In the third reaction, the cleaved lobe is annealed with a specially designed oligonucleotide (FRET cassette) containing a fluorophore tightly linked (resulting in fluorescence quenching) in the FRET pair. The released probe lobes hybridize in a manner that forms an overlapping lobe substrate, which allows the FEN-1 enzyme to cleave the 5'-lobe containing the fluorophore, thereby releasing it from the vicinity of the quencher molecule. The released fluorophore generates the fluorescent signal to be detected. During the second and third reactions, the FEN-1 endonuclease can cleave multiple probes for each target, generating multiple cleaved 5'-lobes per target, and each cleaved 5'-lobe can participate in the cleavage of many FRET cassettes, thus generating additional fluorescent signal amplification throughout the reaction.

[0368] In some configurations, each assay is designed to detect multiple genes, such as reporting three genes for three different 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.

[0369] These implementations can be further understood through the illustrative examples provided below.

[0370] Experimental Examples

[0371] Example 1

[0372] DNA isolation from cells and plasma, and bisulfite conversion.

[0373] DNA isolation

[0374] For cell lines, genomic DNA was isolated from conditioned medium using the Maxwell® RSC ccfDNA Plasma Kit (Promega Corp., Madison, WI). Following the kit protocol, 1 mL of conditioned medium (CCM) was used instead of plasma, and the cells were processed according to the kit procedure. The elution volume was 100 μL, of which 70 μL was used for bisulfite conversion.

[0375] The following is an exemplary procedure for isolating DNA from a 4 mL plasma sample:

[0376] • Add 300 μL of proteinase K (20 mg / mL) to a 4 mL plasma sample and mix.

[0377] • Add 3 μL of 1 μg / μL fish DNA to the plasma-proteinase K mixture.

[0378] • Add 2 mL of plasma lysis buffer to the plasma.

[0379] The plasma lysis buffer is:

[0380] - 4.3 M guanidine thiocyanate

[0381] - 10% IGEPAL CA-630 (branched octylphenoxy poly(ethylene oxy)ethanol)

[0382] (5.3g IGEPAL CA-630 in combination with 45 mL 4.8M guanidine thiocyanate)

[0383] • Incubate the mixture at 55°C for 1 hour while shaking at 500 rpm.

[0384] • Add 3 mL of plasma lysis buffer and mix.

[0385] • Add 200 μL of magnetic silica-bonded beads [16 μg beads / μL] and mix again.

[0386] • Add 2 mL of 100% isopropanol and mix.

[0387] • Incubate at 30°C for 30 minutes with shaking at 500 rpm.

[0388] • Place the tube on the magnet and allow the beads to collect. Suck out and discard the supernatant.

[0389] • Add 750 μL of guanidine hydrochloride-ethanol (GuHCl-EtOH) washing solution to the container containing the bound beads and mix.

[0390] The GuHCl-EtOH washing solution is:

[0391] - 3M GuHCl

[0392] - 57% EtOH.

[0393] Shake at 400 rpm for 1 minute.

[0394] • Transfer the sample to a deep-well plate or a 2 mL microcentrifuge tube.

[0395] • Place the tube on a magnet and allow the magnetic beads to collect for 10 minutes. Aspirate and discard the supernatant.

[0396] • Add 1000 μL of washing buffer (10 mM Tris HCl, 80% EtOH) to the beads and incubate at 30°C with shaking for 3 minutes.

[0397] • Place the tube on the magnet and allow the beads to collect. Suck out and discard the supernatant.

[0398] • Add 500 μL of washing buffer to the beads and incubate with shaking at 30°C for 3 minutes.

[0399] • Place the tube on the magnet and allow the beads to collect. Suck out and discard the supernatant.

[0400] • Add 250 μL of washing buffer and incubate at 30°C with shaking for 3 minutes.

[0401] • Place the tube on the magnet and allow the beads to collect. Draw out and discard any remaining buffer solution.

[0402] • Add 250 μL of washing buffer and incubate at 30°C with shaking for 3 minutes.

[0403] • Place the tube on the magnet and allow the beads to collect. Draw out and discard any remaining buffer solution.

[0404] • Dry the beads at 70°C for 15 minutes and shake.

[0405] • Add 125 μL of elution buffer (10 mM Tris HCl, pH 8.0, 0.1 mM EDTA) to the beads and incubate with shaking at 65°C for 25 minutes.

[0406] • Place the tube on the magnet and let the magnetic beads collect for 10 minutes.

[0407] • Aspirate the supernatant containing DNA and transfer it to a new container or tube.

[0408] Bisulfite conversion

[0409] I. Sulfonation of DNA with ammonium bisulfite

[0410] 1. In each tube, combine 64 μL of DNA, 7 μL of 1 N NaOH, and 9 μL of a vector solution containing 0.2 mg / mL BSA and 0.25 mg / mL fish DNA.

[0411] 2. Incubate at 42℃ for 20 minutes.

[0412] 3. Add 120 μL of 45% ammonium bisulfite and incubate at 66°C for 75 minutes.

[0413] 4. Incubate at 4°C for 10 minutes.

[0414] II. Desulfonate groups using magnetic beads

[0415] Material

[0416] Magnetic beads (Promega MagneSil Paramagnetic Particles, Promega catalog number AS1050, 16 μg / μL).

[0417] Binding buffer: 6.5-7 M guanidine hydrochloride.

[0418] Wash buffer after conversion: 80% ethanol containing 10 mM Tris HCl (pH 8.0).

[0419] Desulfonate buffer: 70% isopropanol and 0.1 N NaOH were selected for desulfonate buffer.

[0420] Mix the sample using any suitable apparatus or technique at the temperature and mixing rate substantially as described below. For example, a thermal mixer (Eppendorf) can be used to mix or incubate the sample. An example of desulfonation is as follows:

[0421] 1. Thoroughly mix the beads by swirling the bottle for 1 minute.

[0422] 2. Divide 50 μL of beads into 2.0 mL tubes (e.g., from USA Scientific).

[0423] 3. Add 750 μL of binding buffer to the beads.

[0424] 4. Add 150 μL of sulfonated DNA from step I.

[0425] 5. Mix (e.g., at 30°C and 1000 RPM for 30 minutes).

[0426] 6. Place the tube on the magnet holder and hold it in place for 5 minutes. For tubes on the holder, remove and discard the supernatant.

[0427] 7. Add 1,000 μL of wash buffer. Mix (e.g., at 30°C and 1000 RPM for 3 minutes).

[0428] 8. Place the tube on the magnet holder and hold it in place for 5 minutes. Remove the tube from the holder and discard the supernatant.

[0429] 9. Add 250 μL of wash buffer. Mix (e.g., at 30°C and 1000 RPM for 3 minutes).

[0430] 10. Place the tube on the magnetic rack; remove it after 1 minute and discard the supernatant.

[0431] 11. Add 200 μL of desulfonate buffer. Mix (e.g., at 30°C and 1000 RPM for 5 minutes).

[0432] 12. Place the tube on the magnetic rack; remove it after 1 minute and discard the supernatant.

[0433] 13. Add 250 μL of wash buffer. Mix (e.g., at 30°C and 1000 RPM for 3 minutes).

[0434] 14. Place the tube on the magnetic rack; remove it after 1 minute and discard the supernatant.

[0435] 15. Add 250 μL of wash buffer to the tube. Mix (e.g., at 30°C and 1000 RPM for 3 minutes).

[0436] 16. Place the tube on the magnetic rack; remove it after 1 minute and discard the supernatant.

[0437] 17. Open the caps on all tubes and incubate at 30°C for 15 minutes.

[0438] 18. Remove the test tube from the magnetic rack and add 70 μL of elution buffer directly to the beads.

[0439] 19. Warm the beads with elution buffer (e.g., at 40°C and 1000 RPM for 45 minutes).

[0440] 20. Place the tube on the magnetic rack for about one minute; remove and preserve the supernatant.

[0441] Then, as described below, the transformed DNA is used for pre-amplification and / or valve endonuclease assays.

[0442] Example 2

[0443] Multiple preamplification-preamplification cycle

[0444] Using a nested approach, the effectiveness of the PCR cycle count was examined by performing 5, 7, or 10 cycles with the outer primer pair for each target sample. A PCR-lobe assay using the inner primer was then used for further amplification and analysis of the pre-amplified products.

[0445] • Experimental conditions:

[0446] 1. Sample source: DNA extracted from the HCT116 cell line and treated with bisulfite as described above;

[0447] 2. 50 μL pre-amplification PCR reaction.

[0448] 3. Target regions tested: NDRG4, BMP3, SFMBT2, VAV3, ZDHHC1, and β-actin (see Figure 5)

[0449] 4. Reaction conditions for pre-amplification PCR and PCR-valve assay:

[0450] 7.5 mM MgCl2

[0451] 10 mM MOPS

[0452] 0.3 mM Tris-HCl, pH 8.0

[0453] 0.8 mM KCl,

[0454] 0.1 μg / μl BSA

[0455] 0.0001% Tween-20

[0456] 0.0001% IGEPAL CA-630

[0457] 250 μM dNTP)

[0458] 0.025 U / μl GoTaq polymerase (Promega Corp., Madison, WI)

[0459] Primer pairs for NDRG4, BMP3, SFMBT2, VAV3, ZDHHC1, and β-actin for bisulfite conversion, such as... Figures 5A-5F As shown, each primer was 500 nM in both pre-amplification and PCR-valve assays.

[0460] Use 10 μL of prepared bisulfite-treated target DNA in each 50 μL PCR reaction mixture. The pre-amplification cycle is shown below:

[0461]

[0462] Following PCR, 10 μL of the amplification reaction was diluted to 100 μL in 10 mM Tris and 0.1 mM EDTA, and the 10 μL diluted amplification product was used for a standard PCR-block assay as described below. Comparative assays were performed directly on bisulfite-treated DNA using a Quarts PCR-block assay without pre-amplification.

[0463] A typical Quarts reaction typically contains approximately 400–600 nM (e.g., 500 nM) of each primer and detection probe, approximately 100 nM of invasive oligonucleotides, approximately 600–700 nM of each FAM (e.g., commercially available from Hologic), HEX (e.g., commercially available from BioSearch Technologies, IDT), and Quasar 670 (e.g., commercially available from BioSearch Technologies) FRET cassette, 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 Corp., Madison, WI) in a 30 μl reaction volume, 10 mM of 3-(n-morpholino)propanesulfonic acid (MOPS), 7.5 mM of MgCl2, and 250 μM of each dNTP.

[0464] An example Quarts loop condition is shown below:

[0465]

[0466] Data shown Figure 6 Furthermore, it was shown that 10 cycles of pre-amplification yielded the most consistent determination of methylation percentage compared to PCR-valve assays without pre-amplification.

[0467] Example 3

[0468] Nested primers vs. non-nested primers; PCR buffer vs. PCR-valve assay buffer

[0469] Assays were performed to compare the use of nested primer arrangements with the use of the same PCR-valve assay primers in both the pre-amplification and PCR-valve assay steps, and to compare the use of common PCR buffers in the pre-amplification step with the use of PCR-valve assay buffers. PCR-valve assay buffers were used. Common PCR buffers consist of 1.5 mM MgCl2, 20 mM Tris-HCl, pH 8, 50 mM KCl, and 250 μM of each dNTP; while PCR-valve assay buffers consist of 7.5 mM MgCl2, 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% IGEPALCA-630, and 250 μM of each dNTP. Primer concentrations of 20 nM, 100 nM, and 500 nM were also compared.

[0470] • Experimental conditions:

[0471] 1. Sample source: DNA extracted from the HCT116 cell line and treated with bisulfite;

[0472] 2. 50 μL PCR reaction mixture.

[0473] 3. Target regions tested: NDRG4, BMP3, SFMBT2, VAV3, ZDHHC1, and β-actin.

[0474] 4. GoTaq polymerase, 0.025 U / μL.

[0475] 5. PCR or PCR-valve assay buffer as described above.

[0476] 6. Primer pairs for NDRG4, BMP3, SFMBT2, VAV3, ZDHHC1, and β-actin for bisulfite conversion, such as... Figures 5A-5F As shown, each primer is available in 20 nM, 100 nM, and 500 nM.

[0477] The pre-amplification cycle is shown below:

[0478]

[0479] 10 μL of prepared bisulfite-treated target DNA was used in each 50 μL PCR reaction. After PCR, as described in Example 2, 10 μL of pre-amplified reaction was diluted to 100 μL in 10 mM Tris and 0.1 mM EDTA, and 10 μL of the diluted amplification product was used for standard PCR-valve assay.

[0480] Data shown Figure 7 The first graph shows the expected yield calculated from the starting DNA amount, and the second graph shows the amount detected using the indicated primer and buffer conditions. These data indicate that the highest nM primer concentration produces the highest amplification efficiency. Surprisingly, primers with relatively high Mg2+ concentrations also yielded higher amplification efficiencies when used for PCR pre-amplification. ++ Compared to using PCR-valve assay buffers with low KCl (7.5 mM and 0.8 mM, respectively), this method is more effective than using buffers with lower Mg2+. ++ Better results were obtained with standard PCR buffers containing higher KCl concentrations (1.5 mM and 50 mM, respectively). Furthermore, the use of PCR-valve assay primers (“inner” primers and…) during pre-amplification PCR yielded better results. Figures 5A-5F The method shown is as good or better than using set outer and inner primer pairs in nested PCR arrangements.

[0481] Example 4

[0482] Cycle pre-amplification was tested in the lobe assay buffer.

[0483] Assays were performed to determine the effect of increasing the number of pre-amplified PCR cycles on background in both target-free control samples and samples containing target DNA.

[0484] Experimental conditions:

[0485] 1. Sample source:

[0486] i) Target-free control = 20 ng / μL fish DNA and / or 10 mM Tris, 0.1 mM EDTA;

[0487] ii) Bisulfite-converted DNA isolated from the plasma of normal patients

[0488] iii) Bisulfite-converted DNA isolated from the plasma of normal patients, combined with DNA extracted from the HCT116 cell line and treated with bisulfite.

[0489] 2. 50 μL PCR reaction mixture,

[0490] 3. Target regions tested: NDRG4, BMP3, SFMBT2, VAV3, ZDHHC1, and β-actin.

[0491] 4. Reaction conditions for pre-amplification and PCR-valve assay:

[0492] 7.5 mM MgCl2

[0493] 10 mM MOPS

[0494] 0.3 mM Tris-HCl, pH 8.0

[0495] 0.8 mM KCl,

[0496] 0.1 μg / μL BSA

[0497] 0.0001% Tween-20

[0498] 0.0001% IGEPAL CA-630

[0499] 250 μM dNTP)

[0500] 0.025 U / μl of GoTaq polymerase,

[0501] Primer pairs for NDRG4, BMP3, SFMBT2, VAV3, ZDHHC1, and β-actin for bisulfite conversion, such as... Figures 5A-5F As shown, each primer is 500 nM.

[0502] The pre-amplification cycle is shown below:

[0503]

[0504] After PCR, as described in Example 1, 10 μL of amplification reaction was diluted to 100 μL in 10 mM Tris and 0.1 mM EDTA, and the 10 μL diluted amplification product was used for standard PCR-valve assay.

[0505] Data shown Figures 8A-8C The results showed that no background was generated in the target-free control reaction, even at the highest cycle number. However, samples pre-amplified for 20 or 25 cycles showed a significant reduction in signal in PCR-valve assays.

[0506] Example 5

[0507] Multiplex targeted pre-amplification of DNA converted from large volumes of bisulfite

[0508] To pre-amplify mostly or all of the bisulfite-treated DNA from an input sample, a large volume of treated DNA can be used in a single, high-volume multiplex amplification reaction. For example, as described above, DNA can be extracted from cell lines (e.g., DFCI032 cell line (adenocarcinoma); H1755 cell line (neuroendocrine)) using, for example, the Maxwell Promega Blood Kit # AS1400. For example, the DNA is bisulfite-converted as described in Example 1.

[0509] Pre-amplification was performed in a reaction mixture containing 7.5 mM MgCl2, 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 dNTPs (e.g., equimolar amounts of 12 primer pairs / 24 primers, or adjusting individual primer concentrations to balance amplification efficiency for different target regions), 0.025 units / μL HotStart GoTaq concentration, and 20 to 50% by volume of bisulfite-treated target DNA (e.g., adding 10 μL of target DNA to 50 μL of reaction mixture, or adding 50 μL of target DNA to 125 μL of reaction mixture). The thermal cycling time and temperature were selected to suit the reaction and amplification vessel volumes. For example, the reaction could be cycled as follows:

[0510]

[0511] After thermal cycling, dilute an aliquot of the pre-amplified reaction (e.g., 10 μL) to 500 μL in 10 mM Tris, 0.1 mM EDTA. Use the diluted aliquot of the pre-amplified DNA (e.g., 10 μL) in a Quarts PCR-valve assay, for example, as described in Example 2.

[0512] Example 6

[0513] Multiplex targeted pre-amplification of bisulfite-converted DNA from fecal samples

[0514] The above multiplex pre-amplification method was used to test DNA isolated from human fecal samples.

[0515] Sample source:

[0516] i) Four DNA samples captured from fecal samples (see, for example, U.S. Patent No. 9,000,146) and treated with bisulfite according to Example 1 above, the samples having the following symptoms:

[0517] 500237 Primary tumor (AA)

[0518] 500621 Adenocarcinoma (ACA)

[0519] 780116 Normal

[0520] 780687 Normal

[0521] ii) Target-free control = 20 ng / μL of native fish DNA and / or 10 mM Tris, 0.1 mM EDTA;

[0522] 2. 50 μL PCR reaction mixture,

[0523] 3. Target regions tested: NDRG4, BMP3, SFMBT2, VAV3, ZDHHC1, and β-actin.

[0524] 4. Reaction conditions for pre-amplification and PCR-valve assay:

[0525] 7.5 mM MgCl2

[0526] 10 mM MOPS

[0527] 0.3 mM Tris-HCl, pH 8.0

[0528] 0.8 mM KCl,

[0529] 0.1 μg / μL BSA

[0530] 0.0001% Tween-20

[0531] 0.0001% IGEPAL CA-630

[0532] 250 μM dNTP)

[0533] 0.025 U / μl of GoTaq polymerase,

[0534] Primer pairs for NDRG4, BMP3, SFMBT2, VAV3, ZDHHC1, and β-actin for bisulfite conversion, such as... Figures 5A-5F As shown, each primer is 500 nM.

[0535] The pre-amplification cycle is shown below:

[0536]

[0537] After PCR, as described in Example 2, 10 μL of amplification reaction was diluted to 100 μL in 10 mM Tris and 0.1 mM EDTA, and the 10 μL diluted amplification product was used for standard PCR-valve assay.

[0538] Data shown Figure 9The results showed no background in the target-free control reaction. No signal of the methylated marker was detected in samples where the expected target biomarker was not methylated (normal samples), while the percentage of methylation detected in samples from subjects with adenomas or adenocarcinomas was consistent with results obtained using standard non-multiplex Quarts PCR-valve assays (i.e., without a separate pre-amplification step).

[0539] Example 6

[0540] Multiplex targeted pre-amplification of bisulfite-converted DNA from plasma samples

[0541] As described in Example 1, the DNA isolated from human plasma samples and treated with bisulfite was tested using the above-described multiplex pre-amplification method.

[0542] Experimental conditions:

[0543] 1. Sample source:

[0544] Seventy-five plasma samples, 2 mL each, were extracted from patients with colorectal or gastric cancer or normal patients and treated with bisulfite.

[0545] 2. 50 μL PCR reaction mixture,

[0546] 3. Target regions tested: NDRG4, BMP3, SFMBT2, VAV3, ZDHHC1, and β-actin.

[0547] 4. Reaction conditions for pre-amplification and PCR-valve assay:

[0548] 7.5 mM MgCl2

[0549] 10 mM MOPS

[0550] 0.3 mM Tris-HCl, pH 8.0

[0551] 0.8 mM KCl,

[0552] 0.1 μg / μL BSA

[0553] 0.0001% Tween-20

[0554] 0.0001% IGEPAL CA-630

[0555] 250 μM dNTP)

[0556] 0.025 U / μl of GoTaq polymerase,

[0557] Primer pairs for NDRG4, BMP3, SFMBT2, VAV3, ZDHHC1, and β-actin for bisulfite conversion, such as... Figures 5A-5F As shown, each primer is 500 nM.

[0558] The pre-amplification cycle is shown below:

[0559]

[0560] After PCR, as described in Example 2, 10 μL of amplification reaction was diluted to 100 μL in 10 mM Tris and 0.1 mM EDTA, and the 10 μL diluted amplification product was used for standard PCR-valve assay.

[0561] Data shown Figure 10A-10I middle. Figures 10A-10C The results obtained using multiple pre-amplification + PCR-valve assays were compared with those from the same samples in which no pre-amplification was performed. Figure 10D-10F The display shows the percentage of methylation calculated for each sample using multiplex pre-amplification + PCR-valve assay, while Figure 10G-10I The data show the percentage recovery of the input strand in multiplex pre-amplified + PCR-valve assays compared to results from the same sample measured using a PCR-valve assay without a pre-amplification step. For colorectal cancer using three biomarkers (VAV3, SFMBT2, ZDHHC1), these data show a sensitivity of 92% (23 / 25) at 100% specificity. Embodiments of the technology disclosed herein provide at least 100-fold or greater sensitivity in detecting DNA from blood compared to 250 copies measured using a Quarts PCR-valve assay without pre-amplification, for example, 2.5 copies from 4 mL of plasma.

[0562] Example 7

[0563] Exemplary scheme for whole blood-to-results analysis of plasma DNA

[0564] This embodiment provides an example of a complete method for isolating DNA from a blood sample for, for example, detection assays. Optional bisulfite conversion and detection methods are also described.

[0565] I. Blood processing

[0566] Collect whole blood in anticoagulant EDTA or Streck cell-free DNA BCT tubes. An exemplary procedure is as follows:

[0567] 1. Draw 10 mL of whole blood into a vacuum blood collection tube (with anticoagulant EDTA or Streck BCT) and collect the full volume to ensure the correct blood-to-anticoagulant ratio.

[0568] 2. After collection, gently mix the blood by turning it over 8 to 10 times to mix the blood and anticoagulant, and keep it at room temperature until centrifuged, which should happen within 4 hours after blood collection.

[0569] 3. Centrifuge the blood sample at 1500 g (±100 g) for 10 minutes in a horizontal rotor (with a swing head) at room temperature. Do not use the brake to stop the centrifuge.

[0570] 4. Carefully aspirate the supernatant (plasma) at room temperature and combine it in a centrifuge tube. Ensure that the cell layer is not disturbed or any cells are transferred.

[0571] 5. Carefully transfer 4 mL of the supernatant into a cryovial.

[0572] 6. Seal the bottle cap and place it on ice immediately after each division. This process should be completed within 1 hour of centrifugation.

[0573] 7. Ensure that the cryovial is fully labeled with relevant information, including detailed information about additives in the blood.

[0574] 8. Samples can be frozen at -20°C for up to 48 hours before being transferred to a -80°C freezer.

[0575] II. Preparation of synthetic control DNA

[0576] Using standard DNA synthesis methods such as phosphoramide addition, a 5-methyl C base was added at a specified position to synthesize a complementary strand of methylated zebrafish DNA with the sequence shown below. The synthesized strand was annealed to produce a double-stranded DNA fragment used as a treatment control.

[0577] A. Annealing and preparation of concentrated zebrafish (ZF-RASS F1 180 polymer) synthesis control

[0578]

[0579] 1. Reconstitute lyophilized single-stranded oligonucleotides at a concentration of 1 μM in 10 mM Tris, pH 8.0, and 0.1 mM EDTA.

[0580] 2. Prepare a 10X annealing buffer consisting of 500 mM NaCl, 200 mM Tris-HCl pH 8.0, and 20 mM MgCl2.

[0581] 3. Anneal the synthetic chain.

[0582] In a total volume of 100 μL, equimolar amounts of each single-stranded oligonucleotide were combined in 1X annealing buffer, as shown in the table below:

[0583]

[0584] 4. Heat the annealing mixture to 98°C for 11-15 minutes.

[0585] 5. Remove the reaction tube from the heat and briefly rotate it to collect the condensate to the bottom of the tube.

[0586] 6. Incubate the reaction tube at room temperature for 10 to 25 minutes.

[0587] 7. Add 0.9 mL of fish DNA diluent (20 ng / mL of native DNA in Te (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA)) to adjust the concentration of the zebrafish RASSF1 DNA fragment to 1.0 x 10⁻⁶. 10 Annealed, double-stranded synthetic zebrafish RASSF1 DNA (in a genomic DNA vector) was prepared at 1 copy / μl.

[0588] 8. For example, as shown in the table below, dilute the treatment control to the desired concentration with 10 mM Tris, pH 8.0, and 0.1 mM EDTA, and store at -20°C or -80°C.

[0589]

[0590] B. Preparation of 100x stock solution treatment control (12,000 copies / μL zebrafish RASSF1 DNA in 200 ng / μL of native fish DNA)

[0591] 1. Thawing reagent

[0592] 2. Reagents for vortexing and co-rotation defreezing

[0593] 3. Add the following reagents to a 50 mL conical tube.

[0594]

[0595] 4. Divide into labeled 0.5 mL tubes and store at -20°C.

[0596] C. Preparation of the stock solution for the 1x treatment control (120 copies / μL zebrafish RASSF1 DNA in 2 ng / μL fish DNA).

[0597] 1. Thawing reagent

[0598] 2. Reagents for vortexing and co-rotation defreezing

[0599] 3. Add the following reagents to a 50 mL conical tube:

[0600]

[0601] 4. Divide 0.3 mL into labeled 0.5 mL tubes and store at -20°C.

[0602] III. Extracting DNA from plasma

[0603] 1. Thaw the plasma, prepare reagents, label test tubes, and clean and set up the biosafety cabinet for extraction.

[0604] 2. Add 300 μL of proteinase K (20 mg / mL) to a 50 mL conical tube for each sample.

[0605] 3. Add 2-4 mL of plasma sample to each 50 mL conical tube (do not vortex).

[0606] 4. Vortex or mix with a pipette and let stand at room temperature for 5 minutes.

[0607] 5. Add 4-6 mL of lysis buffer 1 (LB1) solution to bring the volume to approximately 8 mL.

[0608] LB1 Formula:

[0609] A control was treated with 0.1 mL of zebrafish RASSF1 DNA at 120 copies / μL, as described above;

[0610] 0.9-2.9 mL of 10 mM Tris, pH 8.0, 0.1 mM EDTA (e.g., 2.9 mL for a 2 mL plasma sample).

[0611] 3 mL of 4.3 M guanidine thiocyanate containing 10% IGEPAL (from a stock solution of 5.3 g IGEPAL CA-630 combined with 45 mL of 4.8 M guanidine thiocyanate).

[0612] 6. Flip the tube 3 times.

[0613] 7. Place the tube on a benchtop oscillator (at room temperature) and run it at 500 rpm for 30 minutes at room temperature.

[0614] 8. Add 200 μL of silica-bound beads [16 μg particles / μL] and mix by vortexing.

[0615] 9. Add 7 mL of lysis buffer 2 (LB2) solution and mix by vortexing.

[0616] LB2 Formula:

[0617] 4 mL of 4.3 M guanidine thiocyanate mixed with 10% IGEPAL

[0618] 3 mL 100% isopropanol

[0619] (Lie buffer 2 can be added before, after, or simultaneously with the silica-bound beads)

[0620] 10. Flip the tube 3 times.

[0621] 11. Place the tube on a benchtop oscillator at 500 rpm for 30 minutes at room temperature.

[0622] 12. Place the tube on the capture aspirator and run the program to magnetically collect the beads for 10 minutes, then aspirate. This will collect the beads for 10 minutes, then remove all liquid from the tube.

[0623] 13. Add 0.9 mL of washing solution 1 (3 M guanidine hydrochloride or guanidine thiocyanate, 56.8% EtOH) to resuspend the bound beads and mix by vortexing.

[0624] 14. Place the tube on a benchtop shaker at 400 rpm for 2 minutes at room temperature.

[0625] (All subsequent steps can be performed on the STARlet automation platform.)

[0626] 15. By repeatedly aspirating and mixing, the mixture containing beads is then transferred to a 96-hole plate.

[0627] 16. Place the flat plate on the magnetic rack for 10 minutes.

[0628] 17. Discard the supernatant.

[0629] 18. Add 1 mL of washing solution 2 (80% ethanol, 10 mM Tris pH 8.0).

[0630] 19. Mix for 3 minutes.

[0631] 20. Place the tube on the magnetic rack for 10 minutes.

[0632] 21. Discard the supernatant.

[0633] 22. Add 0.5 mL of washing solution 2.

[0634] 23. Mix for 3 minutes.

[0635] 24. Place the tube on the magnetic rack for 5 minutes.

[0636] 25. Discard the supernatant.

[0637] 26. Add 0.25 mL of washing solution 2.

[0638] 27. Mix for 3 minutes.

[0639] 28. Place the tube on the magnetic rack for 5 minutes.

[0640] 29. Discard the supernatant.

[0641] 30. Add 0.25 mL of washing solution 2.

[0642] 31. Mix for 3 minutes.

[0643] 32. Place the tube on the magnetic rack for 5 minutes.

[0644] 33. Discard the supernatant.

[0645] 34. Place the plate on a 70°C heating block and shake for 15 minutes.

[0646] 35. Add 125 μL of elution buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA).

[0647] 36. Incubate at 65℃ with shaking for 25 minutes.

[0648] 37. Place the plate on the magnet to collect the beads and cool for 8 minutes.

[0649] 38. Transfer the eluent to a 96-well plate and store at -80°C. The recoverable / transferable volume is approximately 100 μL.

[0650] IV. DNA quantification before bisulfite treatment

[0651] To measure DNA in samples using the ACTB gene and to evaluate the recovery of zebrafish treatment controls, DNA can be measured prior to further treatment. The Quarts PCR-valve assay was set up using 10 μL of extracted DNA using the following protocol:

[0652] 1. Prepare a mixture of 10X oligomers containing 2 μM each of forward and reverse primers, 5 μM of probe and FRET cassette, and 250 μM each of dNTPs. (See primer, probe and FRET sequences below)

[0653]

[0654] 2. Prepare the main mixture as follows:

[0655]

[0656] * The 20X enzyme mixture contains 1 unit / μL of GoTaq hot-start polymerase (Promega) and 292 ng / μL of Leavase 2.0 valve endonuclease (Hologic).

[0657] 3. Use a pipette to transfer 10 μL of each sample into the wells of the 96-well plate.

[0658] 4. Add 20 μL of master mixture to each well of the plate.

[0659] 5. Seal the plate and centrifuge at 3000 rpm for 1 minute.

[0660] 6. Run the plate reaction under the following conditions on an ABI 7500 or Light Cycler 480 real-time thermal cycler.

[0661]

[0662] V. Bisulfite conversion and DNA purification

[0663] 1. In the future, all extracted DNA samples from the plasma DNA extraction step will be thawed and the DNA will be decanted.

[0664] 2. Reagent preparation:

[0665]

[0666] 3. Add 5 μL of 100 ng / μL BSA DNA vector solution to each well of the deep well plate (DWP).

[0667] 4. Add 80 μL of each sample to the DWP.

[0668] 5. Add 5 μL of freshly prepared 1.6 N NaOH to each well in the DWP.

[0669] 6. Mix carefully using a pipette set to 30-40 μL to avoid air bubbles.

[0670] 7. Incubate at 42℃ for 20 minutes.

[0671] 8. Add 120 μL of BIS SLN to each well.

[0672] 9. Incubate at 66°C for 75 minutes, mixing during the first 3 minutes.

[0673] 10. Add 750 μL of BND SLN

[0674] 11. Premix silica beads (BND BDS) and add 50 μL of silica beads (BND BDS) into the holes of the DWP.

[0675] 12. Mix at 1,200 rpm for 30 minutes on a heater shaker at 30°C.

[0676] 13. Collect the magnetic beads on a flat magnet for 5 minutes, then remove the waste liquid.

[0677] 14. Add 1 mL of washing buffer (CNV WSH), then transfer the plate to a heated shaker and mix at 1,200 rpm for 3 minutes.

[0678] 15. Collect the beads on a flat magnet for 5 minutes, then remove the solution and discard it.

[0679] 16. Add 0.25 mL of washing buffer (CNV WSH), then transfer the plate to a heated shaker and mix at 1,200 rpm for 3 minutes.

[0680] 17. Collect the beads on a flat magnet, then remove the solution and discard it.

[0681] 18. Add 0.2 mL of desulfonated buffer (DES SLN) and mix at 1,200 rpm for 7 minutes at 30°C.

[0682] 19. Collect the beads on the magnet for 2 minutes, then remove the solution and discard it.

[0683] 20. Add 0.25 mL of washing buffer (CNV WSH), then transfer the plate to a heated shaker and mix at 1,200 rpm for 3 minutes.

[0684] 21. Collect the beads on a magnet for 2 minutes, then remove the solution and discard it.

[0685] 22. Add 0.25 mL of washing buffer (CNV WSH), then transfer the plate to a heated shaker and mix at 1,200 rpm for 3 minutes.

[0686] 23. Collect the beads on the magnet for 2 minutes, then remove the solution and discard it.

[0687] 24. Dry the plate by transferring it to a heater shaker and incubating at 70°C for 15 minutes while mixing at 1,200 rpm.

[0688] 25. Add 80 μL of elution buffer (ELU BFR) to all samples in the DWP.

[0689] 26. Incubate at 65°C for 25 minutes while mixing at 1,200 rpm.

[0690] 27. Manually transfer the eluent to a 96-well plate and store at -80°C.

[0691] 28. The recyclable / transferable volume is approximately 65 μL.

[0692] VI. QuarTS-X for the detection and quantification of methylated DNA

[0693] A. Multiplex PCR (mPCR) settings:

[0694] 1. Prepare a 10X primer mixture containing forward and reverse primers for each target methylation marker (each primer having a final concentration of 750 nM). As described in the examples above, use 10 mM Tris-HCl, pH 8, and 0.1 mM EDTA as diluents.

[0695] 2. Prepare a 10X multiplex PCR buffer containing 100 mM MOPS, pH 7.5, 75 mM MgCl2, 0.08% Tween 20, 0.08% IGEPAL CA-630, and 2.5 mM dNTPs.

[0696] 3. Prepare the multiplex PCR master mixture as follows:

[0697]

[0698] 4. Thaw the DNA and drop the plate.

[0699] 5. Add 25 μL of the master mixture to a 96-well plate.

[0700] 6. Transfer 50 μL of each sample into each well.

[0701] 7. Use aluminum foil seals to seal the flat plate (without using a strip cap).

[0702] 8. Place it in a heated thermal circulator and continue the cycle using the following protocol for approximately 5 to 20 cycles, preferably approximately 10 to 13 cycles:

[0703]

[0704] 9. After thermal cycling, perform a 1:10 dilution of the amplicon as follows:

[0705] a. Transfer 180 μL of 10 mM Tris-HCl, pH 8, 0.1 mM EDTA into each well of the deep-well plate.

[0706] b. Add 20 μL of amplified sample to each pre-filled well.

[0707] c. Mix the diluted sample by repeatedly pipetting using fresh pipette tips and a 200 μL pipette (be careful not to generate aerosols).

[0708] d. Seal the diluted plate with a plastic seal.

[0709] e. Centrifuge the diluted plate at 1000 rpm for 1 minute.

[0710] f. Seal any remaining undiluted multiplex PCR product with a new aluminum foil seal. Store at -80°C.

[0711] B. Quarts assay of multiplexed DNA:

[0712] 1. Thaw the fish DNA diluent (20 ng / μL) and use it to dilute the plasmid calibrator required for the assay (see, for example, U.S. Patent Application Serial No. 15 / 033,803, which is incorporated herein by reference). Use the following table as a dilution guide:

[0713]

[0714] 2. Prepare a mixture of 10X triple Quarts oligomers for markers A, B, and C (e.g., target markers, plus running controls and internal controls such as β-actin or B3GALT6 (see, for example, U.S. Patent Application Serial No. 62 / 364,082, which is incorporated herein by reference) using the table below.

[0715]

[0716] For example, the following can be used to detect bisulfite-treated β-actin, B3GALT6, and zebrafish RASSF1 markers:

[0717]

[0718]

[0719] 3. Prepare the Quarts lobe for measuring the master mixture using the table below:

[0720]

[0721] *The 20X enzyme mixture contains 1 unit / μL of GoTaq hot-start polymerase (Promega) and 292 ng / μL of Leavase 2.0 valve endonuclease (Hologic).

[0722] 4. Using a 96-well ABI plate, pipette 20 μL of the QuARTS master mixture into each well.

[0723] 5. Add 10 μL of appropriate calibrator or diluted mPCR sample.

[0724] 6. Seal the flat plate with ABI clear plastic sealant.

[0725] 7. Centrifuge the plate at 3000 rpm for 1 minute.

[0726] 8. Place the plate in an ABI thermal cycler programmed to run the following thermal program, and then start the instrument.

[0727]

[0728] Example 8

[0729] Comparison of ionized salts in the first wash solution

[0730] During the development of this technology, the effects of using different ionizing salts in the first washing solution, such as guanidine thiocyanate, were compared with those of guanidine hydrochloride.

[0731] As described in Example 7, DNA was extracted from plasma samples using guanidine thiocyanate-ethanol or guanidine hydrochloride-ethanol as the first washing solution (i.e., 57% ethanol containing 3M guanidine hydrochloride or 3M guanidine hydrochloride). The samples were further treated as described in Example 7, and a portion of the DNA was converted to bisulfite. As described above, the amount of unconverted DNA obtained was measured using a Quarts PCR flap assay by detecting the treatment control and β-actin (ACTB), and as described above, the bisulfite-converted DNA was measured using multiplex pre-amplification and a Quarts PCR flap assay to detect the treatment control B3GALT6 and β-actin (BTACT). The results are shown in… Figure 11A-11C (Control data not shown). These data indicate that both solutions produced acceptable DNA yields, with guanidine thiocyanate-ethanol producing a higher yield.

[0732] Example 9

[0733] In the first washing step, ethanol containing guanidine thiocyanate or guanidine hydrochloride is compared with ethanol containing a buffer solution.

[0734] In the development of this technology, the effectiveness of using a mixture of ethanol and a dissociative salt solution (e.g., guanidine thiocyanate (GTC) or guanidine hydrochloride (GuHCl)) in the first washing step of plasma DNA extraction as described in Part III of Example 7 (i.e., using 57% ethanol containing 3 M guanidine hydrochloride (washing solution 1 in Part III of Example 7) or 50% ethanol containing 2.4 M guanidine thiocyanate) in the first washing step was compared with the effectiveness of using 80% ethanol (washing solution 2 in Part III of Example 7) containing 10 mM Tris HCl at pH 8.0 in the first washing step. As described in Example 7, an 80% ethanol-Tris buffer solution was used in subsequent washing steps.

[0735] Each washing condition was repeated 8 times. Samples were further treated as described in Example 7, and the DNA was not treated with bisulfite. The amount of DNA obtained was measured using the Quartz PCR flap assay to detect β-actin (ACTB) as described above. The results (average value of the detected DNA strands) are shown in the table below. These data indicate that using ethanol containing guanidine thiocyanate or guanidine hydrochloride in the first washing step, followed by additional washing with ethanol-buffered buffer, yields a higher yield than using ethanol-buffered buffer for all washing steps.

[0736]

[0737] Example 10

[0738] Tests involving the addition of lysis reagents in one or two steps

[0739] During the development of this technology, the effects of adding lysis reagents in one or two steps of the separation procedure were compared. Using aliquots of 2 mL or 4 mL from six different plasma samples, the first procedure included adding 7 mL of 4.3 M guanidine thiocyanate lysis reagent and 10% IGEPAL containing proteinase K, along with a treatment control, as described in Example 1. The plasma / proteinase / control mixture was incubated at 55°C for 60 minutes, followed by the addition of isopropanol. The second step involved adding an aliquot of 3 mL of 4.3 M guanidine thiocyanate, 10% IGEPAL containing proteinase, and a treatment control, incubating at 55°C, followed by the addition of 4 mL of the aliquot and isopropanol. The sample was then further incubated at 30°C for 30 minutes, and then processed as described in Example 1. A portion of the resulting DNA was converted to bisulfite as described above.

[0740] As described above, the amount of unconverted DNA obtained was measured using the Quarts assay to detect the treatment control and β-actin (ACTB), and the amount of bisulfite-converted DNA was measured using the multiplex pre-amplification and Quarts PCR-valve assay to detect the treatment control B3GALT6 and β-actin (BTACT). Results are shown in... Figures 12A-12C (Treatment control data not shown). The following shows the fold difference in mean yield between each test biomarker and the treatment control (PC):

[0741]

[0742] These data suggest that adding the lysis reagent in two steps (first in the absence of isopropanol, and second with isopropanol) yields a higher yield of detectable DNA.

[0743] All publications and patents mentioned in the foregoing description are incorporated herein by reference in their entirety for all purposes. 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 pertain. Where the definitions of terms in the incorporated references appear to differ from those provided in this teaching, the definitions provided in this teaching shall prevail.

[0744] Various modifications and variations to the composition, methods, and uses of the described technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology. While the technology has been described in conjunction with specific exemplary embodiments, it should be understood that the claimed technology should not be unduly limited to such specific embodiments. Indeed, various modifications to the described modes of implementation of the technology that will be apparent to those skilled in pharmacology, biochemistry, medical science, or related fields are intended to fall within the scope of the following claims.

Claims

1. A method for analyzing samples containing multiple target nucleic acids, the method comprising: a) Provide a sample of volume x, the sample containing bisulfite-treated DNA suspected of containing one or more of a plurality of n distinct target regions, wherein at least one of the target regions is a low-copy target, and if present in the sample, it is present in the sample at a copy number such that: i) Each of the n portions of the sample has a volume of x / n, and one or more of the n portions do not contain the low-copy target, or ii) Each of the n portions of the sample has a volume of x / n, and the low-copy target in one or more of the n portions is below the sensitivity level for the detection of the low-copy target; b) wherein, under the condition that the target regions are amplified to form a pre-amplified mixture having a volume y when the n different target regions are present in the sample, the volume x of the sample is treated to the amplification reaction; c) Dispense the pre-amplified mixture into a plurality of different detection reaction mixtures, wherein each detection reaction mixture contains a portion of the pre-amplified mixture having a volume of y / n or less, and wherein the low-copy target, if present in the sample in step a), is present in each of the detection reaction mixtures; d) Perform multiple detections using the detection reaction mixture, wherein the different target regions, if present in the sample in step a), are detected in the detection reaction mixture.

2. The method of claim 1, wherein the bisulfite-treated DNA is derived from a human subject.

3. The method according to claim 1 or 2, wherein the sample is prepared from body fluid.

4. The method of claim 3, wherein the body fluid comprises plasma.

5. A method for analyzing multiple target nucleic acids in a sample using PCR pre-amplification and PCR-valve assay, the method comprising: a) Bisulfite-treated DNA comprising multiple different target regions is provided in a first reaction mixture containing PCR amplification reagents, wherein the PCR amplification reagents comprise: i) Multiple different primer pairs for amplifying the multiple different target regions from the bisulfite-treated DNA when the target regions are present in the sample; ii) Thermostable DNA polymerase; iii) dNTPs; and iv) Contains Mg ++ buffer solution b) Exposing the first reaction mixture to thermal cycling conditions in which multiple different target regions, if present in the sample, are amplified to produce a pre-amplified mixture, and wherein the thermal cycling conditions are limited to multiple thermal cycles that maintain the amplification in an exponential range, preferably fewer than 20, more preferably fewer than 15, and even more preferably 10 or fewer. c) Dispensing the pre-amplified mixture into multiple PCR-lobe assay reaction mixtures, wherein each PCR-lobe assay reaction mixture contains: i) An additional amount of primer pairs selected from the plurality of different primer pairs described in step a)i); ii) Thermostable DNA polymerase; iii) dNTPs; iv) The one containing Mg ++ buffer solution v) Valvular endonuclease; vi) Valeroid oligonucleotides, and vi) A hairpin oligonucleotide comprising a region complementary to a portion of the valve oligonucleotide; and d) Detect amplification of one or more different target regions from the bisulfite-treated DNA during the PCR-valve assay reaction.

6. The method of claim 5, wherein the pre-amplified mixture is diluted with a diluent prior to the dispensing.

7. A method for processing plasma samples, the method comprising: a) Combine the plasma sample with the following reagents: i) Protease; ii) A first lysis reagent, the first lysis reagent comprising - Guanidine thiocyanate, - Nonionic detergents; To form a mixture in which the proteins are digested by the protease; b) Adding to the mixture of step a) under conditions in which DNA is bound to the silica particles. iii) Silica particles, and iv) A second lysis reagent, comprising: - Guanidine thiocyanate; - Nonionic detergents; and - Isopropanol; c) Separate silica particles with bound DNA from the mixture in b); d) Add a first washing solution to the separated silica particles containing bound DNA, the first washing solution comprising: i) guanidine hydrochloride or guanidine thiocyanate, and ii) ethanol; e) Separate the silica particles containing bound DNA from the first washing solution; f) Add a second washing solution containing a buffer and ethanol to the isolated silica particles containing bound DNA; g) Separate the washed silica particles with bound DNA from the second washing solution; and h) Elute the DNA from the washed silica particles containing bound DNA to produce a separated DNA sample.

8. A kit for isolating DNA from plasma, said kit comprising: a) A first pyrolysis reagent comprising guanidine thiocyanate and a nonionic detergent or a component used to prepare the first pyrolysis reagent; b) A second cleavage reagent comprising guanidine thiocyanate, a nonionic detergent, and isopropanol, or components used to prepare the second cleavage reagent; c) A first washing solution comprising i) guanidine hydrochloride or guanidine thiocyanate and ii) ethanol or components used to prepare the first washing solution; d) A second wash solution containing Tris buffer and ethanol or the components used to prepare the second wash solution; and e) Silica particles.

9. A system for processing plasma samples, the system comprising: a) A first pyrolysis reagent comprising guanidine thiocyanate and a nonionic detergent or a component used to prepare the first pyrolysis reagent; b) A second cleavage reagent comprising guanidine thiocyanate, a nonionic detergent, and isopropanol, or components used to prepare the second cleavage reagent; c) A first washing solution comprising i) guanidine hydrochloride or guanidine thiocyanate and ii) ethanol or components used to prepare the first washing solution; d) A second wash solution containing Tris buffer and ethanol or the components used to prepare the second wash solution; and e) Silica particles.

10. The system of claim 9, further comprising PCR amplification reagents and / or PCR flap assay reagents, wherein the PCR amplification reagents comprise: i) Used to amplify multiple different primer pairs of multiple different target regions when the target region is present in the plasma; ii) Thermostable DNA polymerase; iii) dNTPs; and iv) Contains Mg ++ buffer solution; And the PCR valve assay reagent contains: i) Used to amplify multiple different primer pairs of multiple different target regions when the target region is present in the plasma; ii) Thermostable DNA polymerase; iii) dNTPs; iv) Contains Mg ++ buffer solution v) Valvular endonuclease; vi) Valeroid oligonucleotides, and vi) A hairpin oligonucleotide comprising a region complementary to a portion of the valve oligonucleotide.

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