Detection of target nucleic acid using pre-amplification

Abstract

The present invention provides a method for detecting low-abundance nucleic acids including structural array variants and mutations. The present invention provides pre-amplification of structural variants or mutations that may be present at low concentrations in a sample prior to detection of the structural variants or mutations using other methods such as conventional PCR or digital PCR. After the pre-amplification step, advantageously PCR follows, resulting in a reduction of false negatives due to sample detection. As a result, the method of the present invention enhances the sensitivity for sample detection, particularly for detecting the presence of low-abundance targets in biological samples.

Description

Technical Field

[0001] Technical Field The present invention relates to the field of methods for detecting low-abundance target nucleic acids.

Background Art

[0002] Background The detection of nucleic acids present in very low amounts and / or at low frequencies is desirable for many applications. For example, the detection of gene mutations is important for numerous diseases such as cystic fibrosis, sickle cell anemia, and cancer. In particular, there is an increasing recognition of the need for methods for mutation detection that are highly sensitive and specific, especially in low-input samples such as circulating cell-free DNA (cfDNA) and single-cell analysis. Conventional methods can have various problems, including high requirements for the amount of input sample DNA, high cost per sample, complex and labor-intensive workflows, insufficient sensitivity and / or specificity, and the inability to detect low-abundance mutant sequences (so-called mutant allele frequencies; MAF) within a high background of normal wild-type sequences. Most detection methods rely on nucleic acid amplification using the polymerase chain reaction (PCR), which exponentially copies the region of interest.

[0003] Digital PCR (dPCR) is a method of partitioning a PCR reaction into a large number of smaller individual reactions such that each reaction compartment contains only from zero to a very small number of target sequence molecules. The partitioning of all molecules is random and follows a Poisson distribution. The partitioning converts the situation of extremely low relative abundance of rare variant sequences among a large number of wild-type sequences to a situation where most compartments have only wild-type sequences and some compartments have rare variant sequences with a very high relative abundance compared to the wild-type sequences. The result is a potential improvement in sensitivity. However, dPCR does not necessarily detect the presence of low-abundance target nucleic acids in biological samples. As a result, individuals suffering from diseases that require the detection of low-abundance target nucleic acids in biological samples continue to suffer.

Summary of the Invention

Means for Solving the Problem

[0004] Summary The present invention provides a method for detecting low-abundance nucleic acids including structural variants and mutants. The present invention uses selective pre-amplification to increase the abundance of a target of interest in a sample even when the target is present in very small numbers. The present invention solves certain problems associated with molecular detection assays where targets present in very small amounts cannot be detected due to the probabilistic nature of PCR, and where the "dead volume" of PCR becomes a problem. These problems are addressed by selectively amplifying the target of interest, as described herein as the pre-amplification step. The pre-amplification step can take one of two basic forms. It can be non-exponential, for example, it can be non-PCR. For example, the pre-amplification step can be incremental, which can be taken to mean that the extension product from one round of pre-amplification is not copied using any reverse primer or is not a substrate for copying by any reverse primer. In other preferred embodiments, the pre-amplification step is exponential. In any case, the ratio of rare targets is increased as a result of the pre-amplification step.

[0005] It may be preferable to perform pre-amplification on the selected target while preserving substances that are not targets for subsequent amplification steps. Preferred embodiments use pre-amplification specific to clinically important sequences, such as structural variants associated with tumors. Indeed, embodiments of the present invention involve identifying tumor mutations and selecting one or more structural variants.

[0006] Variant detection according to the methods of the present disclosure is based on selecting a target variant and performing a pre-amplification step directed to the variant. In one aspect, the pre-amplification step is essentially a variant enrichment step, thereby providing a sample with a sufficient amount of the rare variant for detection. Such pre-amplification can amplify only the selected target by a limited amount, a controlled amount, or a known amount. For example, 10 cycles of linear pre-amplification using primers specific for the variant increases the copy of that variant in the sample by about 10-fold. Since the abundance is increased by a known amount, if the sequences in the reaction mixture are later quantified (e.g., by digital PCR), the amount of those sequences in the original sample can be determined (using the known increased amount of the selected variant's abundance). Further, the pre-amplification step significantly increases the probability of detecting rare variants in the sample. The sample can then be assayed by a PCR-based method such as digital PCR (dPCR) for the selected variant and any other variants potentially present in the sample. Indeed, embodiments of the present invention can be multiplexed, for example, using differently labeled fluorescent hydrolysis probes, to examine the sample for multiple variants simultaneously. Since one or more of the selected variants are increased by incremental pre-amplification in relative abundance, they are not lost due to the dead volume or stochastic effects of PCR. Since the increase due to pre-amplification is a known increase, dPCR gives a quantitative readout of the selected one or more variants in the sample. Thus, the assays of the present invention are useful for quantitatively detecting rare targets in a sample, and can be particularly useful for detecting cancer-specific mutations, such as in circulating tumor DNA (ctDNA) in blood or plasma samples (e.g., liquid biopsies). Further, as described later herein, the incremental pre-amplification and exponential amplification of dPCR can proceed without the need for a clean-up step, reagent change, or addition of reagents as the assay progresses, in the presence of the same set of reagents (primers, dNTPs, polymerase, ions, probes...).In fact, the specific embodiments discussed herein use primer pairs that function to incrementally amplify a target under one thermal cycle and exponentially amplify the target for dPCR under different thermal cycles.

[0007] In related embodiments, the present invention provides the identification and ranking of influential or trunk variants among the first variants in tumorigenesis and tumor progression. These first trunk variants or rearrangements are selected and prepared and detected in such a way as to preferentially increase their copy number in a sample. The present invention acknowledges that initial chromosomal rearrangements predominate over point mutations and are detectable by the methods of the present invention. The methods of the present invention enable the detection of low-concentration trunk variants in biological samples.

[0008] The methods of the present invention overcome the probabilistic effects of PCR and the problem of dead volume by pre-amplifying one or more targets of interest to increase the abundance of one or more targets. The increase in abundance is controlled such that the resulting abundance is sufficient to fall within the dynamic range of subsequent quantitative PCR methods, such as digital PCR. The breadth process is performed on the structural sequence rearrangements detected by subsequent digital PCR. These processes are designed to increase the copy number of target biomarkers and reduce noise that often makes the detection of low-frequency sequences in a sample difficult. The methods of the present invention can use the entire pre-amplification reaction product as an input to exponential amplification. In other words, no clean-up is required between amplification steps and all nucleic acids in the sample are used as an input to subsequent processes. In one embodiment, the present invention is performed using pre-amplification of variants in the sample that may be present at low concentrations in the sample prior to exponential amplification. The methods of the present invention improve the sensitivity in sample detection, particularly when detecting the presence of low-abundance targets in biological samples.

[0009] The present invention enables the detection of nucleic acids in any sample containing one or more target nucleic acids. Preferred methods of the present invention include whole genome sequencing of tumor nucleic acids and identifying truncating variants in the tumor sequences. The truncating variants are then ranked based on a number of factors and prioritized in terms of their utility as a timeline of tumor-specific biomarkers of tumor progression. The ranked and filtered truncating variations are selected and analyzed to determine their impact on tumorigenesis. The analysis is preferably achieved via PCR, preferably via digital PCR. The present invention enables the identification of early truncating variants in biological samples as biomarkers for analysis. In one aspect, preamplification sample preparation for preferentially increasing the copy number of a selected biomarker is performed on the nucleic acids in the sample to increase the copy number of the target nucleic acid (e.g., variant) in the sample. The sample is then divided into a plurality of subsamples and PCR / dPCR is performed on the subsamples. The target nucleic acid in the sample is then analyzed and / or detected.

[0010] Preferred methods of the present invention include a preamplification step. By way of example, the preamplification can be an asymmetric incremental amplification or a symmetric exponential amplification, or a combination of the two. Incremental preamplification can involve the use of only a single primer per target of interest or can use primer pairs where only one primer is active in the preamplification. In one example, the active primer is active due to the difference in the melting temperature (Tm) of the primers provided and the annealing temperature.

[0011] Asymmetric incremental amplification can include providing a primer pair capable of amplifying a target nucleic acid. For example, the primer pair can include primer H and primer L. The melting temperature of primer H can be about 10°C to about 22°C higher than the melting temperature of primer L. Primer L can include a sequence complementary to a fragment of the extension product of primer H. The melting temperature of primer H can generally be about 10°C higher than the melting temperature of primer L. For example, the melting temperature of primer H can be about 10°C higher than the melting temperature of primer L, about 12°C higher than the melting temperature of primer L, or about 16°C higher than the melting temperature of primer L.

[0012] The sample preparation reaction (e.g., incremental amplification and / or exponential amplification) can also include a nucleic acid polymerase having polymerase activity, one or more additional primers, and reagents commonly used in standard PCR.

[0013] For example, asymmetric incremental amplification can include a primer set, and at least one primer can specifically amplify only one strand of the target nucleic acid sequence. With the inclusion of the primer, a sample preparation reaction can then be carried out in a solution containing a nucleic acid polymerase having polymerase activity. The sample preparation reaction can also include a nucleic acid polymerase having polymerase activity, one or more primers, and reagents commonly used in standard PCR.

[0014] In the method of the present invention, symmetric exponential amplification can also be used. Symmetric exponential amplification can include providing a primer set capable of specifically amplifying a target nucleic acid sequence. A nucleic acid polymerase having polymerase activity at the extension temperature is then provided to the sample. Then, the sample preparation reaction is prepared to include a portion of the sample, the primer set, the nucleic acid polymerase, and reagents commonly used in standard PCR; and the sample preparation reaction is carried out.

[0015] Asymmetric incremental amplification can be followed by, or combined with, symmetric exponential amplification. Asymmetric incremental amplification can follow symmetric exponential amplification. Asymmetric incremental amplification and symmetric exponential amplification can also be performed in the same reaction. This advantageously enables asymmetric incremental amplification and symmetric exponential amplification, which are performed using the same primer set.

[0016] Asymmetric incremental amplification can be activated at a higher temperature when compared to symmetric exponential amplification. For example, one or more primers for asymmetric incremental amplification can have a different annealing temperature when compared to the annealing temperature of one or more primers for symmetric exponential amplification. Additionally, the thermal cycling conditions for asymmetric incremental amplification can have a different annealing temperature when compared to the annealing temperature for symmetric exponential amplification.

[0017] The invention further includes performing a plurality of PCR reactions on a pre-amplified sample. The PCR step after pre-amplification can include a primer pair capable of specifically amplifying the target nucleic acid and a nucleic acid polymerase having polymerase activity at the extension temperature. Each PCR reaction includes a portion of the sample, a primer set, a nucleic acid polymerase, and PCR reagents. The PCR step can include digital PCR (dPCR).

[0018] Amplification products generated during pre-amplification steps, such as variant enrichment sample preparation reaction steps, can be used as direct sample input for PCR. That is, after pre-amplification, reagents for PCR can be added to the reaction mixture derived from pre-amplification. For example, if pre-amplification is performed in a tube, after pre-amplification, additional reagents useful in PCR can be added to the tube (without cleanup) by topping up the tube. In some embodiments, the primers used in the present invention are part of a plurality of primer pairs capable of amplifying different target nucleic acid sequences. After adding any additional reagents to the tube as needed, pre-amplification can then be performed under a first temperature control, and PCR can be performed under a second temperature control without any cleanup as needed. The method of the present invention can be performed without any cleanup step that could lead to loss of analytical material.

[0019] The method of the present invention may further include the use of a plurality of primer pairs capable of amplifying different target nucleic acid sequences. In certain embodiments, the method of the present invention may include the use of multiplex PCR.

[0020] The method of the present invention can provide that one or more targeted nucleic acid molecules are related to a variant sequence, which can be a variant of a wild-type nucleic acid sequence. The variant sequence can be selected from the group consisting of single nucleotide variants (SNVs), insertions and deletions (indels), duplications, copy number variants (CNVs), inversions, fusions, and translocations. The method can also be used to analyze cfDNA samples from cancer patients and can include variants or structural variants derived from cfDNA as needed.

[0021] The specific method of the invention includes sample preparation for preferentially increasing the copy number of target nucleic acids in a sample prior to PCR. As a result of the targeted sample preparation, an increased copy number of the target is available for the PCR reaction. The advantage of the sample preparation is to reduce the probabilistic bias in samples having a low concentration of the target. Further, the advantage is to reduce the problem of dead volume, since the probability that the target is present only in the dead volume is significantly reduced.

[0022] The present invention also provides a reference assay for the detection of structural variants or mutants of a patient's cfDNA. In particular, the present invention provides a method for detecting wild-type cfDNA as a baseline. Thus, aspects of the present invention provide an assay that includes the use of primer pairs and probes to detect and / or quantify the wild-type (reference) sequence of cfDNA.

[0023] The present invention further provides an assay for calculating the variant allele frequency (VAF) for a target nucleic acid in a sample. Embodiments of the method of the present invention include the calculation of the efficiency of replication, thereby quantifying the amount of target nucleic acid present in the sample. The present invention further provides two positive control reactions between pre-amplifications. The first positive control includes positive control DNA in the assay such that pre-amplification occurs. The second positive control includes an equal amount of positive control DNA without primers. The first positive control and the second positive control are technical replicates, except that the first positive control receives one or more primers. Thereafter, the concentration of the replication containing primers is divided by the replication without primers to estimate the efficiency of the assay. The calculated efficiency can be applied to the measurements performed on the actual sample. As a result, the method of the present invention can be used to quantify the amount of target nucleic acid in a sample.

Brief Description of the Drawings

[0024]

Figure 1

Figure 2

DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description The present invention provides a method for detecting low-abundance nucleic acids, including structural variants and mutants. In one aspect, the present invention provides a sample preparation method that preferentially increases the copy number of rare structural variants or mutants that may be present at low concentrations in a sample prior to digital PCR (dPCR). dPCR following a sample preparation step that functions to incrementally amplify the variant of interest results in a reduction in false-negative results due to the sample. As a result, the method of the present invention improves the sensitivity for sample detection, particularly for detecting the presence of low-abundance targets in biological samples.

[0026] As described above, the method of the present invention is particularly useful for detecting low-abundance target nucleic acids in a sample. The target nucleic acid can be any nucleic acid sequence whose presence it is desirable to detect. The target nucleic acid or variant can be, for example, a nucleic acid sequence associated with a clinical condition. In particular, variants associated with early tumorigenesis (truncated variants) are identified and ranked according to a number of factors. The selected truncated variants are amplified for detection and characterization. Characterization can include reporting on the tumor and the patient's clinical status (e.g., whether it is primary, recurrent, evidence of minimal residual disease).

[0027] As described in FIG. 1, the present invention provides, in one non-limiting example, a method comprising the following steps: (i) providing a sample containing one or more target nucleic acids, (ii) performing pre-amplification to selectively increase the copy number of the target nucleic acids in the sample, (iii) dividing the sample into a plurality of subsamples, each in its own compartment, (iv) performing polymerase chain reaction (PCR) on the subsamples, and (v) detecting the target nucleic acids. Preferably, the PCR step (iv) is digital PCR using a fluorescent probe, such as a hydrolysis probe, that indicates the presence of the amplification product generated in each compartment.

[0028] The method of the present invention is useful for detecting whether a variant sequence is present in a sample that may contain a mixture of target nucleic acids in which only a portion of the target nucleic acids may contain the variant sequence. In particular, the method is useful for detecting the presence of a variant sequence in a sample containing a target nucleic acid in which only a small portion of the target nucleic acid may potentially contain the variant sequence.

[0029] The present invention also provides a method for detecting the presence of a target nucleic acid sequence in a sample. The method may be useful for detecting whether a target nucleic acid sequence is present in the sample. The sample may contain a mixture of template nucleic acids that potentially contain the target nucleic acid sequence. In particular, the method is useful for detecting the presence of the target nucleic acid sequence in the sample, which may potentially contain only a very low level of said target nucleic acid sequence.

[0030] The target nucleic acid sequence can be any target nucleic acid sequence that is desired to be detected. For example, the presence of the target nucleic acid sequence may be related to a clinical condition.

[0031] According to the method of the present invention, a method for detecting the presence of a target nucleic acid sequence, the method comprising providing a sample containing one or more target nucleic acids and performing pre-amplification to increase the copy number of the target nucleic acid in the sample. Thereafter, the pre-amplified sample is divided into a plurality of subsamples, and PCR (preferably dPCR) is performed on the subsamples to detect the target nucleic acid.

[0032] Figure 2 provides an exemplary workflow showing sample acquisition, bioinformatics workflow, and sample processing. This exemplary workflow shows tumor low-pass whole genome sequencing as a first step, followed by filtering and selection of targets that can be structural variants or mutations (or combinations thereof).

[0033] The method of the present invention includes the use of a stepwise nucleic acid pre-amplification step as targeted pre-amplification, i.e., increasing the relative abundance of the target within the sample. The use of targeted pre-amplification increases the copy number of the target nucleic acid in the sample. The pre-amplification can be symmetric or asymmetric, or a combination of both. In asymmetric or stepwise amplification, a single strand of nucleic acid is linearly copied without preferentially copying the other strand between samples. Some embodiments perform stepwise amplification by providing only one primer that copies the target (instead of a pair of forward and reverse PCR primers). Certain embodiments discussed herein perform asymmetric amplification using primer H and primer L. See also U.S. Patent No. 11,066,707 and U.S. Publication No. 2022 / 0056533A1, both of which are incorporated by reference.

[0034] The method of the present invention has a very low detection limit. This enables the detection of target nucleic acid sequences that potentially exist at very low levels and / or the detection of the presence of variant sequences that potentially exist at very low levels in a mixture containing other target nucleic acid sequences.

[0035] Samples and Target Nucleic Acids The sample can be any sample for which it is desirable to detect whether the variant sequence is present. For example, if the variant sequence indicates a clinical condition, the sample can be a sample from an individual at risk of acquiring the clinical condition. The variant sequence can differ from the wild-type sequence by one or more substitutions, one or more deletions, and / or one or more insertions.

[0036] The method of the present invention further provides that one or more targeted nucleic acid molecules are related to the variant sequence, which can be a variant of the wild-type nucleic acid sequence. The variant sequence is selected from the group consisting of single nucleotide variants (SNVs), insertions and deletions (indels), duplications, copy number variants (CNVs), inversions, and translocations. The method of the present invention can also be used to analyze cfDNA samples derived from cancer patients and, optionally, contains variants or structural variants derived from cfDNA.

[0037] For polymorphisms or small indels (usually, < about 50 bases), it may be preferred that both the wild-type sequence and the variant sequence are amplified during the polymerase reaction by a primer pair that can specifically amplify the target nucleic acid sequence. The wild-type sequence and the variant sequence may preferably not differ too much from each other in length. For structural rearrangements (usually at least about 50 bases), the variant is amplified by a stepwise increment prior to digital PCR, and the assay also includes, in parallel, a reference assay that targets a stable region of the genome to quantify the signal. Structural variants are incorporated by reference as described in Mahmoud, 2019, Structural variant calling: the long and the short of it, Genome Biology 20:a246.

[0038] The sample can be any biological sample including a body fluid sample containing bile, blood, plasma, serum, sweat, saliva, urine, feces, sputum, mucus, phlegm, tears, cerebrospinal fluid, synovial fluid, pericardial fluid, lymphatic fluid, semen, vaginal secretion, lactation or physiological products, amniotic fluid, pleural fluid, mucosal secretion, or vomitus.

[0039] Amplification and pre-amplification The method of the present invention involves the use of amplification. Amplification of nucleic acids is the generation of copies of said nucleic acids.

[0040] The method of the present invention also includes a pre-amplification step. The pre-amplification can be described as a preparation reaction step for variant enrichment samples. Such pre-amplification can amplify only a limited, controlled, or known amount of selected targets. For example, 10 cycles of linear pre-amplification using primers specific to the variant increases the abundance of copies of that variant in the sample by about 10-fold. Since the abundance is increased by a known amount, when the sequences in the reaction mixture are later quantified (e.g., by digital PCR), the amount of those sequences in the original sample can be determined (using a known amount by which the abundance of the selected variant is increased). Furthermore, pre-amplification significantly increases the probability of detecting those variants, which helps in detecting very rare sequences in the sample. Therefore, it may be preferable for the pre-amplification to specifically increase the abundance of copies of the selected variant of the sample by a known amount. According to the present invention, the pre-amplification step is a sample preparation for preferentially increasing the copy number of the selected target through asymmetric incremental amplification or symmetric exponential amplification, or a combination of the two. Incremental pre-amplification may be expected to proceed less than linear copy amplification compared to the exponential generation of amplification products in PCR. Incremental amplification can proceed using asymmetric primers (e.g., a single primer) that extend and copy through the target of interest. In some embodiments, incremental pre-amplification includes the use of at least two primers, with only one primer being active in the pre-amplification. The active primer can be active due to the difference in the melting temperature (Tm) and annealing temperature of the primer. The use of these primers for asymmetric incremental amplification is disclosed in U.S. Patent No. 11,066,707, which is hereby incorporated by reference in its entirety.

[0041] The method of the present invention provides that a nucleic acid polymerase enzyme can have different extension temperatures. The extension temperature is the temperature at which the enzymatic activity of the nucleic acid polymerase is recognized after annealing of the primer. Usually, a nucleic acid polymerase has activity over a temperature range, and thus the extension temperature can be any temperature within that range. Most nucleic acid polymerases have temperature optimum conditions, but retain activity at temperatures other than the temperature optimum conditions. In such cases, the extension temperature can be any temperature at which the primer anneals and the nucleic acid polymerase has activity, even if the temperature is not the optimum temperature condition. At the extension temperature of the nucleic acid polymerase, the enzyme can catalyze the synthesis of a new nucleic acid strand complementary to the template strand at the extension temperature. The present invention provides that the extension temperature is near the melting temperature of primer H. Thus, a nucleic acid polymerase having polymerase activity at a temperature near the melting temperature of primer H and / or designed such that primer H has a melting temperature near the extension temperature can be selected.

[0042] Amplification products or products generated during pre-amplification steps, such as the preparation reaction step of the variant enrichment sample, can be used as a direct sample input for PCR. That is, after pre-amplification, reagents for PCR can be added to the reaction mixture derived from pre-amplification. For example, if pre-amplification is performed in a tube, after pre-amplification, the tube can be topped up (without cleanup) to add any additional reagents useful in PCR. In some embodiments, the primers used in the present invention are part of a plurality of primer pairs capable of amplifying different target nucleic acid sequences. Pre-amplification can be performed under a first temperature control, and then, optionally without any cleanup, after adding any further reagents to the tube as needed, PCR can be performed under a second temperature control. The method of the present invention can be performed without any cleanup steps that can lead to loss of the analytical material.

[0043] The method of the present invention involves the use of PCR reagents for a sample preparation reaction. The sample preparation reaction should include at least a part of the sample, a primer set, and sufficient PCR reagents to enable a polymerase reaction. Methods and reagents useful for performing the PCR reaction are well known to those skilled in the art. For example, the PCR reaction may include a nucleic acid polymerase and any of the PCR reagents described in the following "PCR Reagents" section herein. Depending on the mode of detecting whether the sample preparation product contains a variant sequence, the sample preparation reaction may also include detection reagents. PCR reagents are reagents added to the PCR in addition to the nucleic acid polymerase, the sample, and the primer set. PCR reagents include at least nucleotides. Further, the PCR reagents may include one or more salts and other compounds such as one or more buffers.

[0044] For most purposes, PCR reagents contain nucleotides. Thus, the PCR reagents may include all of the deoxynucleoside triphosphates (dNTPs), particularly the four naturally occurring deoxynucleoside triphosphates (dNTPs).

[0045] PCR reagents often contain deoxyribonucleoside triphosphate molecules including dATP, dCTP, dGTP, dTTP. In some cases, dUTP is added.

[0046] PCR reagents may also include compounds useful in assisting the activity of the nucleic acid polymerase. Thus, the PCR reagents may include divalent cations, for example, magnesium ions. The magnesium ions may be added, for example, in the form of magnesium chloride or magnesium acetate (MgCl2), or magnesium sulfate is used.

[0047] PCR reagents may also include one or more of the following: · Nonspecific blockers such as BSA or gelatin from bovine skin, beta-lactoglobulin, casein, dried milk, or other common blockers · Nonspecific background / blocking nucleic acids (e.g., salmon sperm DNA) · Biological preservatives (e.g., sodium azide) · PCR enhancers (e.g., betaine, trehalose, etc.) · Inhibitors (e.g., RNase inhibitor)

[0048] PCR reagents may also contain other additives, such as dimethyl sulfoxide (DMSO), glycerol, betaine (mono) hydrate (N,N,N-trimethylglycine = [carboxy-methyl] trimethylammonium), trehalose, 7-deaza-2'-deoxyguanosine triphosphate (dC7GTP or 7-deaza-2'-dGTP), formamide (methanamide), tetramethylammonium chloride (TMAC), other tetraalkylammonium derivatives (e.g., tetraethylammonium chloride (TEA-Cl) and tetrapropylammonium chloride (TPrA-Cl)), nonionic detergents (e.g., Triton® X-100, Tween® 20, Nonidet P-40 (NP-40)), or PREXCEL-Q.

[0049] PCR reagents may contain buffering agents.

[0050] In some cases, nonionic ethylene oxide / propylene oxide block copolymers are added to the aqueous phase at concentrations of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%. Examples of common biological surfactants include nonionic surfactants such as Pluronic® F-68, Tetronics, Zonyl FSN, etc. Pluronic® F-68 can be present at a concentration of about 0.5% w / v.

[0051] A wide range of commonly available commercial PCR buffers from various suppliers can be used instead of the buffer solution.

[0052] The method of the present invention also involves the use of a nucleic acid polymerase. The nucleic acid polymerase can be any nucleic acid polymerase, such as a DNA polymerase. The nucleic acid polymerase should be active at the extension temperature.

[0053] The nucleic acid polymerase can be a DNA polymerase having 5'-to-3' exonuclease activity. This can be the case particularly in the method of the present invention, and the method or kit involves the use of a detection probe such as a Taqman detection probe.

[0054] Any DNA polymerase, for example, a DNA polymerase having 5'-to-3' exonuclease activity that catalyzes primer extension, can be used. For example, a heat-resistant DNA polymerase can be used. Preferably, the nucleic acid polymerase is Taq polymerase.

[0055] The method of the present invention includes a sample preparation reaction step that can be either an asymmetric incremental amplification or a symmetric exponential amplification, or a combination of the two. Asymmetric incremental amplification can proceed using unpaired, for example, single primers (even if multiple primers are used, each can be "single" in the sense that it is not paired with a reverse primer that anneals to the extension product of the single primer). In some embodiments, the asymmetric incremental pre-amplification can include the use of at least two pairs, with only one primer being active in the sample preparation reaction. For example, the active primer becomes active due to the difference in the melting temperature (Tm) and annealing temperature of the provided primers.

[0056] More preferably, the asymmetric incremental amplification step comprises (i) providing a primer pair capable of amplifying a target nucleic acid, the primer pair comprising primer H and primer L. According to the method of the present invention, the melting temperature of primer H can be about 10°C to about 22°C higher than the melting temperature of primer L, primer L comprises a sequence complementary to a fragment of the extension product of primer H, and a sample preparation reaction is carried out. The sample preparation reaction also includes a nucleic acid polymerase having polymerase activity, one or more primers, and PCR reagents.

[0057] In embodiments using primer H and primer L, the incremental amplification proceeds using an annealing temperature at which primer H anneals (whereas primer L does not anneal). During that incremental amplification, primer H functions as a "single" primer (despite the presence of primer L). Since the reaction mixture is not lowered to the annealing temperature of primer L, primer L does not anneal to any significant extent. The incremental pre-amplification can be carried out for a predetermined number of cycles (e.g., 1, 5, etc.), or for a fixed amount of time, or until the sample shows a result (change in optical density, cleavage of a fluorescent probe, etc.). After the incremental amplification, the reaction mixture can then be subjected to exponential amplification. For exponential amplification, in the annealing step, the temperature is lowered to the annealing temperature of primer L that promotes annealing of both primer H and primer L.

[0058] Since primer H and primer L can function among many other primers, it is important to note that the previous paragraph describes the functions of primer H and primer L. For example, dozens, hundreds, thousands, or more loci can be examined in parallel using the corresponding number of primer pairs. For example, assume that 24 loci are being examined in parallel (however, the number 24 is arbitrary and could equally well be 1, or 2, or 3, or 6, or 17, or 96, or 99, or 384, or 1,000, or 1,536, or any integer multiple of these numbers, etc.). The assay of the present invention can use primer pairs for each locus, for example, 24 primer pairs can be used. Any one or any number of those primer pairs can conform to the description of primer H and primer L. However, although this is important, since it may be desirable to pre-amplify only a specific locus, any number of primer pairs can include a forward primer and a reverse primer each having an annealing temperature essentially the same as that for primer L.

[0059] In other embodiments, pre-amplification proceeds with an unpaired "single" primer (e.g., it operates at the annealing temperature of primer H). Thereafter, the reaction mixture (original sample, additional reagents, further pre-amplification products) can be subjected to conditions for exponential amplification. Exponential amplification can use primer pairs, any one of which can "match" all or part of the single primer, or the primer pair anneals to a target within the length of the extension product of the unpaired single primer. These reactions can proceed at different temperatures, or the paired primers may not be made available until after stepwise pre-amplification. For example, the paired primers can be added (e.g., by microfluidic manipulation) or released from confinement or attachment (e.g., by chemical, temperature, or photodegradation of hydrogel beads).

[0060] Features common among the embodiments are that they enable all reaction reagents to be added first and each step can proceed regardless of the presence of other reaction reagents. For example, in the primer H / primer L embodiment, primer L initially operates at a high temperature where it is essentially not used. In another example, the pair of primers can be isolated from use within the hydrogel beads until release by a change such as in pH or redox or photocleavage. An important feature is that when the final readout is digital PCR in an aqueous compartment, incremental pre-amplification can be performed without the need to change or add reagents as it proceeds through the steps of pre-amplification, compartmentalization, and dPCR in those compartments or in the "bulk phase" prior to compartmentalization.

[0061] Generally, the embodiments can include denaturing the DNA into single-stranded molecules, ii) incubating the molecules with primer H, primer L, and reagents for amplification and dPCR readout at a high annealing temperature where annealing of primer H is possible but annealing of primer L is not, incubating at an extension temperature if necessary (repeating melting, annealing, and extension steps if necessary), compartmentalizing into compartments, performing polymerase chain reaction (PCR) using a low annealing temperature that allows annealing of both primer H and primer L, and detecting whether the compartments contain amplification products that include the target or variant of interest.

[0062] The present invention provides that the melting temperature of primer H can be about 10 °C higher than the melting temperature of primer L. Preferably, the melting temperature of primer H can be about 10 °C higher than the melting temperature of primer L. More preferably, the melting temperature of primer H is about 12 °C higher than the melting temperature of primer L. Even more preferably, the melting temperature of primer H is about 16 °C higher than the melting temperature of primer L.

[0063] In the method of the present invention, the asymmetric incremental amplification is: providing a primer pair capable of amplifying a target nucleic acid, the primer pair including primer H and primer L, the melting temperature of primer H being about 10°C to about 22°C higher than the melting temperature of primer L, and primer L including a sequence complementary to a fragment of the extension product of primer H; providing a nucleic acid polymerase having polymerase activity at an extension temperature; and preparing a sample preparation reaction, each including a part of the sample, a primer set, a nucleic acid polymerase, and PCR reagents. The melting temperature of primer H can be about 10°C higher than the melting temperature of primer L. Preferably, the melting temperature of primer H is about 12°C higher than the melting temperature of primer L. More preferably, the melting temperature of primer H is about 16°C higher than the melting temperature of primer L.

[0064] According to the present invention, the asymmetric incremental amplification may also include a primer set that executes a sample preparation reaction in a solution containing a nucleic acid polymerase having polymerase activity, wherein at least one primer is specifically capable of amplifying only one strand of the target nucleic acid sequence, and the sample preparation reaction is carried out. The sample preparation reaction also includes a nucleic acid polymerase having polymerase activity, one or more primers, and PCR reagents.

[0065] The present invention may further include a step of symmetric exponential amplification, and the symmetric exponential amplification includes: (i) providing a primer set specifically capable of amplifying a target nucleic acid sequence; (ii) providing a nucleic acid polymerase having polymerase activity at an extension temperature; (iii) preparing a sample preparation reaction, each including a part of the sample, a primer set, a nucleic acid polymerase, and PCR reagents; and (iv) executing the sample preparation reaction.

[0066] The method of the present invention provides that symmetric exponential amplification follows asymmetric incremental amplification. The steps involved in asymmetric incremental amplification and symmetric exponential amplification are as described above.

[0067] The method of the present invention further provides that symmetric exponential amplification can be combined with, can follow, or can be followed by asymmetric incremental amplification. The steps involved in asymmetric incremental amplification and symmetric exponential amplification are described above.

[0068] The method of the present invention also provides that the asymmetric incremental amplification and the symmetric exponential amplification can be carried out in the same reaction volume. According to the method of the present invention, the asymmetric incremental amplification and the symmetric exponential amplification can be carried out using the same primer set.

[0069] The method of the present invention also provides that the asymmetric incremental amplification can be activated at a higher temperature when compared to the symmetric exponential amplification. For example, a thermal cycler can be programmed to only lower to a higher annealing temperature for incremental amplification, but to a lower annealing temperature for exponential amplification.

[0070] The present invention further includes the step of performing a plurality of PCR reactions on a sample that has undergone a sample preparation reaction, the steps being a primer pair capable of specifically amplifying a target nucleic acid, a nucleic acid polymerase having polymerase activity at an extension temperature, preparing a PCR reaction, wherein each PCR reaction includes a portion of the sample, a primer set, a nucleic acid polymerase, and PCR reagents; and performing symmetric exponential amplification. The present invention provides that these PCR reactions can be conventional PCR. Preferably, the PCR is digital PCR (dPCR). According to the method of the present invention, a sample containing the product after the sample preparation reaction step can be used as a direct sample input for PCR. Optionally, the primer pair used in the present invention is part of a plurality of primer pairs capable of amplifying different target nucleic acid sequences.

[0071] The method of the present invention may further include the use of a plurality of primer pairs capable of amplifying different target nucleic acid sequences. Preferably, the method of the present invention includes the use of multiplex PCR.

[0072] The method of the present invention may use quantitative PCR, quantitative fluorescence PCR (QF-PCR), multiplex fluorescence PCR (MF-PCR), real-time PCR (RT-PCR), single-cell PCR, restriction fragment length polymorphism PCR (PCR-RFLP), PCR-RFLP / RT-PCR-RFLP, hot start PCR, nested PCR, in situ polony PCR, in situ rolling circle amplification (RCA), digital PCR (dPCR), droplet digital PCR (ddPCR), bridge PCR, picotiter PCR, and emulsion PCR.

[0073] Primer H and Primer L The method of the present invention also includes the use of several primers including the primers named as Primer H and Primer L. In the context of the method of the present invention, Primer H is a primer having a high melting temperature, and Primer L is a primer having a low melting temperature. The melting temperature of a primer is the temperature at which 50% of the primer forms a stable double helix with its complementary sequence and the other 50% is separated into single-stranded molecules. The melting temperature may also be referred to as Tm or Tm. Preferably, when used herein, Tm is calculated using the nearest neighbor method based on the method described in Breslauer, 1986, Predicting DNA duplex stability from the base sequence, PNAS 83:3746-50 (incorporated by reference) using a salt concentration parameter of 50 mM and a primer concentration of 900 nM. For example, the method is performed by the software "Multiple Primer Analyzer" of Life Technologies / Thermo Fisher Scientific Inc.

[0074] The method of the present invention involves the use of a primer set comprising primer H and primer L, where the melting temperature of primer H is about 10°C to about 22°C, preferably at least 10°C, more preferably at least 15°C higher than the melting temperature of primer L, and primer L contains a sequence complementary to the extension product of primer H.

[0075] Primer H is preferably designed as a primer for the amplification of a target sequence or a sequence complementary to the target sequence. Thus, primer H is preferably capable of annealing to either the target nucleic acid sequence or a sequence complementary to the target nucleic acid sequence. For example, primer H may be capable of annealing to the complementary strand of the target nucleic acid sequence at or near the 5'-end of the target nucleic acid sequence, or primer H may be capable of annealing to the target nucleic acid sequence at or near the 3'-end of the target nucleic acid sequence. Thus, primer H may contain the same sequence as the 5'-end of the target nucleic acid sequence. Primer H may even consist of the same sequence as the 5'-end of the target nucleic acid sequence. Primer H may also contain the same sequence as the target nucleic acid sequence. Thus, primer H may contain a sequence complementary to the 3'-end of the target nucleic acid sequence. Primer H may even consist of a sequence complementary to the 3'-end of the target nucleic acid sequence.

[0076] Similarly, primer L is preferably designed as a primer for amplification of a target sequence or a sequence complementary to the target sequence. When primer H is designed for amplification of the target sequence, primer L is preferably designed for amplification of a sequence complementary to the target sequence, and vice versa. Thus, primer L is preferably capable of annealing to either the target nucleic acid sequence or a sequence complementary to the target nucleic acid sequence. When primer H is capable of annealing to the target nucleic acid sequence, then primer L is preferably capable of annealing to a sequence complementary to the target nucleic acid sequence, and vice versa. For example, primer L may be capable of annealing to the complementary strand of the target nucleic acid sequence at or near the 5' end of the target nucleic acid sequence, or primer L may be capable of annealing to the target nucleic acid sequence at or near the 3' end of the target nucleic acid sequence. Thus, primer L may contain the same sequence as the 5' end of the target nucleic acid sequence. Primer L may even consist of the same sequence as the 5' end of the target nucleic acid sequence. Primer L may also contain the same sequence as the target nucleic acid sequence. Thus, primer L may contain a sequence complementary to the 3' end of the target nucleic acid sequence. Primer L may even consist of a sequence complementary to the 3' end of the target nucleic acid sequence.

[0077] Primer H may have the same nucleotide sequence as the sequence at the 5' end of the target nucleic acid sequence, and primer L may contain or consist of the same sequence as the complementary sequence of the 3' end of the target nucleic acid sequence.

[0078] Primer L may have the same nucleotide sequence as the sequence at the 5' end of the target nucleic acid sequence, and primer H may contain or consist of the same sequence as the complementary sequence of the 3' end of the target nucleic acid sequence.

[0079] Primer H and Primer L are designed to have melting temperatures as shown herein. One of ordinary skill in the art can design Primer H and Primer L to have the desired melting temperature by adjusting the primer sequences, the primer lengths, and, if necessary, incorporating nucleotide analogs as described in the section "Primer Sets" herein.

[0080] Primer H is designed to have an annealing temperature that is significantly higher than that of Primer L, for example, at least 10 °C higher. Thus, the melting temperature of Primer H is at least 12 °C higher than that of Primer L, for example, at least 15 °C higher, preferably at least 14 °C higher, more preferably at least 16 °C higher, still more preferably 18 °C higher, for example, at least 20 °C higher, and can be higher in the range of, for example, 15-50 °C, for example, 15-40 °C, for example, 15-25 °C.

[0081] Generally, the melting temperature of Primer H is as high as possible, but preferably not higher than the highest functional extension temperature of at least one nucleic acid polymerase. The extension temperature need not be the optimal temperature for the nucleic acid polymerase, but it is preferred that at least one nucleic acid polymerase is active at the melting temperature of Primer H. Thus, the melting temperature of Primer H can approach 80 °C or even exceed it.

[0082] The melting temperature of primer L should be high enough to ensure specific annealing of primer L to the target nucleic acid sequence / complementary sequence of the target nucleic acid sequence, and it is also preferred that the melting temperature of primer H should be significantly higher than that of primer L. Thus, often, the melting temperature of primer H is at least 60 °C. The melting temperature of primer H can also often be at least 70 °C. The melting temperature of primer H can be, for example, within the range of 60 to 90 °C, for example, within the range of 60 to 85 °C, for example, within the range of 70 to 85 °C, for example, within the range of 70 °C to 80 °C.

[0083] The melting temperature of primer L is preferably high enough to ensure specific annealing of primer L to the target nucleic acid sequence / complementary sequence of the target nucleic acid sequence and is significantly lower than the melting temperature of primer H. Often, the melting temperature of primer L is within the range of 30 to 55 °C, for example, within the range of 35 to 55 °C, preferably within the range of 40 to 50 °C.

[0084] The method of the present invention can also include the use of a set of primers or a plurality of primers. The primer set or the plurality of primers contains two or more different primers. The primer set contains at least one pair of primers that can specifically amplify the target nucleic acid. Further, according to the present invention, the primer set contains at least primer H and primer L. Thus, if the primer set contains only two different primers, then the primer set contains primer H and primer L, and primer H and primer L can amplify the target nucleic acid.

[0085] Detection The method of the present invention generally includes the step of detecting whether the sample contains a variant sequence and / or a target nucleic acid sequence. The detection can be achieved in any suitable manner known to those skilled in the art. For example, numerous useful detection methods that can be used in the method of the present invention are known in the prior art.

[0086] The detection step may include the presence of a detection reagent in the PCR reaction. The detection reagent can be any detectable reagent, for example, a compound containing a detectable label, and the detectable label can be, for example, a dye, radioactivity, fluorophore, heavy metal, or any other detectable label.

[0087] Often, the detection reagent contains a fluorescent compound.

[0088] The detection reagent may include a detection probe. The detection probe can include a nucleotide oligomer or polymer that may contain nucleotide analogs as needed. Often, the detection probe can be a DNA oligomer. Typically, the detection probe is linked to a detectable label, for example, by a covalent bond. The detectable label can be any of the aforementioned detectable labels, but often it is a fluorophore.

[0089] The detection probe is generally capable of specifically binding to a target nucleic acid sequence. For example, the detection probe may be capable of specifically binding to a target nucleic acid containing a variant sequence. Therefore, the detection probe may be capable of annealing to a target nucleic acid sequence or a sequence complementary to the target nucleic acid sequence. Therefore, the detection probe may contain the same sequence as a fragment of the target nucleic acid sequence or a sequence complementary to the target nucleic acid sequence. Generally, it is preferred that the detection probe contains a sequence different from any of the sequences of the primers in the primer set.

[0090] Quantification As described above, the present invention includes providing a sample containing one or more target nucleic acids; performing a sample preparation reaction to increase the copy number of the target nucleic acid in the sample; dividing the sample into a plurality of subsamples; performing a polymerase chain reaction (PCR) on the subsamples; and detecting the target nucleic acid. As a result of targeted preamplification, an increased number of copies of the target are available for the PCR reaction. The advantage of preamplification is to significantly reduce or eliminate false negative cases, which often pose a problem in samples with low target concentration, by reducing the problems of dead volume and probabilistic sampling error.

[0091] The present invention also provides a reference assay for the detection of structural variants or mutants of a patient's cfDNA. In particular, the present invention provides a method for detecting wild-type cfDNA. Advantageously, if a target for a mutant or structural variant is not detected, the present invention enables the detection of cfDNA. As a result, the present invention provides an assay that includes the use of primer pairs and probes for detecting and / or quantifying the wild-type (reference) sequence of cfDNA. The present invention further provides an assay for calculating the variant allele frequency (VAF) for a target nucleic acid in a sample. The target nucleic acid can be a variant of the wild-type nucleic acid sequence in the sample.

[0092] The method of the present invention may further provide for the use of public information to determine regions of the genome that are stable for amplification and to design the assay accordingly. As an example, one of ordinary skill in the art may determine that band 13 of chromosome 2 (2p13) is stable and thus less susceptible to changes in copy number. The present invention further provides that this information can be used to design the assay, particularly to minimize variations in copy number in the sample.

[0093] Advantageously, the present invention provides a method for quantifying the amount of target nucleic acid present in a sample. Replication assays have variability in the pre-amplification step. In particular, the efficiency of pre-amplification is less than 100%. Therefore, a correction factor is applied to back-calculate the original sample concentration. As an example of this problem, the pre-amplification step can result in 50-fold amplification in one case, but only 30-fold amplification in another case, resulting in 50-fold or 30-fold copies of the target sample, respectively. Furthermore, the variable state of the sample also affects how many copies are measured by dPCR. As an example, the same sample can have a two-fold difference in dPCR concentration depending on the variable state (one intact double-stranded DNA molecule in one compartment is measured as one copy, and two single-stranded DNA molecules in two compartments are measured as two copies). The method of the present invention also provides two positive control reactions between pre-amplifications. The first positive control includes positive control DNA and the assay such that pre-amplification occurs. The second positive control does not contain primers and contains an equal amount of positive control DNA. The first positive control and the second positive control are technical replicates except that the first positive control receives one or more primers.

[0094] Thereafter, the concentration of the replication containing primers is divided by the replication without primers to estimate the efficiency of the assay. The calculated efficiency can be applied to the measurements performed on the actual sample. In short, the present invention provides a method for quantifying the amount of target nucleic acid in a sample.

[0095] Incorporation by reference References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web content, publicly accessible databases, have been made throughout this disclosure. All of these documents are hereby incorporated by reference in their entirety for all purposes.

[0096] Equivalents In addition to what is shown and described in this specification, various modifications of the present invention and many further embodiments thereof will be apparent to those skilled in the art from the entire contents of this document, including references to scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be applied to the practice of the present invention in various embodiments thereof and their equivalents.

Claims

1. A method comprising: identifying an array that is present in only a small number in a sample; performing a pre-amplification reaction to increase the abundance of the copy of the identified array in the sample; performing an exponential amplification reaction on the nucleic acid derived from the sample; and analyzing the sample for the identified array A method comprising the above steps.

2. The method according to claim 1, wherein the pre-amplification results in an exponential or linear increase in the abundance.

3. The method according to claim 2, wherein the identified array comprises a structural rearrangement specific to a tumor.

4. The method according to claim 3, wherein the analyzing step detects minimal residual disease (MRD) after treatment.

5. The method according to claim 1, wherein the sample comprises blood or plasma, and the pre-amplification amplifies cell-free DNA.

6. The method according to claim 3, wherein the analyzing step comprises detecting an amplification product comprising the structural rearrangement; and reporting the presence of the tumor in the subject when the amplification product comprising the selected variant is detected.

7. The method according to claim 3, wherein the analyzing step reports the structural rearrangement and genomic mutations.

8. The method according to claim 3, wherein the structural rearrangement is selected as likely to persist within the tumor.

9. The method according to claim 1, further comprising partitioning the sample into compartments and performing the exponential amplification and detection steps within the compartments.

10. The method according to claim 1, wherein the sample comprises blood or plasma derived from a subject, and the nucleic acid comprises cell-free DNA.

11. A method for variant detection, comprising: providing a sample comprising one or more target nucleic acids; performing a pre-amplification to preferentially increase the abundance of the copy of a selected structural sequence rearrangement in the sample; dividing the sample into a plurality of compartments; performing a polymerase chain reaction (PCR) within the compartments; and detecting an amplification product within the compartments comprising at least one of the structural sequence rearrangements A method comprising the above steps.

12. The method according to claim 11, wherein the pre-amplification step is selected from asymmetric incremental amplification and exponential amplification.

13. The asymmetric incremental amplification is Providing a primer pair capable of amplifying a target nucleic acid, wherein the primer pair includes primer H and primer L, the melting temperature of primer H is at least about 10 °C higher than the melting temperature of primer L, and primer L includes a sequence complementary to a fragment of the extension product of primer H; and Thermocycling the sample using an annealing temperature at which primer H anneals but primer L does not anneal The method according to claim 12, comprising:

14. The method according to claim 13, wherein the PCR uses primer H and primer L in a thermocycling with an annealing temperature at which both primer H and primer L anneal.

15. The method according to claim 11, wherein a portion of the pre-amplification reaction mixture is used as an input for the PCR.

16. The method according to claim 15, wherein the portion of the reaction mixture is not subjected to any cleanup step, and the PCR is performed by adding components of a PCR reagent mixture to the portion of the reaction mixture.

17. The method according to claim 11, further comprising determining the amount of the selected structural rearrangement in the sample using (i) the number of compartments in which an amplification product is detected and (ii) the measured increase in the abundance of the copies of the selected structural rearrangement generated by the pre-amplification.

18. The method according to claim 11, wherein the pre-amplification and the PCR are performed using the same primer set.

19. The method according to claim 11, wherein the PCR reaction is digital PCR (dPCR).

20. The method according to claim 11, wherein the step of partitioning comprises diluting the sample such that each compartment contains on average various template molecules for the PCR reaction, and the compartments contain a fluorescent hydrolysis probe.

21. The method according to claim 11, wherein the PCR step comprises multiplex dPCR.

22. The method according to claim 11, wherein the detecting step detects one or more of single nucleotide variants (SNVs), insertions and deletions (indels), duplications, copy number variations (CNVs), inversions, and translocations.

23. The method according to claim 11, wherein the sample comprises cell-free DNA from a cancer patient. Claim 24 The method according to claim 21, further comprising detecting a wild-type cfDNA sequence, and further comprising providing a primer pair capable of binding to the wild-type cfDNA sequence and a probe capable of binding to the wild-type cfDNA sequence.

Images