Compositions and methods for nucleic acid amplification

Abstract

Provided herein is a composition comprising a set of allele-specific primers. Further provided are methods of amplifying a mutant allele, comprising forming a mixture comprising at least one nucleic acid molecule having the mutant allele, and a set of allele-specific primers and subjecting the mixture to a condition sufficient to amplify the at least one nucleic acid molecule. Further provided herein is a method of determining a disease status of a subject.

Description

WSGR Docket No. 69583-701.601COMPOSITIONS AND METHODS FOR NUCLEIC ACID AMPLIFICATIONCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 718,090, filed on November 8, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Detection of subject-specific mutant allele can be informative in determining a disease status of a subject. Several methods exist to amplify specific loci in DNA fragments known to contain patient-specific mutations, which is often obtained via DNA sequencing of tissue biopsies. Typically the amplified fragments can be subjected to DNA sequencing to ascertain whether the DNA fragments contain cancer-associated mutations. These methods, reliant on Next Generation Sequencing (NGS), are expensive, time consuming, inefficient with sample volume, and have low reproducibility or precision. As a result, they are not optimized for monitoring ctDNA levels over time, where cost, precision, and sample efficiency are important.SUMMARY

[0003] Recognized herein is an unmet need to develop a method to detect and quantify mutant alleles, such as single-nucleotide variant (SNV), in non-invasive samples, such as blood and urine, that is cost effective, accurate and precise.

[0004] Provided herein is a method of amplifying a mutant allele comprising a single nucleotide variant (SNV) in a sample comprising a plurality of double-stranded nucleic acid molecules each having a first strand and a second strand, the plurality comprising at least one double-stranded nucleic acid molecule having the mutant allele comprising the SNV, the method comprising: (a) forming a mixture comprising the at least one double-stranded nucleic acid molecule having the mutant allele and a set of allele-specific primers comprising a forward primer and a reverse primer, wherein the forward primer has partial complementarity to a forward primer-binding sequence of the first strand and comprises (i) a 3’ terminal nucleotide that is complementary to a single nucleotide variant of the mutant allele of the first strand and (ii) a first mismatch to the forward primer-binding sequence of the first strand, and wherein the reverse primer has partial complementarity to a reverse primer-binding sequence of the second strand and comprises (i) a 3’ terminal nucleotide that is complementary to an SNV of the mutant allele of the second strand and (ii) a second mismatch to the reverse primer-binding sequence of the second strand; and (b)WSGR Docket No. 69583-701.601 subjecting the mixture to a condition sufficient to amplify the at least one double-stranded nucleic acid molecule.

[0005] In some embodiments, the mixture lacks a universal primer.

[0006] In some embodiments, the forward primer comprises a single mismatch.

[0007] In some embodiments, the forward primer consists of a single mismatch.

[0008] In some embodiments, the reverse primer comprises a single mismatch.

[0009] In some embodiments, the reverse primer consists of a single mismatch.

[0010] In some embodiments, the first mismatch of the forward primer is located at an internal nucleotide.

[0011] In some embodiments, the second mismatch of the reverse primer is located at an internal nucleotide.

[0012] In some embodiments, the method further comprises detecting an amplification product of the at least one double-stranded nucleic acid molecule.

[0013] In some embodiments, detecting comprises contacting the amplification product with a detection agent that non-specifically recognizes polynucleic acids.

[0014] In some embodiments, detecting comprises contacting the amplification product with a nucleic acid intercalating agent.

[0015] In some embodiments, detecting does not comprise using a mutant allele specific probe.

[0016] In some embodiments, detecting does not comprise sequencing the amplification product.

[0017] In some embodiments, the method does not comprise sequencing.

[0018] In some embodiments, the forward primer comprises at most 3 mismatches to the forward primer-binding sequence and / or the reverse primer comprises at most 3 mismatches to the reverse primer-binding sequence.

[0019] In some embodiments, the partial complementarity is at least 90% complementary.

[0020] In some embodiments, the forward primer has a first sequence that includes the 3’ terminal nucleotide and has a length of at least 10 nucleotides, wherein the first sequence has at most 3 mismatches.

[0021] In some embodiments, the first sequence has a length of at least 20 nucleotides.

[0022] In some embodiments, the first sequence has at most 2 mismatches.

[0023] In some embodiments, the first sequence has only one mismatch.

[0024] In some embodiments, the reverse primer has a first sequence that includes the 3’ terminal nucleotide and has a length of at least 10 nucleotides, wherein the first sequence has at most 3 mismatches.

[0025] In some embodiments, the first sequence has a length of at least 20 nucleotides.WSGR Docket No. 69583-701.601

[0026] In some embodiments, the first sequence has at most 2 mismatches.

[0027] In some embodiments, the first sequence has only one mismatch.

[0028] In some embodiments, a melting temperature (Tm) of the forward primer and / or the reverse primer is from 50 degrees Celsius to 70 degrees Celsius.

[0029] In some embodiments, the Tm is from 55 degrees Celsius to 60 degrees Celsius.

[0030] In some embodiments, the Tm is 57 degrees Celsius.

[0031] In some embodiments, the first mismatch to the forward primer-binding sequence of the first strand is at least 6 nucleotides 5’ to the 3’ terminal nucleotide.

[0032] In some embodiments, the first mismatch to the forward primer-binding sequence of the first strand is at most 6 nucleotides 5’ to the 3’ terminal nucleotide.

[0033] In some embodiments, the set of allele-specific primers has a rate of amplification of a corresponding wild-type allele of at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, or less.

[0034] In some embodiments, the set of allele-specific primers has an allele-specific amplification rate of at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or more.

[0035] In some embodiments, the mutant allele is amplified at least 10 times, 100 times, 1000 times, or more than a corresponding wild-type allele is amplified.

[0036] In some embodiments, the plurality of double-stranded nucleic acid molecules further comprises a double-stranded nucleic acid molecule comprising the corresponding wild-type allele.

[0037] In some embodiments, the set of allele-specific primers does not amplify the doublestranded nucleic acid molecule comprising the corresponding wild-type allele or amplifies the double-stranded nucleic acid molecule comprising the corresponding wild-type allele less efficiently than the at least one double-stranded nucleic acid molecule having the mutant allele.

[0038] In some embodiments, the plurality of double-stranded nucleic acid molecules comprises a first double-stranded nucleic acid molecule comprising a first mutant allele, and a second double-stranded nucleic acid molecule comprising a second mutant allele, wherein the first and second mutant alleles are different mutant alleles.

[0039] In some embodiments, the plurality of double-stranded nucleic acid molecules comprises a first double-stranded nucleic acid molecule comprising a first SNV, and a second doublestranded nucleic acid molecule comprising a second SNV, wherein the first and second SNVs are different SNVs.

[0040] In some embodiments, the method further comprises amplifying two or more doublestranded nucleic acid molecules.WSGR Docket No. 69583-701.601

[0041] In some embodiments, the method further comprises amplifying the two or more doublestranded nucleic acid molecules simultaneously.

[0042] In some embodiments, the method further comprises amplifying the two or more doublestranded nucleic acid molecules simultaneously in a same compartment.

[0043] In some embodiments, the method further comprises, prior to amplifying, partitioning the plurality of double-stranded nucleic acid molecules into two or more different compartments.

[0044] In some embodiments, the two or more different compartments are two or more different water-in-oil droplets.

[0045] In some embodiments, the method further comprises, amplifying the two or more double-stranded nucleic acid molecules simultaneously in two or more different compartments, wherein each compartment comprises at least one of the two or more double-stranded nucleic acid molecules and the set of allele-specific primers.

[0046] In some embodiments, the set of allele-specific primers comprises two or more sets of allele-specific primers, and wherein each set of allele-specific primers is specific to a single mutant allele.

[0047] In some embodiments, the set of allele-specific primers comprises two or more sets of allele-specific primers, and wherein each set of allele-specific primers comprises a forward primer that has partial complementarity to a forward primer-binding sequence of a first strand of a double-stranded nucleic acid molecule comprising a different / given mutant allele, and comprises (i) a 3’ terminal nucleotide that is complementary to a single nucleotide variant of the different / given mutant allele of the first strand and (ii) a first mismatch to the forward primerbinding sequence of the first strand, and a reverse primer that has partial complementarity to a reverse primer-binding sequence of the second strand a double-stranded nucleic acid molecule comprising the different / given mutant allele and comprises (i) a 3’ terminal nucleotide that is complementary to an SNV of the different / given mutant allele of the second strand and (ii) a second mismatch to the reverse primer-binding sequence of the second strand.

[0048] In some embodiments, the two or more sets of allele-specific primers comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or more sets of allele-specific primers.

[0049] In some embodiments, the two or more sets of allele-specific primers comprise at most 25, at most 24, at most 23, at most 22, at most 21, at most 20, at most 19, at most 18, at most 17, at most 16, at most 15, at most 14, at most 13, at most 12, at most 11, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3 or less sets of allele-specific primers.WSGR Docket No. 69583-701.601

[0050] In some embodiments, the method comprises detecting amplification products of the two or more double-stranded nucleic acid molecules.

[0051] In some embodiments, the detecting does not differentiate one mutant allele from another different mutant allele.

[0052] In some embodiments, the detecting detects each mutant allele indiscriminately.

[0053] In some embodiments, the method further comprises determining a mean allele frequency of the different mutant alleles or the different SNVs in the sample.

[0054] In some embodiments, the method does not comprise determining an individual allele frequency of a given mutant allele or a given SNV.

[0055] In some embodiments, the method further comprises, prior to (a), obtaining the sample from a subj ect.

[0056] In some embodiments, the subject has cancer.

[0057] In some embodiments, the subject has been treated with a cancer therapy.

[0058] In some embodiments, the subject is suspected of having a minimal residual disease (MRD).

[0059] In some embodiments, the subject has a disease or condition.

[0060] In some embodiments, the disease or condition is cancer.

[0061] In some embodiments, the mutant allele is specific to the subject.

[0062] In some embodiments, the mutant allele is associated with the disease or condition.

[0063] In some embodiments, the method further comprises, prior to (a), identifying one or more mutant alleles in a different sample from the subject.

[0064] In some embodiments, prior to (a), the identity of one or more mutant alleles of the subject is known.

[0065] In some embodiments, the different sample is a tissue sample.

[0066] In some embodiments, the identifying comprises sequencing nucleic acids in the different sample from the subject to identify subject-specific mutant alleles.

[0067] In some embodiments, the mutant allele comprises a SNV of a gene selected from the group consisting of AKT1, APC, BRAF, CTNNB1, EGFR, FGFR3, FLT3, FOXL2, GNA11, GNAQ, GNAS, IDH1, JAK2, KIT, KRAS, MPL, NRAS / CSDE1, PDGFRA, PIK3CA, RET, TP53, ALK, and KRAS.

[0068] In some embodiments, the plurality of double-stranded nucleic acid molecules is a plurality of DNA molecules.

[0069] In some embodiments, the plurality of double-stranded nucleic acid molecules is prepared by reverse transcribing a plurality of RNA molecules.

[0070] In some embodiments, the sample is a fluid sample.WSGR Docket No. 69583-701.601

[0071] In some embodiments, the sample is selected from the group consisting of a blood sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, a pleural or peritoneal fluid sample, a stool sample, a seminal fluid, and any combination thereof.

[0072] In some embodiments, the plurality of double-stranded nucleic acid molecules comprises circulating tumor DNAs or cell-free DNAs.

[0073] In some embodiments, the method further comprises, prior to (a), isolating the plurality of double-stranded nucleic acid molecules from the sample.

[0074] Further provided herein is a composition comprising a set of allele-specific primers, wherein the set of allele-specific primers comprises a forward primer and a reverse primer, wherein the forward primer has partial complementarity to a forward primer-binding sequence of a first strand of a double-stranded nucleic acid molecule comprising a mutant allele comprising an SNV and comprises (i) a 3’ terminal nucleotide that is complementary to a single nucleotide variant of the mutant allele of the first strand and (ii) a first mismatch to the forward primer-binding sequence of the first strand, and wherein the reverse primer has partial complementarity to a reverse primer-binding sequence of the second strand and comprises (i) a 3’ terminal nucleotide that is complementary to a single nucleotide variant of the mutant allele of the second strand and (ii) a second mismatch to the reverse primer-binding sequence of the second strand.

[0075] In some embodiments, the set of allele-specific primers is configured to amplify the mutant allele of the double-stranded nucleic acid molecule.

[0076] In some embodiments, the forward primer comprises a single mismatch.

[0077] In some embodiments, the reverse primer comprises a single mismatch.

[0078] In some embodiments, the first mismatch of the forward primer is located at an internal nucleotide.

[0079] In some embodiments, the second mismatch of the reverse primer is located at an internal nucleotide.

[0080] In some embodiments, the forward primer comprises at most 3 mismatches to the forward primer-binding sequence and / or the reverse primer comprises at most 3 mismatches to the reverse primer-binding sequence.

[0081] In some embodiments, the partial complementarity is at least 90% complementary.

[0082] In some embodiments, the first mismatch to the forward primer-binding sequence of the first strand is at least 6 nucleotides 5’ to the 3’ terminal nucleotide.

[0083] In some embodiments, the first mismatch to the forward primer-binding sequence of the first strand is at most 6 nucleotides 5’ to the 3’ terminal nucleotide.WSGR Docket No. 69583-701.601

[0084] In some embodiments, the set of allele-specific primers has a rate of amplification of a corresponding wild-type allele (false positive rate) of at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, or less.

[0085] In some embodiments, the set of allele-specific primers has an allele-specific amplification rate of at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or more.

[0086] In some embodiments, the set of allele-specific primers does not amplify the doublestranded nucleic acid molecule comprising the corresponding wild-type allele or amplifies the double-stranded nucleic acid molecule comprising the corresponding wild-type allele less efficiently than the at least one double-stranded nucleic acid molecule having the mutant allele.

[0087] Further provided herein is a kit comprising a set of allele-specific primers of the composition of any one of the embodiments disclosed herein, and an instruction for using the kit.

[0088] In some embodiments, the kit further comprises a dNTP, an enzyme, a nucleic acid intercalating agent, and / or a buffer.

[0089] Further provided herein is a use of the composition of any one of the embodiments herein for detecting a disease status in a subject.

[0090] Further provided herein is a method of determining a disease status of a subject, the method comprising: (a) obtaining a first sample comprising a first plurality of nucleic acid molecules from the subject at a first time point or providing the first sample that has been obtained from the subject at the first time point, wherein the first plurality of nucleic acid molecules of the first sample comprises or is suspected of comprising at least one mutant allele; (c) conducting a first allele-specific amplification of the first plurality of nucleic acid molecules using a set of allele-specific primers; (c) detecting amplification products of the first allelespecific amplification using a non-specific polynucleic acid detection agent; and (d) determining the disease status of the subject based on detecting in (c); wherein (i) the set of allele-specific primers has a rate of amplification of a corresponding wild-type allele of at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, or less, (ii) the set of allele-specific primers has an allele-specific amplification rate of at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or more, or (iii) the mutant allele is amplified at least 10 times, 100 times, 1000 times or more than a corresponding wild-type allele is amplified.

[0091] In some embodiments, the method further comprises (a) obtaining a second sample comprising a second plurality of nucleic acid molecules from the subject at a second time point or providing the second sample that has been obtained from the subject at the second time point, wherein the second plurality of nucleic acid molecules of the second sample comprises or is suspected of comprising, or does not comprise a detectable level of the at least one mutant allele;WSGR Docket No. 69583-701.601(b) conducting a second allele-specific amplification of the second plurality of nucleic acid molecules; (c) detecting amplification products of the first allele-specific amplification and amplification products of the second allele-specific amplification using a non-specific polynucleic acid detection agent; and (d) determining the disease status of the subject based on detecting in (e).

[0092] In some embodiments, the set of allele-specific primers comprise an allele-specific forward primer and an allele-specific reverse primer.

[0093] In some embodiments, the allele-specific forward primer and the allele-specific reverse primer each comprises a 3 ’terminal nucleotide that is complementary to a single nucleotide variant of the at least one mutant allele.

[0094] In some embodiments, the at least one mutant allele comprises a SNV.

[0095] Further provided herein is a method of determining a disease status of a subject, the method comprising: (a) obtaining a first sample comprising a first plurality of nucleic acid molecules from a subject at a first time point or providing the first sample that has been obtained from the subject at the first time point, wherein the first plurality of nucleic acid molecules of the first sample comprises or is suspected of comprising at least one mutant allele; (b) conducting a first allele-specific amplification of the first plurality of nucleic acid molecules; (c) obtaining a second sample comprising a second plurality of nucleic acid molecules from the subject at a second time point or providing the second sample that has been obtained from the subject at the second time point, wherein the second plurality of nucleic acid molecules of the second sample comprises or is suspected of comprising, or does not comprise a detectable level of the at least one mutant allele; (d) conducting a second allele-specific amplification of the second plurality of nucleic acid molecules; (e) detecting amplification products of the first allele-specific amplification and amplification products of the second allele-specific amplification using a non-specific polynucleic acid detection agent; and (f) determining the disease status of the subject based on detecting in (e).

[0096] In some embodiments, the detecting in (e) comprises determining a first allele frequency of the at least one mutant allele in the first sample, determining a second allele frequency of the at least one mutant allele in the second sample, and comparing the first allele frequency and the second allele frequency.

[0097] In some embodiments, the at least one mutant allele comprises two or more mutant alleles, and wherein the first allele frequency and / or the second allele frequency comprises a first mean allele frequency and / or a second mean allele frequency.

[0098] In some embodiments, the method does not comprise determining an individual allele frequency for a given mutant allele.WSGR Docket No. 69583-701.601

[0099] In some embodiments, the method does not comprise using a probe for detecting the amplification products.

[0100] In some embodiments, the method does not require using a probe for detecting the amplification products.

[0101] In some embodiments, the method does not comprise using sequencing for detecting the amplification products.

[0102] In some embodiments, determining the disease status comprises determining a tumor fraction (TF).

[0103] In some embodiments, the non-specific polynucleic acid detection agent detects the amplification products irrespective of sequences of the amplification products.

[0104] In some embodiments, the non-specific polynucleic acid detection agent detects any double-stranded nucleic acid molecules.

[0105] In some embodiments, the non-specific polynucleic acid detection agent comprises a nucleic acid intercalating agent.

[0106] In some embodiments, the first allele-specific amplification and / or the second allelespecific amplification comprises using the composition of any one of any one of the embodiments disclosed herein.

[0107] Further provided herein is a use of a set of allele-specific primers in a method for determining a disease status of a subject, wherein the method comprises (a) obtaining a first sample comprising a first plurality of nucleic acid molecules from the subject at a first time point or providing the first sample that has been obtained from the subject at the first time point, wherein the first plurality of nucleic acid molecules of the first sample comprises or is suspected of comprising at least one mutant allele; (b) conducting a first allele-specific amplification of the first plurality of nucleic acid molecules using the set of allele-specific primers; (c) detecting amplification products of the first allele-specific amplification using a non-specific polynucleic acid detection agent; and (d) determining the disease status of the subject based on detecting in (c); wherein (i) the set of allele-specific primers has a rate of amplification of a corresponding wild-type allele of at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, or less, (ii) the set of allele-specific primers has an allele-specific amplification rate of at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or more, or (iii) the mutant allele is amplified at least 10 times, 100 times, 1000 times or more than a corresponding wild-type allele is amplified.

[0108] Further provided herein is a use of a non-specific polynucleic acid detection agent in a method of determining a disease status of a subject, wherein the method comprises: (a) obtaining a first sample comprising a first plurality of nucleic acid molecules from a subject at a first time point or providing the first sample that has been obtained from the subject at the first time point,WSGR Docket No. 69583-701.601 wherein the first plurality of nucleic acid molecules of the first sample comprises or is suspected of comprising at least one mutant allele; (b) conducting a first allele-specific amplification of the first plurality of nucleic acid molecules; (c) obtaining a second sample comprising a second plurality of nucleic acid molecules from the subject at a second time point or providing the second sample that has been obtained from the subject at the second time point, wherein the second plurality of nucleic acid molecules of the second sample comprises or is suspected of comprising, or does not comprise a detectable level of the at least one mutant allele; (d) conducting a second allele-specific amplification of the second plurality of nucleic acid molecules; (e) detecting amplification products of the first allele-specific amplification and amplification products of the second allele-specific amplification using the non-specific polynucleic acid detection agent; and (f) determining the disease status of the subject based on detecting in (e).INCORPORATION BY REFERENCE

[0109] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0110] The novel features of the inventive concepts are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present inventive concepts will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the inventive concepts are utilized, and the accompanying drawings of which:

[0111] FIGs. 1A-1E illustrate forward and reverse primers targeting DNA fragment. FIG. 1A shows double stranded DNA template comprising wild-type nucleotide. FIG. IB shows double stranded DNA template comprising mutant nucleotide. FIG. 1C shows forward and reverse non allele-specific (NAS) primers complementary to either strand of the template, but not to the mutant or wild-type nucleotide. FIG. ID shows forward and reverse allele-specific (AS) primers complementary to a target region, wherein a 3’ terminal nucleotide is complementary to a mutant nucleotide and a first mismatch to a corresponding wild-type nucleotide. FIG. IE showsWSGR Docket No. 69583-701.601 forward and reverse allele specific multiple mismatch (ASMM) primers, wherein a 3’ terminal nucleotide is complementary to a mutant nucleotide and first mismatch to a corresponding wildtype nucleotide, and a second mismatch upstream of the first mismatch (e.g., second mismatch positioned five nucleotides away from the 3’ end of the primer).

[0112] FIG. 2 shows false positive rate (or specificity) of primer combinations targeting wildtype DNA template.

[0113] FIG. 3 shows allele specific amplification (or sensitivity) of primer combinations targeting mutant DNA template.DETAILED DESCRIPTION

[0114] Provided herein are compositions, kits, and methods for amplifying a mutant allele. Further provided herein are methods of determining a disease status of a subject by detecting one or more mutant alleles.I. DEFINITIONS

[0115] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0116] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure.Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0117] As used in the specification and claims, the singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.

[0118] The term “dNTP” refers to an individual or combination of deoxynucleotides containing a phosphate, sugar and organic base in the triphosphate form, that provide precursors requiredWSGR Docket No. 69583-701.601 by a DNA polymerase for DNA synthesis. A dNTP mixture may include each of the naturally occurring deoxynucleotides (i.e., adenine (A), guanine (G), cytosine (C), uracil (U), and Thymine (T)). In some embodiments, each of the naturally occurring deoxynucleotides may be replaced or supplemented with a synthetic analog (e.g., inosine, isoG, IsoC, deaza G, deaza A).

[0119] The term “ddNTP” refers to an individual or combination of dideoxynucleotides containing a phosphate, sugar and organic base in the triphosphate form, that provide precursors required by a DNA polymerase for DNA synthesis. A ddNTP mixture may include each of the naturally occurring dideoxynucleotides (i.e., adenine (A), guanine (G), cytosine (C), uracil (U), and Thymine (T)). In some embodiments, each of the naturally occurring dideoxynucleotides may be replaced or supplemented with a synthetic analog (e.g., inosine, isoG, IsoC, deazaG, deaza A).

[0120] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0121] The term “primer binding sequence” refers to a complete or partial sequence in a nucleic acid molecule, such as in a double-stranded nucleic acid molecules, to which a primer hybridizes or binds.

[0122] The term “amplification product” refers to a copy of a template nucleic acid sequence or molecule. The copy can be a fragment of a template nucleic acid sequence or molecule (e.g., DNA) amplified by a polymerase using a set of primers in an amplification method, such as PCR or dPCR.

[0123] The term “mismatch” refers to a nucleotide in one strand of nucleic acid does not or cannot pair through Watson-Crick base pairing with a nucleotide in an opposing complementary nucleic acid strand. Examples of mismatches are but not limited to AA, AG, AC, GG, CC, TT, TG, TC, UU, UG, UC, and UT base pairs. Mismatches can happen between DNA and DNA molecules, DNA and RNA molecules, RNA and RNA molecules, and among other natural or artificial nucleic acid analogs.

[0124] The terms “single nucleotide variant,” or “SNV” refer to a change in a single nucleotide in a nucleic acid sequence (e.g., DNA sequence).WSGR Docket No. 69583-701.601

[0125] The terms “multi-nucleotide variant” or “MNV” refer to two or more single nucleotide variants that are close together on the same strand of a nucleic acid sequence and haplotype in an individual.

[0126] The term “indel” refers to a genomic variant that occurs when nucleotides are deleted or inserted in a nucleic acid sequence (e.g., DNA sequence).

[0127] The term “specificity” refers to a measure of how well a test or method can identify true negatives. Thus, high specificity can refer to low false positive rate.

[0128] The term “sensitivity” refers to a measure of how well a test or method can identify true positives. Thus, high sensitivity can refer to low false negative rate.

[0129] The terms “subject,” “individual,” or “patient” are often used interchangeably herein. A “subject” can be a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.

[0130] As used herein, the term “about” a number refers to that number plus or minus 10% of that number. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.

[0131] As used herein, the terms “treatment” or “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0132] The melting temperature (Tm) refers to the temperature at which one-half (50%) of a nucleic acid duplex (e.g., double-stranded DNA) of an oligonucleotide (such as a primer) and itsWSGR Docket No. 69583-701.601 perfect complement dissociates and becomes single-strand nucleic acid molecules (e.g., singlestranded DNAs).

[0133] Minimal residual disease (MRD), as used herein, refers to the small number of cancer cells that remain in the body after treatment.

[0134] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.II. OVERVIEW

[0135] As cancerous solid tumors originate, develop, and grow in a healthy organism (e.g., humans), the tumors can shed DNA into the blood and urine (e.g., in the case of bladder cancer). Detecting the presence and quantity of this circulating tumor DNA (ctDNA) in bodily fluids, such as blood and urine of patients, has been shown to inform cancer diagnosis, staging, and treatment and decisions.

[0136] However, accurate and precise detection and quantitation of ctDNA in blood and urine can involve many technical challenges. Several methods exist to amplify specific loci in DNA fragments known to contain patient-specific mutations. Often, patient-specific mutations can be obtained via DNA sequencing of tissue biopsies. Once amplified, the amplified fragments can be subjected to DNA sequencing to ascertain whether the DNA fragments contain cancer- associated mutations. These methods, reliant on Next Generation Sequencing (NGS), can be expensive, time consuming, inefficient with sample volume, and have low reproducibility (precision). As a result, they are not optimized for monitoring ctDNA levels over time, where cost, precision, and sample efficiency are important.

[0137] Disclosed herein are methods to cost effectively detect and quantify ctDNA mutant alleles (e.g., single nucleotide variant (SNV) alleles, indels, and multi -nucleotide variant (MNV) alleles) in fluid samples, such as blood and urine. For example, known tumor-associated mutations can be amplified using an innovative primer design and can be quantified using digital PCR instruments. The detection and quantitation of ctDNA alleles can be done without the use of NGS. This new approach to ctDNA detection and quantitation can be highly valuable to cancer patients and clinical development work where frequent, cost effective, accurate, and precision ctDNA monitoring helps inform tumor progression and treatment efficacy.

[0138] An example of methods disclosed herein includes a method of amplifying a mutant allele comprising a SNV in a sample comprising a plurality of double-stranded nucleic acid molecules each having a first and a second strand, the plurality comprising at least one double-stranded nucleic acid molecule having the mutant allele comprising the SNV. Another example of methods disclosed herein includes a method of determining a disease status of a subject.WSGR Docket No. 69583-701.601Disclosed herein is also, for example, a composition comprising a set of allele-specific primers, wherein the set of allele-specific primers comprise a forward primer and a reverse primer. Further disclosed herein is, for example, a kit comprising any one of the compositions herein.III. COMPOSITIONS

[0139] In some aspects, provided here is a composition comprising a set of allele-specific primers. The compositions described herein can be used in the methods described herein to amplify mutant alleles in liquid (or fluid) samples for allele-specific amplifications. In some embodiments, the set of allele-specific primers is used for amplifying a mutant allele. In some embodiments, the mutant allele comprises an indel, a multi -nucleotide variant (MNV), or a single nucleotide variant (SNV). In some embodiments, the mutant allele comprises a SNV. In some embodiments, the set of allele-specific primers comprises a forward primer and a reverse primer disclosed herein.

[0140] In some embodiments, the set of allele-specific primers comprises two or more sets of allele-specific primers. In some embodiments, the two or more sets of allele-specific primers comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or more sets of allele-specific primers. In some embodiments, the two or more sets of allele-specific primers comprise at most 25, at most 24, at most 23, at most 22, at most 21, at most 20, at most 19, at most 18, at most 17, at most 16, at most 15, at most 14, at most 13, at most 12, at most 11, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3 or less sets of allele-specific primers. In some embodiments, each set of allele-specific primers comprises a forward primer and a reverse primer.

[0141] In some embodiments, the set of allele-specific primers binds to a target nucleic acid molecule from a plurality of nucleic acid molecules. A target nucleic acid molecule or a plurality of nucleic acid molecules can be RNA (e.g., viral, microRNA, mRNA, cRNA, rRNA, hnRNA, cfRNA), or DNA (e.g., genomic, somatic, cfDNA, cffDNA, or cDNA). In some embodiments, a plurality of nucleic acid molecules comprises a target nucleic acid molecule comprising a mutant allele (e.g., SNV) and a nucleic acid molecule comprising the corresponding wild-type allele. In some embodiments, the plurality of nucleic acid molecules comprises a first double-stranded nucleic acid molecule comprising a first mutant allele, and a second double-stranded nucleic acid molecule comprising a second mutant allele, wherein the first and second mutant alleles are different mutant alleles. In some embodiments, the plurality of nucleic acid molecules comprises a first double-stranded nucleic acid molecule comprising a first SNV, and a second doublestranded nucleic acid molecule comprising a second SNV, wherein the first and second SNVsWSGR Docket No. 69583-701.601 are different SNVs. In some embodiments, the target nucleic acid molecule is a double-stranded nucleic acid molecule. In some embodiments, the double-stranded nucleic acid molecule comprises a mutant allele (e.g., SNV). In some embodiments, the double-stranded nucleic acid molecule comprises a wild-type allele. A target nucleic acid molecule can comprise a forward primer-binding sequence or reverse primer-binding sequence for the set of allele-specific primers to bind.

[0142] In some embodiments, the set of allele-specific primers is configured to amplify the mutant allele (e.g., SNV) of a target nucleic acid molecule (e.g., double-stranded nucleic acid molecule). In some embodiments, the set of allele-primers does not amplify a target nucleic acid molecule (e.g., double-stranded nucleic acid molecule comprising a mutant allele) comprising a corresponding wild-type allele or amplifies a nucleic acid molecule (e.g., double-stranded nucleic acid molecule) comprising a corresponding wild-type allele less efficiently than at least one a target nucleic acid molecule (e.g., double-stranded nucleic acid molecule) having the mutant allele. In some embodiments, the mutant allele is amplified at least 10 times, 100 times, 1000 times or more than a corresponding wild-type allele. In some embodiments, the set of allele-specific primers has a rate of amplification of a corresponding wild-type (false positive rate) of at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, or less. To determine the false positive rate, amplification of the wild-type alleles of a sample obtained from a healthy donor using the allele-specific primers can be measured. The copy number of the genome equivalence of the wild-type alleles can be determined by using a housekeeping gene. The wildtype alleles can then be subject to the amplification methods described herein using digital PCR and the set of primers of interest to determine the number of positive droplets relative to the genome equivalence. In some embodiments, the set of allele-specific primers has an allelespecific amplification rate (sensitivity) of at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or more. To determine the allele-specific amplification, amplification of the mutant alleles (e.g., templates having the SNVs) using the allele-specific primers can be measured. The mutant alleles can be subject to the amplification methods described herein using digital PCR and the set of primers of interest to determine the number of positive droplets relative to the total copy number of the mutant alleles.A. Forward and Reverse Primer

[0143] In some embodiments, the forward and reverse primer are designed to amplify the mutant allele. Primers can be designed with Primer XL, a primer design webtool. Primer designs can be generated with Primer3 (Untergrasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) “Primer3 - new capabilities and interfaces.” Nucleic AcidsWSGR Docket No. 69583-701.601Research 40(15):e 115 and Koressaar T, Remm M (2007) “Enhancements and modifications of primer design program Primer3.” Bioinformatics 23(10): 1289-91) source code available at primer3.sourceforge.net). Primer specificity can be evaluated by BLAST and added to existing primer design pipeline.

[0144] Plus (+) strand primers can be generated for selected target regions. Target region sequences can be targeted in windows every 20-50 bp. Each primer design window can be 20-40 bp long from the window start. Primers can be searched in two consecutive windows for pairing nested Outer and Inner primers. Outer primers can be designed that target the right most, 5' (or leftmost on minus strand) coordinate of each region using Primer3. Primers can be generated using RunPrimer3.java with one_sided=true option. This mode of the program can generate only one set of primers without generating a paired minus primer. Primer specificities can be determined using the BLASTn program from the ncbi-blast-2.2.29+ package. The task option “blastn-short” can be used to map the primers against hgl9 human genome. Primer designs can be determined as “specific” if the primer has less than 100 hits to the genome and the top hit is the target complementary primer binding region of the genome and is at least two scores higher than other hits (score is defined by BLASTn program).

[0145] In some embodiments, the forward and reverse primer disclosed herein amplify a mutant allele comprising a SNV of a gene selected from the group consisting of AKT1, APC, BRAF, CTNNB1, EGFR, FGFR3, FLT3, FOXL2, GNA11, GNAQ, GNAS, IDH1, JAK2, KIT, KRAS, MPL, NRAS / CSDE1, PDGFRA, PIK3CA, RET, TP53, ALK, and KRAS. In some embodiments, the forward and reverse primer disclosed herein amplify a mutant allele comprising a SNV as set forth in Table 1. * in Table 1 represents a change to a stop codon. The mutant allele can be a subject-specific mutant allele. The subject-specific mutant can be identified by sequencing a sample (e.g., a solid sample) obtained from the subject.WSGR Docket No. 69583-701.601Table 1. Examples of SNV of various genes

[0146] In some embodiments, a forward primer has a sequence complementarity to its primerbinding sequence of a target nucleic acid molecule (e.g., a strand of a double-stranded nucleic acid molecule). Complementarity or complementary of a primer (e.g., forward or reverse primer) to its primer-binding sequence refers to the alignment of the respective nucleotides to one another. For example, complementary nucleotides in DNA can be the pairing of A with T, and C with G, and in RNA, C with G, and U with A (Watson-Crick pairings). Full complementarity or full complementary can mean that all nucleotides of two strands (e.g., between a primer and a primer-binding sequence) form Watson-Crick pairs to form a stable hybrid complex. Partial complementarity or partial complementary can mean that most, but not necessarily all nucleotides of two strands (e.g., between a primer and a primer binding sequence)WSGR Docket No. 69583-701.601 form Watson-Crick pairs to form a stable hybrid complex. In some embodiments, the forward primer has partial complementarity to a forward primer-binding sequence. In some embodiments, the forward primer has partial complementarity to a forward primer-binding sequence of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. In some embodiments, the forward primer has partial complementarity to a forward primer-binding sequence of at least 90%. In some embodiments, the forward primer has full complementarity to a forward primerbinding sequence. In some embodiments, the forward primer comprises a 3’ terminal nucleotide that is complementary to a single nucleotide variant of a mutant allele. In some embodiments, the forward primer comprises a mismatch to a forward primer-binding sequence. In some embodiments, the forward primer comprises (i) a 3’ terminal nucleotide that is complementary to a single nucleotide variant of a mutant and (ii) a mismatch to the forward primer-binding sequence. In some embodiments, the forward primer comprises a single mismatch. In some embodiments, the forward primer consists of a single mismatch. In some embodiments, the mismatch of the forward primer is located at an internal nucleotide. In some embodiments, the forward primer comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 mismatches. In some embodiments, the forward primer comprises at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 mismatches. In some embodiments, the forward primer comprises at most 3 mismatches to the forward primer-binding sequence. In some embodiments, the mismatch to the forward primer-binding sequence is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides 5’ to the 3’ terminal nucleotide. In some embodiments, the mismatch to the forward primer-binding sequence is at least 6 nucleotides 5’ to the 3’ terminal nucleotide. In some embodiments, the forward primer has a first sequence that includes the 3’ terminal nucleotide and has a length of at least 5, at least 10, at least 15, or at least 20 nucleotides, wherein the first sequence has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 mismatches. In some embodiments, the forward primer has a first sequence that includes the 3’ terminal nucleotide and has a length of at least 10 nucleotides, wherein the first sequence has at most 3 mismatches. In some embodiments the first sequence has at most 2 mismatches. In some embodiments the first sequence has only one mismatch. In some embodiments, the first sequence has a length of at least 20 nucleotides. In some embodiments, the mismatch to the forward primer-binding sequence is at the last nucleotide at the 3’ end of the forward primer. In some embodiments, the mismatch to the forward primer-binding sequence is at a position that isWSGR Docket No. 69583-701.601 at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 from the last nucleotide at the 3’ end of the forward primer.

[0147] In some embodiments, a melting temperature (Tm) of the forward primer is from 50 degrees to 55 degrees Celsius, from 55 degrees to 60 degrees Celsius, from 60 degrees to 65 degrees Celsius, from 65 degrees to 70 degrees Celsius, or from 70 degrees to 75 degrees Celsius. In some embodiments, a Tm of the forward primer is from 50 degrees to 60 degrees Celsius, or from 60 degrees to 70 degrees Celsius. In some embodiments, a Tm of the forward primer is from 50 degrees Celsius to 70 degrees Celsius. In some embodiments, the Tm of the forward primer is from 55 degrees Celsius to 60 degrees Celsius. In some embodiments, the Tm of the forward primer is at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 degrees Celsius. In some embodiments, the Tm of the forward primer is 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 degrees Celsius. In some embodiments, the Tm of the forward primer is 57 degrees Celsius.

[0148] In some embodiments, a forward primer is at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, or at least about 40 nucleotides long. In some embodiments, a forward primer is no more than about 25, no more than about 30, no more than about 40, no more than about 50, or no more than about 75 nucleotides long. In some embodiments, a forward primer is 15-50 nucleotides, 20-30 nucleotides, or 30-40 nucleotides. In some embodiments, a forward primer has a 5’ tail complementary to a target nucleic acid (e.g., strand of a double-stranded nucleic acid molecule). In some embodiments, the 5’ tail is used for attaching a fluorophore or a quencher, or for linking discontinuous segments of primer complementary to its primer-binding sequence.

[0149] In some embodiments, a reverse primer has a sequence complementary to its primerbinding sequence. In some embodiments, the reverse primer has partial complementarity to a reverse primer-binding sequence. In some embodiments, the reverse primer has partial complementarity to a reverse primer-binding sequence of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. In some embodiments, the reverse primer has partial complementarity to a reverse primer-binding sequence of at least 90%. In some embodiments, the reverse primer has full complementarity to a reverse primer-binding sequence. In some embodiments, the reverse primer comprises a 3’ terminal nucleotide that is complementary to a single nucleotide variant of a mutant allele. In some embodiments, the reverse primer comprises a mismatch to a reverse primer-binding sequence. In some embodiments, the reverse primer comprises (i) a 3’ terminal nucleotide that is complementary to a single nucleotide variant of a mutant allele and (ii) a mismatch to the reverse primer-binding sequence. In some embodiments, the reverse primerWSGR Docket No. 69583-701.601 comprises a single mismatch. In some embodiments, the reverse primer consists of a single mismatch. In some embodiments, the mismatch of the reverse primer is located at an internal nucleotide. In some embodiments, the reverse primer comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 mismatches. In some embodiments, the reverse primer comprises at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 mismatches. In some embodiments, the reverse primer comprises at most 3 mismatches to the reverse primer-binding sequence. In some embodiments, the mismatch to the reverse primer-binding sequence is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides 5’ to the 3’ terminal nucleotide. In some embodiments, the mismatch to the reverse primer-binding sequence is at least 6 nucleotides 5’ to the 3’ terminal nucleotide. In some embodiments, the reverse primer has a first sequence that includes the 3’ terminal nucleotide and has a length of at least 10 nucleotides, wherein the first sequence has at most 3 mismatches. In some embodiments the first sequence has a most 2 mismatches. In some embodiments the first sequence has only one mismatch. In some embodiments, the first sequence has a length of at least 20 nucleotides. In some embodiments, the mismatch to the reverse primer-binding sequence is at the last nucleotide at the 3’ end of the reverse primer. In some embodiments, the mismatch to the reverse primer-binding sequence is at the last nucleotide at the 3’ end of the reverse primer. In some embodiments, the mismatch to the reverse primer-binding sequence is at a position that is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 from the last nucleotide at the 3’ end of the reverse primer.

[0150] In some embodiments, a melting temperature (Tm) of the forward primer is from 50 degrees to 55 degrees Celsius, from 55 degrees to 60 degrees Celsius, from 60 degrees to 65 degrees Celsius, from 65 degrees to 70 degrees Celsius, or from 70 degrees to 75 degrees Celsius. In some embodiments, a Tm of the forward primer is from 50 degrees to 60 degrees Celsius, or from 60 degrees to 70 degrees Celsius. In some embodiments, a Tm of the forward primer is from 50 degrees Celsius to 70 degrees Celsius. In some embodiments, the Tm of the forward primer is from 55 degrees Celsius to 60 degrees Celsius. In some embodiments, the Tm of the forward primer is at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 degrees Celsius. In some embodiments, the Tm of the forward primer is 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 degrees Celsius. In some embodiments, the Tm of the forward primer is 57 degrees Celsius.

[0151] In some embodiments, a reverse primer is at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, or at least about 40 nucleotides long. In some embodiments, a reverse primer is no more than about 25, no more than about 30, no more thanWSGR Docket No. 69583-701.601 about 40, no more than about 50, or no more than about 75 nucleotides long. In some embodiments, a reverse primer is 15-50 nucleotides, 20-30 nucleotides, or 30-40 nucleotides. In some embodiments, a reverse primer has a 5’ tail complementary to a target nucleic acid. In some embodiments, the 5’ tail is used for attaching fluorophore or quenches, or for linking discontinuous segments of primer complementary to its primer-binding sequence.

[0152] In some embodiments, the forward and reverse primers bind to opposite strands of a target nucleic acid (e.g., double-stranded nucleic acid molecule), wherein the forward primer has full or partial complementarity to a forward primer-sequence of a first strand, and the reverse primer has full or partial complementarity to a reverse primer-binding sequence of a second strand. In some embodiments, the forward primer comprises a 3’ terminal nucleotide that is complementary to a mutant allele (e.g., SNV) of the first strand. In some embodiments, the forward primer comprises a first mismatch to the forward primer-binding sequence of the first strand. In some embodiments, the reverse primer comprises a 3’ terminal nucleotide that is complementary to a mutant allele (e.g., SNV) of the first strand. In some embodiments, the forward primer comprises a second mismatch to the reverse primer-binding sequence of the second strand. In some embodiments, the forward primer and / or the reverse primer comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 mismatches. In some embodiments, the forward primer and the reverse primer comprises at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 mismatches. In some embodiments, the forward primer and / or the reverse primer comprises at most 3 mismatches. In some embodiments, the forward and reverse primer binding sites are on the same strand. For example, linked forward and reverse primers can bind to binding sites on the same strand and amplify by a rolling circle mechanism. Some pairs of three way junction primers can also bind to sites on the same nucleic acid strand such that one primer serves as a template for the other.

[0153] In some embodiments, complement of the forward primer-binding site (e.g., forward primer) and complement of the reverse primer-binding site (e.g., reverse primer) can be positioned contiguous with one another or separated by intervening nucleotides in the target nucleic acid (e.g., double-stranded nucleic acid molecule). In some embodiments, the forward primer-binding site and the reverse primer-binding site can be contiguous with one another or separated by intervening nucleotides in the target nucleic acid (e.g., double-stranded nucleic acid molecule). In some embodiments, the length of the intervening nucleotides is no more than about 100, no more than about 500, no more than about 1000, or no more than about 10000 nucleotides. In some embodiments, the length of the intervening nucleotides is at least about 1, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at leastWSGR Docket No. 69583-701.601 about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 1000 nucleotides.

[0154] In some embodiments, a set of allele-specific primers can be used to amplify a mutant allele having an indel or a MNV. For example, the set of allele-specific primers can be used to amplify at least one double-stranded nucleic acid molecule having the mutant allele comprising an indel or MNV. The forward primer can have partial complementarity to a forward primerbinding sequence of the first strand of the at least one double-stranded nucleic acid molecule and comprise (i) a 3’ terminal sequence that is complementary to the indel or the MNV of the first strand and (ii) a first mismatch to the forward primer-binding sequence of the first strand. The reverse primer can have partial complementarity to a reverse primer-binding sequence of the second strand and comprise (i) a 3’ terminal sequence that is complementary to the indel or the MNV of the mutant allele of the second strand and (ii) a second mismatch to the reverse primerbinding sequence of the second strand. The forward primer and the reverse primer may not have complementarity to each other at the 3’ terminal for two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) nucleotides. The forward primer and the reverse primer may only be complementary to each other at a single nucleotide at the 3’ terminal.

[0155] In some embodiments, a composition comprises a set of allele-specific primers, wherein the set of allele-specific primers comprises a forward primer and a reverse primer, wherein the forward primer has partial complementarity to a forward primer-binding sequence of a first strand of a double-stranded nucleic acid molecule comprising a mutant allele comprising an SNV and comprises (i) a 3’ terminal nucleotide that is complementary to a single nucleotide variant of the mutant allele of the first strand and (ii) a first mismatch to the forward primerbinding sequence of the first strand, and wherein the reverse primer has partial complementarity to a reverse primer-binding sequence of the second strand and comprises (i) a 3’ terminal nucleotide that is complementary to a single nucleotide variant of the mutant allele of the second strand and (ii) a second mismatch to the reverse primer-binding sequence of the second strand.B. PCR Components

[0156] In some embodiments, the composition herein comprises a mixture comprising at least one nucleic acid molecule (e.g., double-stranded nucleic acid molecule), and a set of allelespecific primers disclosed herein. In some embodiments, the mixture lacks a universal primer. The universal primer can be a common primer that does not differentiate a mutant allele template from a wild-type template. In some embodiments, the double-stranded nucleic acid molecule has a mutant allele (e.g., SNV). In some embodiments, the mixture further comprises one or more PCR components necessary to amplify at a mutant allele (e.g., SNV). In someWSGR Docket No. 69583-701.601 embodiments, the mixture comprise nucleotides (e.g., dNTPs, ddNTPs), a thermostable polymerase, a Tris buffer, a monovalent salt, Mg2+, or a combination thereof. In some embodiments, the mixture comprises ethylenediaminetetraacetic acid (EDTA), magnesium, tetramethyl ammonium chloride (TMAC), or any combination thereof. In some embodiments, Tris buffer is used at, for example, a concentration of between 10 and 100 mM, such as between 10 and 25 mM, 25 and 50 mM, 50 and 75 mM, or 25 and 75 mM, inclusive. In some embodiments, any of these concentrations of Tris are used at a pH between 7.5 and 8.5. In some embodiments, the mixture comprises a combination of KC1 and (NH^SCU. In some embodiments, the mixture comprises between 50 and 150 mM KC1 and between 10 and 90 mM (NH4)2SO4, inclusive. In some embodiments, the mixture comprises concentration of KC1 that is between 0 and 30 mM, between 50 and 100 mM, or between 100 and 150 mM, inclusive. In some embodiments, the mixture comprises concentration of (NH4)2SO4 that is between 10 and 50 mM,50 and 90 mM, 10 and 20 mM, 20 and 40 mM, 40 mM and 60, or 60 mM and 80 mM (NH4)2SO4, inclusive. In some embodiments, the mixture comprises ammonium [NH4+] with a concentration that is between 0 and 160 mM, such as between 0 to 50, 50 to 100, or 100 to 160 mM, inclusive. In some embodiments, the mixture comprises a sum of a potassium and ammonium concentration ([K+] + [NH4+]) that is between 0 and 160 mM, such as between 0 to 25, 25 to 50, 50 to 150, 50 to 75, 75 to 100, 100 to 125, or 125 to 160 mM, inclusive. An exemplary buffer with [K+] + [NH4+] = 120 mM is 20 mM KC1 and 50 mM (NH4)2SO4. In some embodiments, the buffer includes 25 to 75 mM Tris, pH 7.2 to 8, 0 to 50 mM KCL, 10 to 80 mM ammonium sulfate, and 3 to 6 mM magnesium, inclusive. In some embodiments, the buffer includes 25 to 75 mM Tris pH 7 to 8.5, 3 to 6 mM MgC12, 10 to 50 mM KC1, and 20 to 80 mM (NH4)2SO4, inclusive.

[0157] In some embodiments, a crowding agent is used, such as polyethylene glycol (e.g., PEG, e.g., PEG 8,000) or glycerol. In some embodiments, the amount of PEG (e.g., PEG 8,000) is between 0.1 to 20%, such as between 0.5 to 15%, 1 to 10%, 2 to 8%, or 4 to 8%, inclusive. In some embodiments, the amount of glycerol is between 0.1 to 20%, such as between 0.5 to 15%, 1 to 10%, 2 to 8%, or 4 to 8%, inclusive. In some embodiments, a crowding agent allows either a low polymerase concentration and / or a shorter annealing time to be used.

[0158] In some embodiments, the mixture comprises a polymerase. Non-limiting examples of polymerase include a DNA polymerase, an RNA polymerase or a reverse transcriptase. Examples of DNA polymerases include: E. coli DNA polymerase I, Taq DNA polymerase, S. pneumoniae DNA polymerase I, Tfl DNA polymerase, D. radiodurans DNA polymerase I, Tth DNA polymerase, Tth XL DNA polymerase, M. tuberculosis DNA polymerase I, M. thermoautotrophicum DNA polymerase I, Herpes simplex- 1 DNA polymerase, T4 DNAWSGR Docket No. 69583-701.601 polymerase, thermosequenase or a wild-type or modified T7 DNA polymerase, 29 Polymerase, Bst Polymerase, Vent Polymerase, 9° Nm Polymerase, Klenow fragment of DNA Polymerase I. Examples of reverse transcriptase: AMV Reverse Transcriptase, MMLV Reverse Transcriptase, HIV Reverse Transcriptase. Examples of RNA polymerases include: T7 RNA polymerase or SP6 RNA polymerase, bacterial RNA polymerases and eukaryotic RNA polymerases.IV. KITS

[0159] Provided here is a kit comprising a composition, comprising a set of allele-specific primers disclosed herein. In some embodiments, the kit comprises a composition disclosed herein, reagents, devices, and instructions on how to perform the methods or test on a particular biological sample type. In some embodiments, the kit comprises one or more of the following components: reagents, primers, reaction mixtures, buffers, enzymes (e.g., endonucleases, exonucleases, ligases, polymerases, RNA polymerases, DNA polymerases, Hot-start polymerases, reverse transcriptases, topoisomerases, kinases, phosphatases), antibodies, probes, dyes, and / or experimental standards. In some embodiments, the kit comprises one or more mixtures disclosed herein. In some embodiments, the kit comprises one or more set of primers disclosed herein (e.g., allele-specific primers, housekeeping gene specific primers). In some embodiments, the kit comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 primer pairs. The primer pairs in a kit may be capable of use in the same multiplex reaction. In some embodiments, the kit further comprises a computer software (e.g. computer-executable logic that instructs a processor) to drive and instruct the devices, and instructions for the user or technical staff for implementing the methods provided herein. The DNA polymerase and primers disclosed herein and used in an assay can be stored in a state where they exhibit long-term stability, e.g., in suitable storage buffers or in a lyophilized or freeze dried state. In some embodiments, the kits can further comprise a buffer for the DNA polymerase.

[0160] In some embodiments, the kit further comprises reagents or devices to enable the detection by additional downstream methods that can enhance or add in further clinical detection, prognosis, drug response determination, and diagnosis of a patient suffering from a disease.

[0161] In some embodiments, the kit further comprises a software package for data analysis of genetic profiling, which can include reference genetic profiles for comparison. In someWSGR Docket No. 69583-701.601 embodiments, the software package comprises connection to a central server to conduct for data analysis and where a report is generated which comprises with recommendation on disease state, drug interactions, or treatment suggestions to a health care provider. In some embodiments, the report provided with the kit can be a paper or electronic report. It can be generated by computer software provided with the kit, or by a computer sever which the user uploads to a website wherein the computer server generates the report.

[0162] In some embodiments, the report may include prognosis such as predicted overall survival, predicted response to therapy, predicted disease-free survival, predicted progression- free survival, or predicted non-reoccurrence survival. The report may include a diagnosis of a condition. The report may include a recommendation for a treatment modality such as treatment or stopping treatment with of a particular drug.V. METHODS

[0163] Provided here are methods of amplifying a mutant allele. Further provided herein are methods of determining a disease status of a subject.A. Method of Amplifying a Mutant Allele

[0164] The present disclosure provides a method of amplifying a mutant allele comprising a single nucleotide variant (SNV), an indel, or a multiple nucleotide variant (MNV) in a sample comprising a plurality of double-stranded nucleic acid molecules. Each nucleic acid molecule of the plurality of double-stranded nucleic acid molecules can have a first strand and a second strand. The plurality can comprise at least one double-stranded nucleic acid molecule having the mutant allele comprising a SNV.

[0165] In some embodiments, the method disclosed herein comprises forming a mixture comprising the at least one double-stranded nucleic acid molecule having the mutant allele and a set of allele-specific primers. The set of allele-specific primers can comprise a forward primer and a reverse primer. In some embodiments, the forward primer disclosed herein has partial complementarity to a forward primer-binding sequence of the first strand. In some embodiments, the forward primer disclosed herein has a 3’ terminal nucleotide that is complementary to a single nucleotide variant of the mutant allele of the first strand. In some embodiments, the forward primer disclosed herein has a first mismatch to the forward primerbinding sequence of the first strand. In some embodiments, the reverse primer disclosed herein has partial complementarity to a reverse primer-binding sequence of the second strand. In some embodiments, the reverse primer disclosed herein has a 3’ terminal nucleotide that is complementary to an SNV of the mutant allele of the second strand. In some embodiments, theWSGR Docket No. 69583-701.601 reverse primer disclosed herein has a second mismatch to the reverse primer-binding sequence of the second strand.

[0166] In some embodiments, the method disclosed herein comprises subjecting the mixture to a condition sufficient to amplify the at least one double-stranded nucleic acid molecule. In some embodiments, the mixture lacks a universal primer.

[0167] In some embodiments, the forward primer disclosed herein comprises a single mismatch. In some embodiments, the forward primer disclosed herein consists of a single mismatch. In some embodiments, the reverse primer disclosed herein comprises a single mismatch. In some embodiments, the reverse primer disclosed herein consists of a single mismatch. In some embodiments, the first mismatch of the forward primer disclosed herein is located at an internal nucleotide. In some embodiments, the second mismatch of the forward primer disclosed herein is located at an internal nucleotide.

[0168] The method disclosed herein can further comprise detecting an amplification product of the at least one double-stranded nucleic acid molecule. In some embodiments, detecting comprises contacting the amplification product with a detection agent that non-specifically recognizes polynucleic acids. In some embodiments, detecting comprises contacting the amplification product with a nucleic acid intercalating agent. In some embodiments, detecting does not comprise using a mutant allele specific probe. In some embodiments, detecting does not comprise sequencing the amplification product. In some embodiments, the method disclosed herein does not comprise sequencing. For example, the method disclosed herein does not comprise sequencing the amplification product for monitoring the disease status (e.g., minimum residual disease or MRD) of a subject.

[0169] In some embodiments, the forward primer disclosed herein comprises at most 3 mismatches to the forward primer-binding sequence. In some embodiments, the reverse primer disclosed herein comprises at most 3 mismatches to the reverse primer-binding sequence. In some embodiments, the partial complementarity of a primer (e.g., forward or reverse primer) disclosed herein is at least 90% complementary to a primer-binding sequence (e.g., forward or reverse primer-binding sequence).

[0170] In some embodiments, the forward primer disclosed herein has a first sequence that includes the 3’ terminal nucleotide and has a length of at least 10 nucleotides. In some embodiments, the first sequence has at most 3 mismatches. In some embodiments, the first sequence has a length of at least 20 nucleotides. In some embodiments, the first sequence has at most 2 mismatches. In some embodiments, the first sequence has only one mismatch.

[0171] In some embodiments, the reverse primer disclosed herein has a first sequence that includes the 3’ terminal nucleotide and has a length of at least 10 nucleotides. In someWSGR Docket No. 69583-701.601 embodiments, the first sequence has at most 3 mismatches. In some embodiments, the first sequence has a length of at least 20 nucleotides. In some embodiments, the first sequence has at most 2 mismatches. In some embodiments, the first sequence has only one mismatch.

[0172] In some embodiments, a melting temperature (Tm) of the forward primer disclosed herein is from 50 degrees Celsius to 70 degrees Celsius. In some embodiments, the Tm of the forward primer disclosed herein is from 55 degrees Celsius to 60 degrees Celsius. In some embodiments, the Tm of the forward primer disclosed herein is 57 degrees Celsius. In some embodiments, a Tm of the reverse primer disclosed herein is from 50 degrees Celsius to 70 degrees Celsius. In some embodiments, the Tm of the reverse primer disclosed herein is from 55 degrees Celsius to 60 degrees Celsius. In some embodiments, the Tm of the reverse primer disclosed herein is 57 degrees Celsius.

[0173] In some embodiments, the first mismatch of the forward primer disclosed herein to the forward primer-binding sequence of the first strand is at least 6 nucleotides 5’ to the 3’ terminal nucleotide. In some embodiments, the first mismatch of the forward primer disclosed herein to the forward primer-binding sequence of the first strand is at most 6 nucleotides 5’ to the 3’ terminal nucleotide.

[0174] In some embodiments, the set of allele-specific primers disclosed herein has a rate of amplification of a corresponding wild-type allele of at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, or less. In some embodiments, the set of allele-specific primers disclosed herein has an allele-specific amplification rate of at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or more. In some embodiments, the mutant allele disclosed herein is amplified at least 10 times, at least 100 times, at least 1000 times or more than a corresponding wild-type allele is amplified.

[0175] In some embodiments, the plurality of double-stranded nucleic acid molecules further comprises a double-stranded nucleic acid molecule comprising the corresponding wild-type allele. In some embodiments, the set of allele-specific primers disclosed herein does not amplify the double-stranded nucleic acid molecule comprising the corresponding wild-type allele. In some embodiments, the set of allele-specific primers disclosed herein amplifies the doublestranded nucleic acid molecule comprising the corresponding wild-type allele less efficiently than the at least one double-stranded nucleic acid molecule having the mutant allele. In some embodiments, the plurality of double-stranded nucleic acid molecules comprises a first doublestranded nucleic acid molecule comprising a first mutant allele, and a second double-stranded nucleic acid molecule comprising a second mutant allele, wherein the first and second mutant alleles are different mutant alleles. In some embodiments, the plurality of double-stranded nucleic acid molecules comprises a first double-stranded nucleic acid molecule comprising aWSGR Docket No. 69583-701.601 first SNV. In some embodiments, the plurality of double-stranded nucleic acid molecules comprises a second double-stranded nucleic acid molecule comprising a second SNV. In some embodiments, the first and second SNVs are different SNVs.

[0176] The method disclosed herein can further comprise amplifying two or more doublestranded nucleic acid molecules. The two or more double-stranded nucleic acid molecules can be amplified simultaneously. The two or more double-stranded nucleic acid molecules can be amplified simultaneously in a same compartment. Prior to amplifying, the plurality of doublestranded nucleic acid molecules can be partitioned into two or more different compartments. In some embodiments, the two or more different compartments are two or more different water-in- oil droplets. The two or more double-stranded nucleic acid molecules can be amplified simultaneously in two or more different compartments. Each compartment can comprise at least one of the two or more double-stranded nucleic acid molecules and the set of allele-specific primers. In some embodiments, the method disclosed herein can comprise amplifying doublestranded nucleic acid molecules via a multiplex amplification. For example, multiplex amplification can comprise amplifying at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more double-stranded nucleic acid molecules. In some embodiments, the multiplex amplification can be performed simultaneously. The multiplex amplification can be amplified simultaneously in a same compartment or one or more different compartments.

[0177] In some embodiments, the set of allele-specific primers disclosed herein comprises two or more sets of allele-specific primers. Each set of allele-specific primers can be specific to a single mutant allele. Each set of allele-specific primers can comprise a forward primer that has partial complementarity to a forward primer-binding sequence of a first strand of a doublestranded nucleic acid molecule comprising a different / given mutant allele. The forward primer can comprise a 3’ terminal nucleotide that is complementary to a single nucleotide variant of the different / given mutant allele of the first strand. The forward primer can also comprise a first mismatch to the forward primer-binding sequence of the first strand. Each set of allele-specific primers can comprise a reverse primer that has partial complementarity to a reverse primerbinding sequence of a second strand of a double-stranded nucleic acid molecule comprising a different / given mutant allele. The reverse primer can comprise a 3’ terminal nucleotide that is complementary to a single nucleotide variant of the different / given mutant allele of the second strand. The reverse primer can also comprise a second mismatch to the reverse primer-binding sequence of the second strand.

[0178] In some embodiments, the two or more sets of allele-specific primers comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or moreWSGR Docket No. 69583-701.601 sets of allele-specific primers. In some embodiments, the two or more sets of allele-specific primers comprise at most 25, at most 24, at most 23, at most 22, at most 21, at most 20, at most 19, at most 18, at most 17, at most 16, at most 15, at most 14, at most 13, at most 12, at most 11, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3 or less sets of allele-specific primers.

[0179] The method disclosed herein can comprise detecting amplification products of the two or more double-stranded nucleic acid molecules. In some embodiments, the detecting does not differentiate one mutant allele from another different mutant allele. In some embodiments, the detecting detects each mutant allele indiscriminately.

[0180] The method disclosed herein can determining a mean allele frequency of the different mutant alleles or the different SNVs in the sample. In some embodiments, the method disclosed herein does not comprise determining an individual allele frequency of a given mutant allele or a given SNV.

[0181] The method disclosed herein can further comprise, prior to (a) obtaining the sample from a sample. In some embodiments, the subject has cancer. In some embodiments, the subject has been treated with a cancer therapy. In some embodiments, the subject is suspected of having a minimal residual disease (MRD). In some embodiments, the subject has a disease or condition. The disease or condition can be a cancer. In some embodiments, the mutant allele is specific to the subject. In some embodiments, the mutant allele is associated with the disease or condition.

[0182] The method disclosed herein can further comprise prior to (a), identifying one or more mutant alleles in a different sample from the subject. Prior to (a), the identity of one or more mutant alleles of the subject can be known. In some embodiments, the different sample is a tissue sample. The tissue sample can be a tumor tissue sample. In some embodiments, identifying comprises sequencing nucleic acids in the different sample from the subject to identify subject-specific mutant alleles. In some embodiments, the mutant allele comprises a SNV of a gene selected from the group consisting of AKT1, APC, BRAF, CTNNB1, EGFR, FGFR3, FLT3, FOXL2, GNA11, GNAQ, GNAS, IDH1, JAK2, KIT, KRAS, MPL, NRAS / CSDE1, PDGFRA, PIK3CA, RET, TP53, ALK, and KRAS.

[0183] In some embodiments, the plurality of double-stranded nucleic acid molecules are a plurality of DNA molecules. In some embodiments, the plurality of double-stranded nucleic acid molecules are prepared by reverse transcribing a plurality of RNA molecules.

[0184] In some embodiments, the sample is a fluid sample. In some embodiments, the sample is selected from the group consisting of a blood sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, a pleural or peritoneal fluid sample, a stool sample, a seminal fluid, and any combination thereof. In some embodiments, the plurality of double-stranded nucleicWSGR Docket No. 69583-701.601 acid molecules comprises circulating tumor DNAs. In some embodiments, the plurality of double-stranded nucleic acid molecules comprises cell-free DNAs.

[0185] The method disclosed herein can further comprise, prior to (a), isolating the plurality of double-stranded nucleic acid molecules from the sample.Samples

[0186] A nucleic acid molecule or a plurality of nucleic acid molecules (e.g., a plurality of double-stranded nucleic acid molecule) can be prepared from various biological samples of interest to amplify a mutant allele (e.g., SNV, indel, and MNV).

[0187] In some embodiments, the sample is obtained from a subject (e.g., human). In some embodiments, the subject has cancer. In some embodiments, the subject has been treated with a cancer therapy. In some embodiments, the subject is suspected of having a minimal residual disease (MRD). In some embodiments, the subject has a disease or condition. In some embodiments, the subject comprises a mutant allele specific to the subject. In some embodiments, the mutant allele is associated with a disease or condition. In some embodiments, the disease or condition is a cancer. Types of cancers include but are not limited to, acute myeloid leukemia, bladder cancer, including upper tract tumors and urothelial carcinoma of the prostate, bone cancer, including chondrosarcoma, Ewing's sarcoma, and osteosarcoma, breast cancer, including noninvasive, invasive, phyllodes tumor, Paget's disease, and breast cancer during pregnancy, central nervous system cancers, adult low-grade infiltrative supratentorial astrocytoma / oligodendroglioma, adult intracranial ependymoma, anaplastic astrocytoma / anaplastic oligodendroglioma / glioblastoma multiforme, limited (1-3) metastatic lesions, multiple (>3) metastatic lesions, carcinomatous lymphomatous meningitis, nonimmunosuppressed primary CNS lymphoma, and metastatic spine tumors; cervical cancer; chronic myelogenous leukemia (CML); colon cancer, rectal cancer, anal carcinoma; esophageal cancer; gastric (stomach) cancer; head and neck cancers, including ethmoid sinus tumors, maxillary sinus tumors, salivary gland tumors, cancer of the lip, cancer of the oral cavity, cancer of the oropharynx, cancer of the hypopharynx, occult primary, cancer of the glottic larynx, cancer of the supraglottic larynx, cancer of the nasopharynx, and advanced head and neck cancer; hepatobiliary cancers, including hepatocellular carcinoma, gallbladder cancer, intrahepatic cholangiocarcmoma, and extrahepatic cholangiocarcmoma, Hodgkin disease / lymphoma, kidney cancer, melanoma, multiple myeloma, systemic light chain amyloidosis, Waldenstrom's macro globulinemia, myelodysplasia syndromes; neuroendocrine tumors, including multiple endocrine neoplasia, type 1, multiple endocrine neoplasia, type 2, carcinoid tumors, islet cell tumors, pheochromocytoma, poorly differentiated / small cell / atypicalWSGR Docket No. 69583-701.601 lung carcinoids; Non-Hodgkin's Lymphomas, including chronic lymphocytic leukemia / small lymphocytic lymphoma, follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, diffuse large B-Cell lymphoma, Burkitt's lymphoma, lymphoblastic lymphoma, AIDS-Related B-Cell lymphoma, peripheral T-Cell lymphoma, and mycosis fungoides / Sezary Syndrome; nonmelanoma skin cancers, including basal and squamous cell skin cancers, dermatofibrosarcoma protuberans, Merkel cell carcinoma; non-small cell lung cancer (NSCLC), including thymic malignancies; occult primary; ovarian cancer, including epithelial ovarian cancer, borderline epithelial ovarian cancer (Low Malignant Potential), and less common ovarian histologies; pancreatic adenocarcinoma; prostate cancer; small cell lung cancer and lung neuroendocrine tumors; soft tissue sarcoma, including soft-tissue extremity, retroperitoneal, intra-abdominal sarcoma, and desmoid; testicular cancer; thymic malignancies, including thyroid carcinoma, nodule evaluation, papillary carcinoma, follicular carcinoma, Hiirthle cell neoplasm, medullary carcinoma, and anaplastic carcinoma; uterine neoplasms, including endometrial cancer and uterine sarcoma.

[0188] In some embodiments, one or more mutant alleles is identified in a sample from a subject that is different from a sample that will be obtained to amplify a mutant allele (e.g., SNV). For example, the different sample can be a tissue sample, and the sample used to amplify a mutant allele can be a fluid sample. In some embodiments, identifying the one or more mutant alleles comprises sequencing nucleic acids in the different sample from the subject to identify subjectspecific mutant alleles. In some embodiments, the one or more mutant alleles of the subject is known.

[0189] In some embodiments, the sample is a fluid sample. In some embodiments, the fluid sample comprises circulating tumor DNAs or cell-free DNAs. In some embodiments, the sample is selected form the group consisting of a blood sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, a pleural or peritoneal fluid sample, a stool sample, a seminal fluid, and any combination thereof. Examples of samples include but are not limited to, blood, plasma, urine, saliva, cerebrospinal fluid, pleural or peritoneal, stool, seminal, or a biopsy sample of a subject having a cancer or being suspect of having a cancer, or any subject of known or unknown status with respect to genetic variations, blood cells, bone marrow derived mononuclear cells, placenta cells, umbilical cord sample, fetal tissue, fetal fibroblasts or blood cells, tissue from infant or child, neonatal tissue, non-cellular entity comprising nucleic acid (e.g. virus), cell-based organisms (e.g. plant, fungi, eubacteria, archaebacteria, protist, or animal), plants or food products. Examples of biological samples include but not limited to, tissues of lung, respiratory tract, nasal cavity, gastrointestinal tract, mouth, skin, heart, lung,WSGR Docket No. 69583-701.601 kidney, breast, pancreas, liver, blood, muscle, smooth muscle, bladder, gall bladder, colon, intestine, brain, prostate, esophagus, or thyroid.Nucleic Acid Molecule Isolation

[0190] In some embodiments, the method disclosed herein comprises isolating nucleic acid molecules from a sample disclosed herein. In some embodiments, the sample disclosed herein is processed before analysis to obtain a plurality of nucleic acid molecules (e.g.,. a plurality of double-stranded nucleic acid molecules). Examples of sample preparation operations may include extraction of intracellular material from a cell, tissue, fluid (e.g., blood, plasma, urine) or microorganisms. Extracted intracellular material can include nucleic acids, protein, or other macromolecules from the samples. In some embodiments, a sample is prepared using formalin- fixed, paraffin embedded (FFPE), or frozen section for sectioning. In some embodiments, the sample is microdissected with a laser before any extraction methods are used. In some embodiments, extraction of cell free DNA (cfDNA) from samples (e.g., blood, plasma, urine) is performed. In some embodiments, the sample disclosed herein is obtained by swabbing, scraping, phlebotomy, a biopsy (e.g., excisional, fine needle aspiration, incisional, core needle), or any other suitable method particularly for subjects having or suspected of having a disease or infection. In some embodiments, serial biopsies are obtained from a diseased tissue or organ.

[0191] In some embodiments, a plurality of nucleic acid molecules (e.g., a plurality of doublestranded nucleic acid molecules or RNA) are isolated from a sample disclosed herein. For example, in some embodiments, DNA or RNA can be extracted from a sample by the use of detergent lysates, sonication, or vortexing with beads (e.g., glass beads). In some embodiments, a plurality of nucleic acid molecules are isolated by using standard extraction kits. In some embodiments, a plurality of nucleic acid molecules are isolated using gradient centrifugation protocols, boiling, purification kits, or liquid extraction protocols using Trizol or DNAzol. . In some embodiments, a plurality of nucleic acid molecules (e.g., a plurality of double-stranded nucleic acid molecules) is prepared by reverse transcribing a plurality of RNA molecules obtained or extracted from a sample disclosed herein.

[0192] In some embodiments, a plurality of nucleic acid molecules (e.g., a plurality of doublestranded nucleic acid molecule) is prepared from an RNA using RT-PCR. In some embodiments, the plurality of nucleic acid molecules is prepared by an RT-PCR, followed by dPCR in two distinct steps or a single step. In some embodiments, the plurality of nucleic acid molecules is pre-amplified in a separate reaction to specifically or non-specifically enrich the target sequences of interest. In some embodiments, the plurality of nucleic acid molecules is not pre-amplified.WSGR Docket No. 69583-701.601Am pH lien tion

[0193] In some embodiments, the method disclosed herein comprises amplification. In some embodiments, the method disclosed herein comprises allele-specific amplification. In some embodiments, the method disclosed herein comprises amplifying a mutant allele of a nucleic acid molecule (e.g., double-stranded nucleic acid molecule). In some embodiments, the mutant allele comprises an indel, a multi -nucleotide variant (MNV), or a single nucleic nucleotide variant (SNV). In some embodiments, the method disclosed herein comprises amplifying a mutant allele comprising a single nucleic nucleotide variant (SNV). In some embodiments, the method disclosed herein comprises amplifying a mutant allele comprising a single nucleic nucleotide variant (SNV) as set forth in Table 1. In some embodiments, the method disclosed herein comprises amplifying two or more nucleic acid molecules. In some embodiments, the method disclosed herein comprises amplifying two or more double-stranded nucleic acid molecules. In some embodiments, the method disclosed herein comprises amplifying two or more nucleic acid molecules simultaneously. In some embodiments, the method disclosed herein comprises amplifying two or more double-stranded nucleic acid molecules simultaneously. In some embodiments, the method disclosed herein comprises amplifying two or more nucleic acid molecules (e.g., double-stranded molecules) simultaneously in a same compartment. In some embodiments, the method disclosed herein comprises, prior to amplifying, partitioning a plurality of nucleic acid molecules (e.g., a plurality of double-stranded molecules) into two or more compartments. In some embodiments, the method disclosed herein comprises, amplifying the two or more nucleic acid molecules (e.g., double-stranded nucleic acid molecules) simultaneously in two or more different compartments, wherein each compartment comprises at least one of the two or more nucleic acid molecules (e.g., double-stranded nucleic acid molecules) and the set of allele-specific primers disclosed herein. In some embodiments, the two or more different compartments are two or more different water-in-oil droplets. For example, the plurality of nucleic acid molecules (e.g., a plurality of double-stranded molecules) from a sample from a subject can be partitioned into at least about 100, at least about 10,000, at least about 100,000, at least about 1,000,000, at least about 10,000,000, at least about 100,000,000, at least about 1,000,000,000, at least about 10,000,000,000, at least about 100,000,000,000, or more compartments. The plurality of nucleic acid molecules (e.g., a plurality of double-stranded molecules) from a sample from a subject can comprise at least about 100, at least about 10,000, at least about 100,000, at least about 1,000,000, at least about 10,000,000, at least about 100,000,000, at least about 1,000,000,000, at least about 10,000,000,000, at least about 100,000,000,000, or more template molecules. The compartments may be substantially uniform in size, or may have different sizes (e.g., a set of two or more individual uniform-sizedWSGR Docket No. 69583-701.601 compartments). A non-limiting example of a compartment is a droplet. The compartments may also vary continuously in size with a predetermined size distribution or with a random size distribution. The droplet can be a small, typically spherical, liquid encapsulated by an immiscible fluid, such as the continuous phase of an emulsion. The volume of the droplets in the emulsion and / or the average volume of the droplets in the emulsion can be, for example, less than about 1 microliter, less than about 1 nanoliter, or less than about 1 picoliter. The droplets (or emulsion droplets) may especially have a diameter (or average diameter) of less than about 1000, 100, or 10 micrometers, or about 1000 to 10 micrometers. Droplets may be spherical or non-spherical. The droplet may be a simple droplet or a composite droplet, that is, a droplet in which at least one droplet encapsulates at least one other droplet.

[0194] Amplification refers to either producing an additional copy or copies of all or a segment of a target nucleic acid comprising a mutant allele by template-directed primer extension (target amplification) or amplifying detection signal for qualitatively / quantitatively measurement (signal amplification) or both. Amplification can be performed under temperature cycled or isothermal conditions or combined. Amplification can be linear or exponential. In some embodiments, amplifying is performed with PCR. Non-limiting examples of PCR include multiplex PCR, long-range PCR, qPCR, single-cell PCR, digital PCR (dPCR), fast-cycling PCR, methylation-specific PCR, hot start PCR, high-fidelity PCR, differential display PCR, or in situ PCR. In some embodiments, amplifying is performed with dPCR.

[0195] PCR (e.g., digital PCR) can comprise the steps of denaturing, annealing, elongation, or a combination thereof. In some embodiments, PCR comprises at least 5 PCR cycles, at least 10 PCR cycles, at least 20 PCR cycles, at least 30 PCR cycles, or at least 40 PCR cycles, wherein each cycle comprises the steps of denaturing, annealing, elongation, or a combination thereof. In some embodiments, the denaturing step occurs between 30 seconds to 1 minute, 1 minute to 1.5 minutes, 1.5 minutes to 2 minutes, or 2 minutes to 2.5 minutes. In some embodiments, the denaturing step occurs at a temperature between 80 degrees Celsius to 85 degrees Celsius, 85 degrees Celsius to 90 degrees Celsius, 90 degrees Celsius to 95 degrees Celsius, between 95 degrees Celsius to 100 degrees Celsius, or between 100 degrees Celsius to 105 degrees Celsius. In some embodiments, the primer annealing step occurs between 10 seconds to 15 seconds, 15 seconds to 20 seconds, 20 seconds to 25 seconds, 25 seconds to 30 seconds, 30 seconds to 1 minute, 1 minute to 1.5 minutes, 1.5 minutes to 2 minutes, or 2 minutes to 2.5 minutes. In some embodiments, the primer annealing step occurs at a temperature between 50 degrees Celsius to 55 degrees Celsius, 55 degrees Celsius to 60 degrees Celsius, 60 degrees Celsius to 65 degrees Celsius, between 65 degrees Celsius to 70 degrees Celsius, or between 70 degrees Celsius to 75 degrees Celsius. In some embodiments, the elongation or extension step occurs between 10WSGR Docket No. 69583-701.601 seconds to 15 seconds, 15 seconds to 20 seconds, 20 seconds to 25 seconds, 25 seconds to 30 seconds, 30 seconds to 1 minute, 1 minute to 1.5 minutes, 1.5 minutes to 2 minutes, or 2 minutes to 2.5 minutes. In some embodiments, the primer annealing step occurs at a temperature between 50 degrees Celsius to 55 degrees Celsius, 55 degrees Celsius to 60 degrees Celsius, 60 degrees Celsius to 65 degrees Celsius, between 65 degrees Celsius to 70 degrees Celsius, or between 70 degrees Celsius to 75 degrees Celsius. In some embodiments, the elongation or extension step occurs between 10 seconds to 15 seconds, 15 seconds to 20 seconds, 20 seconds to 25 seconds, 25 seconds to 30 seconds, 30 seconds to 1 minute, 1 minute to 1.5 minutes, 1.5 minutes to 2 minutes, or 2 minutes to 2.5 minutes. In some embodiments, the elongation or extension step occurs at a temperature between 50 degrees Celsius to 55 degrees Celsius, 55 degrees Celsius to 60 degrees Celsius, 60 degrees Celsius to 65 degrees Celsius, between 65 degrees Celsius to 70 degrees Celsius, between 70 degrees Celsius to 75 degrees Celsius, between 75 degrees Celsius to 80 degrees Celsius. PCR can further comprise a final extension step. In some embodiments, the final extension step occurs between 30 seconds to 1 minute, 1 minute to 2 minutes, 2 minutes to 3 minutes, 3 minutes to 4 minutes, 4 minutes to 5 minutes, or 5 minutes to 6 minutes. In some embodiments, the final extension step occurs at a temperature between 50 degrees Celsius to 55 degrees Celsius, 55 degrees Celsius to 60 degrees Celsius, 60 degrees Celsius to 65 degrees Celsius, between 65 degrees Celsius to 70 degrees Celsius, between 70 degrees Celsius to 75 degrees Celsius, between 75 degrees Celsius to 80 degrees Celsius.

[0196] Hybridization or annealing conditions during the amplification step disclosed herein can include chemical components and their concentrations (e.g., salts, chelating agents, formamide) of an aqueous or organic solution containing the nucleic acids, and the temperature of a mixture disclosed herein in which one nucleic acid strand bonds to a second nucleic acid strand by complementary strand interactions to produce a hybridization complex.

[0197] In some embodiments, amplification comprises forming a mixture. In some embodiments, the mixture comprises at least one nucleic acid molecule (e.g., double-stranded nucleic acid molecule) and a set of allele-specific primers disclosed herein. In some embodiments, the mixture lacks a universal primer. In some embodiments, the mixture comprises a set of allele primers comprising a forward primer and a reverse primer. In some embodiments, the forward and reverse primers bind to opposite strands of a target nucleic acid (e.g., double-stranded nucleic acid molecule), wherein the forward primer has full or partial complementarity to a forward primer-sequence of a first strand, and the reverse primer has full or partial complementarity to a reverse primer-binding sequence of a second strand. In some embodiments, the forward primer comprises a 3’ terminal nucleotide that is complementary to a mutant allele (e.g., SNV) of the first strand. In some embodiments, the forward primerWSGR Docket No. 69583-701.601 comprises a first mismatch to the forward primer-binding sequence of the first strand. In some embodiments, the reverse primer comprises a 3’ terminal nucleotide that is complementary to a mutant allele (e.g., SNV) of the first strand. In some embodiments, the forward primer comprises a second mismatch to the reverse primer-binding sequence of the second strand. In some embodiments, the forward primer and / or the reverse primer comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 mismatches. In some embodiments, the forward primer and / or the reverse primer comprises at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 mismatches. In some embodiments, the forward primer and / or the reverse primer comprises at most 3 mismatches. In some embodiments, the forward primer comprises a single mismatch. In some embodiments, the forward primer consists of a single mismatch. In some embodiments, the reverse primer comprises a single mismatch. In some embodiments, a reverse primer consists of a single mismatch.

[0198] In some embodiments, the mixture further comprises one or more PCR components necessary to amplify at a mutant allele (e.g., SNV). In some embodiments, the mixture comprise nucleotides (e.g., dNTPs, ddNTPs), a thermostable polymerase, a Tris buffer, a monovalent salt, Mg2+, or a combination thereof. In some embodiments, the mixture comprises ethylenediaminetetraacetic acid (EDTA), magnesium, tetramethyl ammonium chloride (TMAC), or any combination thereof. In some embodiments, Tris buffer is used at, for example, a concentration of between 10 and 100 mM, such as between 10 and 25 mM, 25 and 50 mM, 50 and 75 mM, or 25 and 75 mM, inclusive. In some embodiments, any of these concentrations of Tris are used at a pH between 7.5 and 8.5. In some embodiments, the mixture comprises a combination of KC1 and (NH4)2SO4. In some embodiments, the mixture comprises between 50 and 150 mM KC1 and between 10 and 90 mM (NH4)2SO4, inclusive. In some embodiments, the mixture comprises concentration of KC1 that is between 0 and 30 mM, between 50 and 100 mM, or between 100 and 150 mM, inclusive. In some embodiments, the mixture comprises concentration of (NH4)2SO4 that is between 10 and 50 mM,50 and 90 mM, 10 and 20 mM, 20 and 40 mM, 40 mM and 60, or 60 mM and 80 mM (NH4)2SO4, inclusive. In some embodiments, the mixture comprises ammonium [NH4+] with a concentration that is between 0 and 160 mM, such as between 0 to 50, 50 to 100, or 100 to 160 mM, inclusive. In some embodiments, the mixture comprises a sum of a potassium and ammonium concentration ([K+] + [NH4+]) that is between 0 and 160 mM, such as between 0 to 25, 25 to 50, 50 to 150, 50 to 75, 75 to 100, 100 to 125, or 125 to 160 mM, inclusive. An exemplary buffer with [K+] + [NH4+] = 120 mM is 20 mM KC1 and 50 mM (NH4)2SO4. In some embodiments, the buffer includes 25 to 75 mM Tris, pH 7.2 to 8, 0 to 50 mM KCL, 10 to 80 mM ammonium sulfate, and 3 to 6 mMWSGR Docket No. 69583-701.601 magnesium, inclusive. In some embodiments, the buffer includes 25 to 75 mM Tris pH 7 to 8.5, 3 to 6 mM MgC12, 10 to 50 mM KC1, and 20 to 80 mM (NH4)2SO4, inclusive.

[0199] In some embodiments, a crowding agent is used, such as polyethylene glycol (e.g., PEG, e.g., PEG 8,000) or glycerol. In some embodiments, the amount of PEG (e.g., PEG 8,000) is between 0.1 to 20%, such as between 0.5 to 15%, 1 to 10%, 2 to 8%, or 4 to 8%, inclusive. In some embodiments, the amount of glycerol is between 0.1 to 20%, such as between 0.5 to 15%, 1 to 10%, 2 to 8%, or 4 to 8%, inclusive. In some embodiments, a crowding agent allows either a low polymerase concentration and / or a shorter annealing time to be used.

[0200] In some embodiments, the mixture comprises a polymerase. A polymerase is an enzyme that can perform template directed extension of a primer hybridized to the template. Nonlimiting examples of polymerase include a DNA polymerase, an RNA polymerase or a reverse transcriptase. Examples of DNA polymerases include: E. coli DNA polymerase I, Taq DNA polymerase, S. pneumoniae DNA polymerase I, Tfl DNA polymerase, D. radiodurans DNA polymerase I, Tth DNA polymerase, Tth XL DNA polymerase, M. tuberculosis DNA polymerase I, M. thermoautotrophicum DNA polymerase I, Herpes simplex- 1 DNA polymerase, T4 DNA polymerase, thermosequenase or a wild-type or modified T7 DNA polymerase, 029 Polymerase, Bst Polymerase, Vent Polymerase, 9° Nm Polymerase, Klenow fragment of DNA Polymerase I. Examples of reverse transcriptase: AMV Reverse Transcriptase, MMLV Reverse Transcriptase, HIV Reverse Transcriptase. Examples of RNA polymerases include: T7 RNA polymerase or SP6 RNA polymerase, bacterial RNA polymerases and eukaryotic RNA polymerases.

[0201] In some embodiments, the method disclosed herein results in a rate of amplification of a wild-type allele (false positive rate, or specificity) of at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, at most 0.5%, at most 0.4%, at most 0.3%, at most 0.2%, at most 0.1%, or less. In some embodiments, the method disclosed herein results in an allele-specific amplification rate (sensitivity) of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more. In some embodiments, a mutant allele is amplified at least 10 times, 100 times, 1000 times, or more than a corresponding wild-type allele is amplified.Digital PCR

[0202] In some embodiments, the methods disclosed herein comprises amplification of a target nucleic acid molecule (e.g., double-stranded nucleic acid molecule comprising a mutant allele) with a digital polymerase chain reaction (dPCR). The term “digital polymerase chain reaction” or “dPCR” or “digital PCR” refers to a refined version of polymerase chain reaction (PCR)WSGR Docket No. 69583-701.601 methods used to directly quantify and clonally amplify nucleic acids including DNA, cDNA or RNA, such that the amount of target nucleic acid can be directly quantitatively measured without the use of standard curves. dPCR can achieve the direct quantitative measurement by partitioning individual target nucleic acid molecules present in a sample into multiple aliquots within many separate reaction compartments that are able to localize and concentrate the amplification product to detectable levels. In some embodiments, the sample is partitioned into one or more different compartments such that most aliquots (e.g., at least 50%, 75%, 90%, 95% or 99%) receive zero or one molecule of each target nucleic acid to be detected. The partitions are amplified to the terminal plateau phase of PCR (or end-point) and then read to determine the fraction of positive partitions. The presence of a signal in any chamber is an indication the target nucleic is present and a count of chambers containing the PCR end-product is a direct measure of the absolute nucleic acid quantity. The capture or isolation of individual nucleic acid molecules, typically by way of dilution, may be effected in capillaries, microemulsions, arrays of miniaturized chambers, or on nucleic acid binding surfaces.

[0203] dPCR can include a variety of formats, including droplet digital PCR, BEAMing (beads, emulsion, amplification, and magnetic), and microfluidic chips. "Droplet digital PCR" (ddPCR) refers to a digital PCR assay that measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions that support PCR amplification (Hinson et al., 2011, Anal. Chem. 83 :8604-8610; Pinheiro et al., 2012, Anal. Chem. 84: 1003-1011). A single ddPCR reaction may be comprised of at least 20,000 partitioned droplets per well. A "droplet" or "water-in-oil droplet" refers to an individual partition of the droplet digital PCR assay. A droplet supports PCR amplification of template molecule(s) using homogenous assay chemistries and workflows similar to those widely used for real-time PCR applications (Hinson et al., 2011, Anal. Chem. 83:8604-8610; Pinheiro et al., 2012, Anal. Chem. 84: 1003-1011). ddPCR may be performed using any platform that performs a dPCR assay that measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions that support PCR amplification. The strategy for ddPCR may be summarized as follows: a sample is diluted and partitioned into thousands to millions of separate reaction compartments (water-in-oil droplets) so that each contains one or no copies of the nucleic acid molecule of interest. The number of "positive" droplets detected, which contain the target amplicon (i.e., nucleic acid molecule of interest), versus the number of "negative" droplets, which do not contain the target amplicon (i.e., nucleic acid molecule of interest), may be used to determine the number of copies of the nucleic acid molecule of interest that were in the original sample. Examples of droplet digital PCR systems include but not limited to the QX100™ Droplet Digital PCR System by Bio-Rad,WSGR Docket No. 69583-701.601 which partitions samples containing nucleic acid template into 20,000 nanoliter-sized droplets, and the RainDrop™ digital PCR system by RainDance, which partitions samples containing nucleic acid template into 1,000,000 to 10,000,000 picoliter-sized droplets.Detection

[0204] In some embodiments, the method disclosed herein comprises detecting an amplification product of a target nucleic acid molecule (e.g., double-stranded nucleic acid molecule comprising a mutant allele). In some embodiments, the method disclosed herein comprises detecting amplification products of two or more nucleic acid molecules (e.g., double-stranded nucleic acid molecule). In some embodiments, detecting does not differentiate one mutant allele from another mutant allele. In some embodiments, detecting detects each mutant allele indiscriminately. Amplification reactions used with the methods, compositions, and kits disclosed herein can generate one or more signals. In some embodiments, labels are used in or after the amplification reaction to generate the signal for detection of one or more amplification products. In some embodiments, dyes are used in or after the amplification reaction to generate the signal.

[0205] In some embodiments, detecting comprises contacting the amplification product with a detection agent that non-specifically recognizes polynucleic acids. In some embodiments, a nonspecific polynucleic acid detection agent detects the amplification products irrespective of sequences of the amplification products. In some embodiments, the non-specific polynucleic acid detection agent detects any nucleic acid molecules (e.g., double-stranded nucleic acid molecules). In some embodiments, a detection agent that non-specifically recognizes polynucleic acids is a nucleic acid intercalating dye. Examples of intercalating dyes that can be used with the disclosure include but are not limited to, ethidium bromide, propidium iodide, acridine orange, 9-amino-6-chloro-2-methoxyacridine (ACMA), SYBR™ Green, SYBR™ Green II, SYBR™ Gold, YO (Oxazole Yellow), TO (Thiazole Orange), PG (PicoGreen®), or EvaGreen®. In some embodiments, the DNA intercalating dye is EvaGreen. Positive dPCR reactions can be distinguished from negative dPCR reactions by their elevated signal intensity over the background signal generated by primers. In some embodiments, detecting does not comprise using a mutant allele specific probe.

[0206] In some embodiments, detecting comprises using a mutant allele specific probe. In some embodiments, the amplification product is detected with fluorophore labeled probes, such as Taqman probe, Molecular Beacon, and fluorophore labeled primers provided with a quencher labeled complementary oligo. In another embodiment, the target nucleic acid of the dPCR is detected with a combination of DNA intercalating dyes and fluorophore labeled probes.WSGR Docket No. 69583-701.601Examples of fluorophores used with the disclosure include but are not limited to, 5-FAM (also called 5-carboxyfluorescein; also called Spiro(isobenzofuran-1(3H)' 9'-(9H)xanthene)-5- carboxylic aci',3',6'-dihydroxy-3-oxo-6- carboxyfluorescein); 5-Hexachloro-Fluorescein; ([4,',2',4',5',7'-hexachloro'(3',6'-dipivaloyl- fluoresceinyl)-6-carboxyli- c acid]); 6-Hexachloro- Fluorescein; ([4,',2',4',5',7'-hexachloro'(3',6'- dipivaloylfluoresceinyl)-5-carboxylic acid]); 5- Tetrachloro-Fluorescein; ([4,',2',7'-tetra-chloro'(3',6'-dipivaloylfluoresceinyl)-5-carboxylic acid]); 6-Tetrachloro-Fluorescein; ([4, ',2', 7'- tetrachloro'(3',6'-dipivaloylfluoresceinyl)-6- carboxylic acid]); 5-TAMRA (5-carboxytetramethylrhodamine); Xanthylium, 9-(2,4- di carb oxy phenyl) -3 ,6-bis(dimethyl-amino); 6-TAMRA (6-carboxytetramethylrhodamine); 9- (2,5-dicarboxyphenyl)-3,6-bis(dimethylamino); EDANS -5 -((2-aminoethyl)amino)naphthalene- 1 -sulfonic acid); 1,5-IAEDANS (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-l -sulfonic acid); Cy5 (lndodicarbocyanine-5); Cy3 (lndo-dicarbocyanine-3); and BODIPY FL (2,6- dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pr- oprionic acid); Quasar™-670 dye (Biosearch Technologies); Cal Fluor™ Orange dye (Biosearch Technologies); Rox dyes; Max dyes (Integrated DNA Technologies), or derivatives thereof.

[0207] In some embodiments, detecting does not comprise sequencing the amplification product. In some embodiments, detecting may comprise sequencing the amplification product.

[0208] In some embodiments, sequencing is performed to identify one or more mutant alleles in a subject, prior to monitoring a disease status of the subject. For example, a tumor tissue sample can be obtained from the subject and subject to sequencing to identify subject-specific mutant alleles, which can be used to design the allele-specific primers for future detection of the subject-specific mutant alleles during the course of a treatment, and / or to monitor the disease status of the subject. Non-limiting examples of sequencing methods include Sanger sequencing, next generation sequencing, massively parallel sequencing, exome sequencing, whole genome sequencing, nanopore sequencing, pyrosequencing, ATAC-seq, RNA sequencing, single-cell RNA sequencing or single nuclei RNA sequencing.

[0209] In some embodiments, detection of one or more amplification products is analyzed to determine a mean allele frequency of one or more different mutant alleles in a sample. In some embodiments, the methods described herein does not involve determining allele frequency of each individual mutant allele. Mean allele frequency, for example, can be calculated by the total number of one or more mutant alleles (across the one or more genomic locations) detected divided by the total number of alleles at the one or more genomic locations analyzed. The total number of one or more mutant alleles detected can be the total copy number of the one or more mutant alleles detected. The total number of alleles at the one or more genomic locations analyzed can be the total copy number at the one or more genomic locations analyzed. In someWSGR Docket No. 69583-701.601 embodiments, the total number of alleles at the one or more genomic locations analyzed can be quantified by using one or more housekeeping genes. In some cases, the expression levels of the mutant allele can be compared to the wild-type allele, and the expression data can be normalized to a housekeeping gene to account for potential differences in overall gene expression levels. The housekeeping genes can be used to determine a gene equivalence in a given sample. For example, to determine gene equivalence using housekeeping genes, the expression level of a target gene can be quantified relative to the expression level of a housekeeping gene, and then compare these ratios across different samples to assess relative gene expression. Non-limiting examples of housekeeping genes include, ACTB, ARBP, GAPDH, HPRT, SDHA, UBC, YWHAZ, HPRT1, RPL13A, RPL17, RPSD, TBP, ATP5fl / ATP5PB, B2M, PGK1, RER1, TRT, OTX2, or RPP30. In some embodiments, the housekeeping gene used in any one of the methods disclosed herein is RPP30. In some embodiments, detection of one or more amplification products is analyzed to determine a mean allele frequency of one or more different SNVs in a sample. In some embodiments, detection of one or more amplification products is analyzed to determine tumor fraction. Tumor fraction can refer to a measurement of the amount of circulating tumor DNA (ctDNA) in a sample (e.g., blood sample) relative to the total cell-free DNA. The tumor fraction can indicate a percentage of the DNA fragments that are from the tumor within the total cell-free DNA. The tumor fraction can be a number between 0 and 1. For example, 0.5 can indicate that 50% of the DNA fragments in a sample containing cell-free DNAs are from tumor.

[0210] In one aspect, a method of amplifying a mutant allele comprises a single nucleotide variant (SNV) in a sample comprising a plurality of double-stranded nucleic acid molecules each having a first strand and a second strand, the plurality comprising at least one double-stranded nucleic acid molecule having the mutant allele comprising the SNV, the method comprising: forming a mixture comprising the at least one double-stranded nucleic acid molecule having the mutant allele and a set of allele-specific primers comprising a forward primer and a reverse primer, wherein the forward primer has partial complementarity to a forward primer-binding sequence of the first strand and comprises (i) a 3’ terminal nucleotide that is complementary to a single nucleotide variant of the mutant allele of the first strand and (ii) a first mismatch to the forward primer-binding sequence of the first strand, and wherein the reverse primer has partial complementarity to a reverse primer-binding sequence of the second strand and comprises (i) a 3’ terminal nucleotide that is complementary to an SNV of the mutant allele of the second strand and (ii) a second mismatch to the reverse primer-binding sequence of the second strand; and subjecting the mixture to a condition sufficient to amplify the at least one double-stranded nucleic acid molecule.WSGR Docket No. 69583-701.601B. Method of Determining Disease Status

[0211] Provided herein is a method of determining a disease status of a subject disclosed herein.

[0212] In some embodiments, the method comprises obtaining a first sample comprising a first plurality of nucleic acid molecules from the subject at a first time point. In some embodiments, the method comprises providing a first sample that has been obtained from a subject at the first time point. In some embodiments, the first plurality of nucleic acid molecules of a first sample comprises or is suspected of comprising at least one mutant allele (e.g., SNV).

[0213] In some embodiments, the method comprises conducting a first allele-specific amplification of the first plurality of nucleic acid molecules using a set of allele-specific primers disclosed herein. In some embodiments, the method further comprises detecting amplification products of the first allele-specific amplification using a non-specific polynucleic acid detection agent disclosed herein. In some embodiments, the detection of the amplification products is used to determine a disease status of a subject.

[0214] In some embodiments, the method further comprises obtaining a sample comprising a second plurality of nucleic acid molecules from the subject at a second time point. In some embodiments, the method further comprises providing a second sample that has been obtained from the subject at a second time point. In some embodiments, the second plurality of nucleic acid molecules of the second sample comprises or is suspected of comprising a detectable level of at least one mutant allele. In some embodiments, the second plurality of nucleic acid molecules of the second sample does not comprise a detectable level of at least one mutant allele. In some embodiments, the method further comprises detecting amplification products of the first allele-specific amplification and amplification products of the second allele-specific amplification using a non-specific polynucleic acid detection agent disclosed herein to determine the disease status of the subject.

[0215] In some embodiments, detecting comprises determining a first allele frequency of the at least one mutant allele in the first sample, determining a second allele frequency of the at least one mutant allele in the second sample, and comparing the first allele frequency and the second allele frequency. In some embodiments, at least one mutant allele comprises two or more mutant alleles, and wherein the first allele frequency and / or the second allele frequency comprises a first mean allele frequency and / or a second mean allele frequency.

[0216] In some embodiments, the method disclosed herein does not comprise determining any individual allele frequency for a given mutant allele. In some embodiments, determining the disease status comprises determining a tumor fraction.

[0217] In one aspect, provided herein is a method of determining a disease status of a subject, the method comprising: obtaining a first sample comprising a first plurality of nucleic acidWSGR Docket No. 69583-701.601 molecules from the subject at a first time point or providing the first sample that has been obtained from the subject at the first time point, wherein the first plurality of nucleic acid molecules of the first sample comprises or is suspected of comprising at least one mutant allele; conducting a first allele-specific amplification of the first plurality of nucleic acid molecules using a set of allele-specific primers; detecting amplification products of the first allele-specific amplification using a non-specific polynucleic acid detection agent; and determining the disease status of the subject based on detecting in (c); wherein (i) the set of allele-specific primers has a rate of amplification of a corresponding wild-type allele of at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, or less, (ii) the set of allele-specific primers has an allele-specific amplification rate of at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or more, or (iii) the mutant allele is amplified at least 10 times, 100 times, 1000 times or more than a corresponding wild-type allele is amplified.

[0218] In one aspect, provided herein is a method of determining a disease status of a subject, the method comprising: obtaining a first sample comprising a first plurality of nucleic acid molecules from a subject at a first time point or providing the first sample that has been obtained from the subject at the first time point, wherein the first plurality of nucleic acid molecules of the first sample comprises or is suspected of comprising at least one mutant allele; conducting a first allele-specific amplification of the first plurality of nucleic acid molecules; obtaining a second sample comprising a second plurality of nucleic acid molecules from the subject at a second time point or providing the second sample that has been obtained from the subject at the second time point, wherein the second plurality of nucleic acid molecules of the second sample comprises or is suspected of comprising, or does not comprise a detectable level of the at least one mutant allele; conducting a second allele-specific amplification of the second plurality of nucleic acid molecules; detecting amplification products of the first allele-specific amplification and amplification products of the second allele-specific amplification using a nonspecific polynucleic acid detection agent; and determining the disease status of the subject based on detecting in (e).VI. EXAMPLES

[0219] The following examples are included for illustrative purposes only and are not intended to limit the scope of the inventive concepts.Example 1: Sensitive Allele-Specific PCR

[0220] To build a sensitive test to detect rare somatic variants, publicly available allele-specific primer designs were evaluated and extended. Briefly, the allele-specific primers were designed with Primer XL, a primer design webtool. Primers as shown in Table 2 and FIGs. 1A-1E wereWSGR Docket No. 69583-701.601 subjected to PCR conditions with either wild-type (wt) or mutant template DNA to assess primer pairs’ sensitivity and specificity in amplifying the mutant DNA. Underline in Table 2 represents the target SNV. 100 pM primer stocks were prepared by IDT, suspended in IDTE pH 8.0.Primer stocks were diluted to 4 uM as an intermediate dilution, and further diluted 10-fold to yield a 400 nM final reaction concentration comprising wt or mutant template DNA.

[0221] The wt template was obtained from either healthy donor plasma or cell lines, as outlined below:1. Healthy donor plasma protocol: DNA was extracted from healthy donor plasma using the Applied Biosystems MagMAX™ Cell-Free DNA Isolation Kit (Catalog number: A29319) obtained from Thermo-Fisher. cfDNA fraction was quantified using an Agilent BioAnalyzer.2. Cell line DNA protocol: lOOug of Human Genomic DNA derived from cell lines was obtained from Promega (Catalog number: G3041) acoustically sheared by Covaris (Woburn, MA) to yield fragments of ~180bp to mimic cell free DNA (cfDNA); fragment size was confirmed using an Agilent (Santa Clara, CA) BioAnalyzer.3. Either healthy donor plasma and sheared cell line DNA were diluted (based on weight) to yield 1000 genome equivalents per dPCR reaction.

[0222] For obtaining mutant template, oligos with complete amplicon for each assay, with the mutant allele, were ordered from IDT. The oligos were resuspending and diluted in TE buffer to obtain a concentration of approximately 1000 copies per reaction.

[0223] To accurately assess the concentration of each oligos or wt template, dPCR was run with non-allele specific (NAS) primers at about 5-10 degrees centigrade below the calculated annealing temperature of the primers. For performing dPCR, QIAcuity One Digital PCR System (Qiagen) was used with a preincubation at 95°C for 120 seconds, followed by 40 cycles of 95°C for 15 seconds and 60°C for 30 seconds. All reactions were performed in either 8-well or 24- well Qiagen 26K partition nanoplates, with a reaction volume of 40pL. Next, dPCR assays were performed using different combinations of NAS primers, allele-specific (AS) primers, and allele-specific multiple mismatch (ASMM) primer, and the results were compared to the total number of oligo copies identified. QIAcuity software was used to call positive partitions. Five replicates were performed.Table 2. Template and Primer SequencesWSGR Docket No. 69583-701.601

[0224] As shown in FIG. 2, a first primer combination, an AS forward primer and a NAS reverse primer yielded high amplification of the wt template. An ASMM primer, which has an artificial mismatch, was combined with a NAS primer to magnify the amplification efficiency of the mutant as compared to the wt template. The combination of ASMM and NAS primers showed lower wt template amplification. The combination of AS / AS primers and ASMM / AS primers lead to similar false positive rates. To further reduce the false positive rate, the approach was extended by using ASMM primers in both directions: ASMM primers in both forward and reverse strands. As shown in FIG. 2, the combination of ASMM / ASMM primers increased the specificity to < 1 false positive partitions per thousand genome equivalents, an order of magnitude improvement relative to the one ASMM primer approach. The results are also shown in Table 3 for each of the five replicates performed.WSGR Docket No. 69583-701.601Table 3. False Positive Rate (Specificity)

[0225] These same primer combinations in the same dPCR conditions were tested against mutant template to assess their amplification efficiency of mutant template (sensitivity). As shown in FIG. 3, mutant template was replicated similarly across the primer combinations. The results are also shown in Table 4 for each of the five replicates performed.Table 4. Allele Specific Amplification (Sensitivity)

[0226] Correctively, the results shown in FIG. 2 and FIG. 3 showed that the ASMM / ASMM primer combination amplified mutant template at high efficiency with low false positive rate.Example 2: Multiplex Sensitive Allele-Specific PCR

[0227] Multiplex sensitive allele-specific PCR was performed to identify multiple mutant alleles in a sample.

[0228] Similar experiments are performed as described in Example 1, but two set of primers were included in a sample with wild-type (WT) or mutant DNA template for multiplexing. Table 5 and Table 6 shows the sequences for the WT template, mutant template, and the primers used. The target SNV relative to the WT template is underlined and bolded. The additional target mismatch relative to the mutant template is bolded.WSGR Docket No. 69583-701.601Table 5. Template and Primer Sequences for Multiplexing - Set 1Table 6. Template and Primer Sequences for Multiplexing - Set 2WSGR Docket No. 69583-701.601

[0229] The primers listed in Table 5 and Table 6 were tested in varying combinations as presented in Table 7 and Table 8. A primer combination of AS1 / AS2 primers, ASMM1 / AS2, AS1 / ASMM2 primers were tested with the WT templates (WT Template 1 and WT Template 2) in the same run to measure false positive rates via dPCR. The combination of ASMM1 / ASMM2 primers were also tested, and showed that the combination lowered the false positive rate. The subscript represents the set of primers used for the experiment. For example, AS1 / AS2 primers means that forward AS 1, reverse AS 1, forward AS 2 and reverse AS 2 from Table 7 and Table 8 were used. The results are shown in Table 7 for each of the five replicates performed.

[0230] These same primer combinations in the same dPCR conditions were tested against mutant templates (Mutant Template 1 and Mutant Template 2) in the same run to assess their amplification efficiency of mutant template (sensitivity). As shown in Table 8, mutant template was replicated similarly across the primer combinations.Table 7. False Positive Rate (Specificity, WT DNA)Table 8. Sensitivity (% Positive Partitions, Mutant DNA)

[0231] While preferred embodiments of the present inventive concepts have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the inventive concepts. It should be understood that various alternatives to the embodiments of the inventive concepts described herein may be employed in practicing the inventive concepts. It is intended that the following claims define the scope of the inventive concepts and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WSGR Docket No. 69583-701.601CLAIMSWhat is claimed is:

1. A method of amplifying a mutant allele comprising a single nucleotide variant (SNV) in a sample comprising a plurality of double-stranded nucleic acid molecules each having a first strand and a second strand, the plurality comprising at least one double-stranded nucleic acid molecule having the mutant allele comprising the SNV, the method comprising:(a) forming a mixture comprising the at least one double-stranded nucleic acid molecule having the mutant allele and a set of allele-specific primers comprising a forward primer and a reverse primer, wherein the forward primer has partial complementarity to a forward primerbinding sequence of the first strand and comprises (i) a 3’ terminal nucleotide that is complementary to a single nucleotide variant of the mutant allele of the first strand and (ii) a first mismatch to the forward primer-binding sequence of the first strand, and wherein the reverse primer has partial complementarity to a reverse primerbinding sequence of the second strand and comprises (i) a 3’ terminal nucleotide that is complementary to an SNV of the mutant allele of the second strand and (ii) a second mismatch to the reverse primer-binding sequence of the second strand; and(b) subjecting the mixture to a condition sufficient to amplify the at least one doublestranded nucleic acid molecule.

2. The method of claim 1, wherein the mixture lacks a universal primer.

3. The method of claim 1 or 2, wherein the forward primer comprises a single mismatch.

4. The method of any one of claims 1- 3, wherein the forward primer consists of a single mismatch.

5. The method of any one of claims 1-4, wherein the reverse primer comprises a single mismatch.

6. The method of any one of claims 1-5, wherein the reverse primer consists of a single mismatch.

7. The method of any one of claims 1-6, wherein the first mismatch of the forward primer is located at an internal nucleotide.

8. The method of any one of claims 1-7, wherein the second mismatch of the reverse primer is located at an internal nucleotide.WSGR Docket No. 69583-701.6019. The method of any one of claims 1-8, further comprising detecting an amplification product of the at least one double-stranded nucleic acid molecule.

10. The method of claim 9, wherein detecting comprises contacting the amplification product with a detection agent that non-specifically recognizes polynucleic acids.

11. The method of claim 9, wherein detecting comprises contacting the amplification product with a nucleic acid intercalating agent.

12. The method of any one of claims 9-11, wherein detecting does not comprise using a mutant allele specific probe.

13. The method of any one of claims 9-12, wherein detecting does not comprise sequencing the amplification product.

14. The method of any one of claims 1-13, wherein the method does not comprise sequencing.

15. The method of any one of claims 1-14, wherein the forward primer comprises at most 3 mismatches to the forward primer-binding sequence and / or the reverse primer comprises at most 3 mismatches to the reverse primer-binding sequence.

16. The method of any one of claims 1-15, wherein the partial complementarity is at least 90% complementary.

17. The method of any one of claims 1-16, wherein the forward primer has a first sequence that includes the 3’ terminal nucleotide and has a length of at least 10 nucleotides, wherein the first sequence has at most 3 mismatches.

18. The method of claim 17, wherein the first sequence has a length of at least 20 nucleotides.

19. The method of claim 17 or 18, wherein the first sequence has at most 2 mismatches.

20. The method of any one of claims 17-19, wherein the first sequence has only one mismatch.

21. The method of any one of claims 1-20, wherein the reverse primer has a first sequence that includes the 3’ terminal nucleotide and has a length of at least 10 nucleotides, wherein the first sequence has at most 3 mismatches.

22. The method of claim 21, wherein the first sequence has a length of at least 20 nucleotides.

23. The method of claim 21 or 22, wherein the first sequence has at most 2 mismatches.

24. The method of any one of claims 21-23, wherein the first sequence has only one mismatch.WSGR Docket No. 69583-701.60125. The method of any one of claims 1-24, wherein a melting temperature (Tm) of the forward primer and / or the reverse primer is from 50 degrees Celsius to 70 degrees Celsius.

26. The method of claim 25, wherein the Tm is from 55 degrees Celsius to 60 degrees Celsius.

27. The method of claim 25 or 26, wherein the Tm is 57 degrees Celsius.

28. The method of any one of claims 1-27, wherein the first mismatch to the forward primerbinding sequence of the first strand is at least 6 nucleotides 5’ to the 3’ terminal nucleotide.

29. The method of any one of claims 1-27, wherein the first mismatch to the forward primerbinding sequence of the first strand is at most 6 nucleotides 5’ to the 3’ terminal nucleotide.

30. The method of any one of claims 1-29, wherein the set of allele-specific primers has a rate of amplification of a corresponding wild-type allele of at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, or less.

31. The method of any one of claims 1-30, wherein the set of allele-specific primers has an allele-specific amplification rate of at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or more.

32. The method of any one of claims 1-31, wherein the mutant allele is amplified at least 10 times, 100 times, 1000 times, or more than a corresponding wild-type allele is amplified.

33. The method of claim 32, wherein the plurality of double-stranded nucleic acid molecules further comprises a double-stranded nucleic acid molecule comprising the corresponding wild-type allele.

34. The method of claim 32 or 33, wherein the set of allele-specific primers does not amplify the double-stranded nucleic acid molecule comprising the corresponding wild-type allele or amplifies the double-stranded nucleic acid molecule comprising the corresponding wild-type allele less efficiently than the at least one double-stranded nucleic acid molecule having the mutant allele.

35. The method of any one of claims 1-34, wherein the plurality of double-stranded nucleic acid molecules comprises a first double-stranded nucleic acid molecule comprising a first mutant allele, and a second double-stranded nucleic acid molecule comprising a second mutant allele, wherein the first and second mutant alleles are different mutant alleles.

36. The method of any one of claims 1-35, wherein the plurality of double-stranded nucleic acid molecules comprises a first double-stranded nucleic acid molecule comprising a firstWSGR Docket No. 69583-701.601SNV, and a second double-stranded nucleic acid molecule comprising a second SNV, wherein the first and second SNVs are different SNVs.

37. The method of any one of claims 1-36, further comprising amplifying two or more double-stranded nucleic acid molecules.

38. The method of claim 37, further comprising amplifying the two or more double-stranded nucleic acid molecules simultaneously.

39. The method of claim 37 or 38, further comprising amplifying the two or more doublestranded nucleic acid molecules simultaneously in a same compartment.

40. The method of any one of claims 1-39, further comprising, prior to amplifying, partitioning the plurality of double-stranded nucleic acid molecules into two or more different compartments.

41. The method of claim 40, wherein the two or more different compartments are two or more different water-in-oil droplets.

42. The method of any one of claims 37-39, further comprising amplifying the two or more double-stranded nucleic acid molecules simultaneously in two or more different compartments, wherein each compartment comprises at least one of the two or more double-stranded nucleic acid molecules and the set of allele-specific primers.

43. The method of any one of claims 1-42, wherein the set of allele-specific primers comprises two or more sets of allele-specific primers, and wherein each set of allelespecific primers is specific to a single mutant allele.

44. The method of any one of claims 1-42, wherein the set of allele-specific primers comprises two or more sets of allele-specific primers, and wherein each set of allelespecific primers comprises a forward primer that has partial complementarity to a forward primerbinding sequence of a first strand of a double-stranded nucleic acid molecule comprising a different / given mutant allele, and comprises (i) a 3’ terminal nucleotide that is complementary to a single nucleotide variant of the different / given mutant allele of the first strand and (ii) a first mismatch to the forward primer-binding sequence of the first strand, and a reverse primer that has partial complementarity to a reverse primer-binding sequence of the second strand a double-stranded nucleic acid molecule comprising the different / given mutant allele and comprises (i) a 3’ terminal nucleotide that is complementary to an SNV of the different / given mutant allele of the second strand and (ii) a second mismatch to the reverse primer-binding sequence of the second strand.WSGR Docket No. 69583-701.60145. The method of claim 43 or 44, wherein the two or more sets of allele-specific primers comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or more sets of allele-specific primers.

46. The method of claim 43 or 44, wherein the two or more sets of allele-specific primers comprise at most 25, at most 24, at most 23, at most 22, at most 21, at most 20, at most 19, at most 18, at most 17, at most 16, at most 15, at most 14, at most 13, at most 12, at most 11, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3 or less sets of allele-specific primers.

47. The method of any one of claims 37-46, comprising detecting amplification products of the two or more double-stranded nucleic acid molecules.

48. The method of claim 47, wherein the detecting does not differentiate one mutant allele from another different mutant allele.

49. The method of claim 47 or 48, wherein the detecting detects each mutant allele indiscriminately.

50. The method of any one of claims 36-49, further comprising determining a mean allele frequency of the different mutant alleles or the different SNVs in the sample.

51. The method of any one of claims 1-50, wherein the method does not comprise determining an individual allele frequency of a given mutant allele or a given SNV.

52. The method of any one of claims 1-51, further comprising, prior to (a), obtaining the sample from a subject.

53. The method of claim 52, wherein the subject has cancer.

54. The method of claim 52 or 53, wherein the subject has been treated with a cancer therapy.

55. The method of any one of claims 52-54, wherein the subject is suspected of having a minimal residual disease (MRD).

56. The method of claim 52, wherein the subject has a disease or condition.

57. The method of claim 56, wherein the disease or condition is cancer.

58. The method of any one of claims 52-57, wherein the mutant allele is specific to the subject.

59. The method of any one of claims 56-58, wherein the mutant allele is associated with the disease or condition.

60. The method of any one of claims 52-59, further comprising, prior to (a), identifying one or more mutant alleles in a different sample from the subject.WSGR Docket No. 69583-701.60161. The method of claim 60, wherein, prior to (a), the identity of one or more mutant alleles of the subject is known.

62. The method of claim 60 or 61, wherein the different sample is a tissue sample.

63. The method of any one of claims 60-62, wherein the identifying comprises sequencing nucleic acids in the different sample from the subject to identify subject-specific mutant alleles.

64. The method of any one of claims 1-63, wherein the mutant allele comprises a SNV of a gene selected from the group consisting of AKT1, APC, BRAF, CTNNB1, EGFR, FGFR3, FLT3, FOXL2, GNA11, GNAQ, GNAS, IDH1, JAK2, KIT, KRAS, MPL, NRAS / CSDE1, PDGFRA, PIK3CA, RET, TP53, ALK, and KRAS.

65. The method of any one of claims 1-64, wherein the plurality of double-stranded nucleic acid molecules is a plurality of DNA molecules.

66. The method of any one of claims 1-65, wherein the plurality of double-stranded nucleic acid molecules is prepared by reverse transcribing a plurality of RNA molecules.

67. The method of any one of claims 1-66, wherein the sample is a fluid sample.

68. The method of any one of claims 1-67, wherein the sample is selected from the group consisting of a blood sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, a pleural or peritoneal fluid sample, a stool sample, a seminal fluid, and any combination thereof.

69. The method of any one of claims 1-68, wherein the plurality of double-stranded nucleic acid molecules comprises circulating tumor DNAs or cell-free DNAs.

70. The method of any one of claims 1-69, further comprising, prior to (a), isolating the plurality of double-stranded nucleic acid molecules from the sample.

71. A composition comprising a set of allele-specific primers, wherein the set of allelespecific primers comprises a forward primer and a reverse primer, wherein the forward primer has partial complementarity to a forward primer-binding sequence of a first strand of a double-stranded nucleic acid molecule comprising a mutant allele comprising an SNV and comprises (i) a 3’ terminal nucleotide that is complementary to a single nucleotide variant of the mutant allele of the first strand and (ii) a first mismatch to the forward primerbinding sequence of the first strand, and wherein the reverse primer has partial complementarity to a reverse primer-binding sequence of the second strand and comprises (i) a 3’ terminal nucleotide that is complementary to a single nucleotide variant of the mutantWSGR Docket No. 69583-701.601 allele of the second strand and (ii) a second mismatch to the reverse primerbinding sequence of the second strand.

72. The composition of claim 71, wherein the set of allele-specific primers is configured to amplify the mutant allele of the double-stranded nucleic acid molecule.

73. The composition of claim 71 or 72, wherein the forward primer comprises a single mismatch.

74. The composition of claim 71 or 72, wherein the reverse primer comprises a single mismatch.

75. The composition of any one of claims 71-74, wherein the first mismatch of the forward primer is located at an internal nucleotide.

76. The composition of any one of claims 71-75, wherein the second mismatch of the reverse primer is located at an internal nucleotide.

77. The composition of any one of claims 71-76, wherein the forward primer comprises at most 3 mismatches to the forward primer-binding sequence and / or the reverse primer comprises at most 3 mismatches to the reverse primer-binding sequence.

78. The composition of any one of claims 71-77, wherein the partial complementarity is at least 90% complementary.

79. The composition of any one of claims 71-78, wherein the first mismatch to the forward primer-binding sequence of the first strand is at least 6 nucleotides 5’ to the 3’ terminal nucleotide.

80. The composition of any one of claims 71-78, wherein the first mismatch to the forward primer-binding sequence of the first strand is at most 6 nucleotides 5’ to the 3’ terminal nucleotide.

81. The composition of any one of claims 71-80, wherein the set of allele-specific primers has a rate of amplification of a corresponding wild-type allele (false positive rate) of at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, or less.

82. The composition of any one of claims 71-80, wherein the set of allele-specific primers has an allele-specific amplification rate of at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or more.

83. The composition of claim 81 or 82, wherein the set of allele-specific primers does not amplify the double-stranded nucleic acid molecule comprising the corresponding wildtype allele or amplifies the double-stranded nucleic acid molecule comprising the corresponding wild-type allele less efficiently than the at least one double-stranded nucleic acid molecule having the mutant allele.WSGR Docket No. 69583-701.60184. A kit comprising a set of allele-specific primers of the composition of any one of claims 71-83, and an instruction for using the kit.

85. The kit of claim 84, wherein the kit further comprises a dNTP, an enzyme, a nucleic acid intercalating agent, and / or a buffer.

86. Use of the composition of any one of claims 71-83 for detecting a disease status in a subject.

87. A method of determining a disease status of a subject, the method comprising:(a) obtaining a first sample comprising a first plurality of nucleic acid molecules from the subject at a first time point or providing the first sample that has been obtained from the subject at the first time point, wherein the first plurality of nucleic acid molecules of the first sample comprises or is suspected of comprising at least one mutant allele;(b) conducting a first allele-specific amplification of the first plurality of nucleic acid molecules using a set of allele-specific primers;(c) detecting amplification products of the first allele-specific amplification using a nonspecific polynucleic acid detection agent; and(d) determining the disease status of the subject based on detecting in (c); wherein(i) the set of allele-specific primers has a rate of amplification of a corresponding wild-type allele of at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, or less,(ii) the set of allele-specific primers has an allele-specific amplification rate of at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or more, or(iii) the mutant allele is amplified at least 10 times, 100 times, 1000 times or more than a corresponding wild-type allele is amplified.

88. The method of claim 87, further comprising(e) obtaining a second sample comprising a second plurality of nucleic acid molecules from the subject at a second time point or providing the second sample that has been obtained from the subject at the second time point, wherein the second plurality of nucleic acid molecules of the second sample comprises or is suspected of comprising, or does not comprise a detectable level of the at least one mutant allele;(f) conducting a second allele-specific amplification of the second plurality of nucleic acid molecules;(g) detecting amplification products of the first allele-specific amplification and amplification products of the second allele-specific amplification using a nonspecific polynucleic acid detection agent; andWSGR Docket No. 69583-701.601(h) determining the disease status of the subject based on detecting in (e).

89. The method of claim 87 or 88, wherein the set of allele-specific primers comprise an allele-specific forward primer and an allele-specific reverse primer.

90. The method of claim 89, wherein the allele-specific forward primer and the allelespecific reverse primer each comprises a 3 ’terminal nucleotide that is complementary to a single nucleotide variant of the at least one mutant allele.

91. The method of any one of claims 87-90, wherein the at least one mutant allele comprises a SNV.

92. A method of determining a disease status of a subject, the method comprising:(a) obtaining a first sample comprising a first plurality of nucleic acid molecules from a subject at a first time point or providing the first sample that has been obtained from the subject at the first time point, wherein the first plurality of nucleic acid molecules of the first sample comprises or is suspected of comprising at least one mutant allele;(b) conducting a first allele-specific amplification of the first plurality of nucleic acid molecules;(c) obtaining a second sample comprising a second plurality of nucleic acid molecules from the subject at a second time point or providing the second sample that has been obtained from the subject at the second time point, wherein the second plurality of nucleic acid molecules of the second sample comprises or is suspected of comprising, or does not comprise a detectable level of the at least one mutant allele;(d) conducting a second allele-specific amplification of the second plurality of nucleic acid molecules;(e) detecting amplification products of the first allele-specific amplification and amplification products of the second allele-specific amplification using a nonspecific polynucleic acid detection agent; and(f) determining the disease status of the subject based on detecting in (e).

93. The method of claim 92, wherein the detecting in (e) comprises determining a first allele frequency of the at least one mutant allele in the first sample, determining a second allele frequency of the at least one mutant allele in the second sample, and comparing the first allele frequency and the second allele frequency.

94. The method of claim 93, wherein the at least one mutant allele comprises two or more mutant alleles, and wherein the first allele frequency and / or the second allele frequency comprises a first mean allele frequency and / or a second mean allele frequency.

95. The method of any one of claims 87-94, wherein the method does not comprise determining an individual allele frequency for a given mutant allele.WSGR Docket No. 69583-701.60196. The method of any one of claims 87-95, wherein the method does not comprise using a probe for detecting the amplification products.

97. The method of any one of claims 87-95, wherein the method does not require using a probe for detecting the amplification products.

98. The method of any one of claims 87-97, wherein the method does not comprise using sequencing for detecting the amplification products.

99. The method of any one of claims 87-98, wherein determining the disease status comprises determining a tumor fraction (TF).

100. The method of any one of claims 87- 99, wherein the non-specific polynucleic acid detection agent detects the amplification products irrespective of sequences of the amplification products.

101. The method of any one of claims 87-100, wherein the non-specific polynucleic acid detection agent detects any double-stranded nucleic acid molecules.

102. The method of any one of claims 87-101, wherein the non-specific polynucleic acid detection agent comprises a nucleic acid intercalating agent.

103. The method of any one of claims 87-102, wherein the first allele-specific amplification and / or the second allele-specific amplification comprises using the composition of any one of claims 71-83.

104. Use of a set of allele-specific primers in a method for determining a disease status of a subject, wherein the method comprises (a) obtaining a first sample comprising a first plurality of nucleic acid molecules from the subject at a first time point or providing the first sample that has been obtained from the subject at the first time point, wherein the first plurality of nucleic acid molecules of the first sample comprises or is suspected of comprising at least one mutant allele; (b) conducting a first allele-specific amplification of the first plurality of nucleic acid molecules using the set of allele-specific primers; (c) detecting amplification products of the first allele-specific amplification using a nonspecific polynucleic acid detection agent; and (d) determining the disease status of the subject based on detecting in (c); wherein(i) the set of allele-specific primers has a rate of amplification of a corresponding wildtype allele of at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, or less,(ii) the set of allele-specific primers has an allele-specific amplification rate of at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or more, or(iii) the mutant allele is amplified at least 10 times, 100 times, 1000 times or more than a corresponding wild-type allele is amplified.WSGR Docket No. 69583-701.601105. Use of a non-specific polynucleic acid detection agent in a method of determining a disease status of a subject, wherein the method comprises: (a) obtaining a first sample comprising a first plurality of nucleic acid molecules from a subject at a first time point or providing the first sample that has been obtained from the subject at the first time point, wherein the first plurality of nucleic acid molecules of the first sample comprises or is suspected of comprising at least one mutant allele; (b) conducting a first allelespecific amplification of the first plurality of nucleic acid molecules; (c) obtaining a second sample comprising a second plurality of nucleic acid molecules from the subject at a second time point or providing the second sample that has been obtained from the subject at the second time point, wherein the second plurality of nucleic acid molecules of the second sample comprises or is suspected of comprising, or does not comprise a detectable level of the at least one mutant allele; (d) conducting a second allele-specific amplification of the second plurality of nucleic acid molecules; (e) detecting amplification products of the first allele-specific amplification and amplification products of the second allele-specific amplification using the non-specific polynucleic acid detection agent; and (f) determining the disease status of the subject based on detecting in (e).

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