Multiplexed method for identification and quantification of secondary alleles and polymorphisms

Abstract

This application relates to multiplexed methods for the identification and quantification of minor alleles and polymorphisms. Products and methods are provided for detecting the presence of minor nucleic acid substances in a sample containing a mixture of minor nucleic acid substances and one or more major nucleic acid substances, wherein the amount (frequency or copy number) of the minor nucleic acid substances is less than the amount of the major nucleic acid substances. Some methods involve amplifying the mixture and extending the resulting amplicons with a chain terminator and an extension primer that hybridizes specifically to the amplicons, wherein the chain terminator specific to the major nucleic acid substances has a lower concentration than the chain terminator specific to the minor nucleic acid substances. A higher concentration of the chain terminator specific to the minor nucleic acid substances, relative to the chain terminator specific to the major nucleic acid substances, improves the detection limit (sensitivity) for detecting minor nucleic acid substances present in the mixture at low frequency or copy number. Additionally, the signal generated from the extension product of the major nucleic acid substance amplicons can be used as a positive control and allows for the quantification of the minor nucleic acid substances relative to the major nucleic acid substances in the mixture.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680013462.4.

[0002] Related applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 280951, filed January 20, 2016, entitled "Multiplexing Method for Identification and Quantification of Minor Alleles and Polymorphisms," inventor Anders Nygren, attorney file number AGB-7001-PV2. This application also claims priority to U.S. Provisional Application No. 62 / 152697, filed April 24, 2015, entitled "Multiplexing Method for Identification and Quantification of Minor Alleles and Polymorphisms," inventor Anders Nygren, attorney file number AGB-7001-PV. This application relates to U.S. Patent Application No. 13 / 718,758, filed December 18, 2012, by inventors Martin Beaulieu and Dirk Johannes van den Boom, entitled "Method for High-Level Multiplexed Polymerase Chain Reaction and Uniform Mass Extension Reaction," Agent's File No. AGB-2079-CT2, which is a successor to U.S. Patent Application No. 13 / 193,390, filed July 28, 2011, by inventors Martin Beaulieu and Dirk Johannes van den Boom, entitled "Method for High-Level Multiplexed Polymerase Chain Reaction and Uniform Mass Extension Reaction," Agent's File No. AGB-2079-CT, and now a successor to U.S. Patent No. 8,349,566, filed July 30, 2004, by inventors Martin Beaulieu and Dirk Johannes van den Boom. Boom, entitled "Method for High-Level Multiplex Polymerase Chain Reaction and Uniform Mass Extension Reaction", U.S. Patent Application No. 10 / 903,268, Agent File No. SEQ-2079-UT, now a successor to U.S. Patent No. 8,003,317, which claims priority under 35 U.S. SC §119(e) to U.S. Provisional Application No. 60 / 492,102, filed July 31, 2003, by Martin Beaulieu and Dirk van den Boom, entitled "Method for High-Level Multiplex Polymerase Chain Reaction and Uniform Mass Extension Reaction", Agent File No. 17082-087P01(P2079). This application also relates to International PCT Application No. PCT / US2004 / 024953, filed December 18, 2012, by Martin Beaulieu and Dirk van den Boom, entitled "Method for High-Level Multiplex Polymerase Chain Reaction and Uniform Mass Extension Reaction". The contents of these applications are incorporated herein by reference, including text, tables, and figures. Technical Field

[0004] This technology involves identifying and / or quantifying nucleic acid variants, such as mutant variants or polymorphisms of wild-type alleles. Background Technology

[0005] Detection of specific nucleic acids is a crucial tool in diagnostic medicine and molecular biology research. Nucleic acid assays can, for example, identify infectious organisms such as bacteria and viruses in host organisms, detect the expression of normal genes and identify mutated genes such as oncogenes, perform tissue typing for compatibility before tissue transplantation, match tissue or blood samples for forensic medicine, analyze homology between genes of different substances, and identify alleles and polymorphisms of gene variants. These applications typically require the ability to detect and / or identify small amounts of minor nucleic acids of interest (e.g., minor allelic variants of wild-type alleles, or oncogenes) in nucleic acid samples or mixtures containing large amounts of non-target (major) nucleic acids. This capability can be further enhanced (e.g., in terms of efficiency) if nucleic acid assays can be performed in a multiplexed manner, i.e., screening for multiple nucleic acids.

[0006] Existing methods, including multiplexing methods, that reliably and reproducibly detect low-frequency (copy number) variants, polymorphisms, or other mutations among alleles typically have lower detection sensitivity or limits of detection than may be required to identify low-frequency variants (e.g., a limit of detection for approximately 10%–15% frequencies would fail to identify mutation frequencies below that range). The lower limit of detection is generally due to the low-frequency variant detection signal being masked by the larger detection signal of the dominant wild-type material. Other methods overcome this problem by removing the “wild-type” signal, thereby improving the detection of low-frequency variants. However, removing the wild-type signal can lead to difficulties in quantifying the relative amount of variants in a nucleic acid sample or in definitively confirming the absence of low-frequency variants. Methods, including multiplexing methods, that combine high detection sensitivity and high accuracy can provide improved identification and / or quantification of certain low-frequency variants that are undetectable or not optimally detected by previous methods. Summary of the Invention

[0007] This paper presents methods, including multiplexing methods, that combine high detection sensitivity and high accuracy to provide improved identification and / or quantification of certain low-frequency variants that are undetectable or cannot be optimally detected or quantified.

[0008] In some embodiments, this document provides a multiplexing method for identifying the presence or absence of one or more minor nucleic acid substances in a nucleic acid cluster, said nucleic acid cluster comprising a mixture of said one or more minor nucleic acid substances and one or more major nucleic acid substances, wherein each minor nucleic acid substance is a variant of a corresponding major nucleic acid substance and has a copy number less than that of its corresponding major nucleic acid substance, wherein the method comprises:

[0009] (a) Amplify the target region of the mixture simultaneously with amplification primers under amplification conditions including dNTPs, thereby producing an amplified mixture of nucleic acids containing major and minor nucleic acid substances.

[0010] (b) The amplification mixture is contacted with the extension primers under extension conditions including a chain terminator, wherein:

[0011] (i) One or more major nucleic acid substances share a common chain terminator that is specific to the major nucleic acid substance but non-specific to the minor nucleic acid substance, and

[0012] (ii) Each of one or more minor nucleic acid substances has a chain terminator that is specific to the minor nucleic acid substance and non-specific to the major nucleic acid substance, wherein the chain terminator that is specific to the minor nucleic acid substance is: (A) unique to the specific minor nucleic acid substance in the amplification mixture and not shared by other minor nucleic acid substances in the amplification mixture, or (B) at least one of one or more minor nucleic acid substances shares a common chain terminator with at least one other minor nucleic acid substance in the amplification mixture.

[0013] The primer is extended to or through different nucleotide positions in the minor nucleic acid material relative to the major nucleic acid material, thereby producing extension products corresponding to chain terminations of the minor and major nucleic acid materials, respectively, wherein the concentration of the chain terminator specific to the major nucleic acid material is less than the concentration of each chain terminator specific to one or more minor nucleic acid materials; and

[0014] (c) Analyze the extended products of (b) to identify the presence or absence of one or more minor nucleic acid substances.

[0015] In some embodiments, the nucleic acid group comprises multiple minor nucleic acid substances, which are variants of a single major nucleic acid substance, and the multiple minor nucleic acid substances are identified in a single multiplexing reaction. In some embodiments, part (b) of the method is carried out in a set of at least two reaction vessels or compartments, wherein:

[0016] The first container or compartment includes extended conditions containing a chain terminator specific to the primary nucleic acid substance and free from chain terminators specific to one or more secondary nucleic acid substances; and

[0017] Each remaining container or compartment includes extended conditions containing a single chain terminator specific to and common to one or more minor nucleic acid substances, and not containing chain terminators specific to the major nucleic acid substance or specific to minor nucleic acid substances but not common to a single chain terminator. In some embodiments, the concentration of each chain terminator is known.

[0018] This article also provides a method for quantifying one or more minor nucleic acid substances in a nucleic acid group, said nucleic acid group comprising a mixture of said one or more minor nucleic acid substances and a major nucleic acid substance, wherein said minor nucleic acid substances are variants of the same major nucleic acid substance and each exists at a copy number lower than that of the major nucleic acid substance, wherein the method comprises:

[0019] (a) Amplify the target region of the mixture simultaneously with amplification primers under amplification conditions including dNTPs, thereby producing an amplified mixture of nucleic acids containing major and minor nucleic acid substances.

[0020] (b) The amplification mixture is contacted with extension primers under extension conditions, said extension conditions including for (i) each of one or more minor nucleic acid substances, and (ii) a major nucleic acid substance-specific chain terminator, whereby the primers extend to or through different nucleotide positions in the minor nucleic acid substances relative to the major nucleic acid substances, thereby producing extension products corresponding to chain terminations of the minor nucleic acid substances and the major nucleic acid substances, respectively, wherein: (1) the concentration of each chain terminator is known; and (2) the concentration of the chain terminator specific to the major nucleic acid substance is less than the concentration of the chain terminator specific to one or more minor nucleic acid substances;

[0021] (c) Determine the ratio of the amount of extension product corresponding to each of one or more minor nucleic acid substances to the amount of extension product corresponding to the major nucleic acid substance; and

[0022] (d) Based on the proportion of (c), and based on the concentration of one or more chain terminators specific to the primary nucleic acid substance relative to the concentration of the primary nucleic acid substance, the amount of secondary nucleic acid substance is quantified relative to the amount of the primary nucleic acid substance.

[0023] This article also provides a method for multiplexing one or more minor nucleic acid substances in a quantitative nucleic acid population, the nucleic acid population comprising a mixture of the one or more minor nucleic acid substances and a major nucleic acid substance, wherein each minor nucleic acid substance is a variant of the corresponding major nucleic acid substance and exists at a lower copy number than its corresponding major nucleic acid substance, wherein the method includes:

[0024] (a) Amplify the target region of the mixture simultaneously with amplification primers under amplification conditions including dNTPs, thereby producing an amplified mixture of nucleic acids containing major and minor nucleic acid substances.

[0025] (b) The amplification mixture is contacted with the extension primers under extension conditions including a chain terminator, wherein:

[0026] (i) One or more major nucleic acid substances share a common chain terminator that is specific to the major nucleic acid substance but non-specific to the minor nucleic acid substance, and

[0027] (ii) Each of one or more minor nucleic acid substances has a chain terminator that is specific to the minor nucleic acid substance and non-specific to the major nucleic acid substance, wherein the chain terminator that is specific to the minor nucleic acid substance is: (A) unique to the specific minor nucleic acid substance in the amplification mixture and not shared by other minor nucleic acid substances in the amplification mixture, or (B) at least one of one or more minor nucleic acid substances shares a common chain terminator with at least one other minor nucleic acid substance in the amplification mixture.

[0028] The primer is extended to or through different nucleotide positions in the minor nucleic acid material relative to the major nucleic acid material, thereby producing extension products corresponding to chain terminations of the minor and major nucleic acid materials, respectively, wherein: (1) the concentration of each chain terminator is known; and (2) the concentration of the chain terminator specific to the major nucleic acid material is less than the concentration of the chain terminator specific to one or more minor nucleic acid materials.

[0029] (c) Determine the ratio of the amount of extension product corresponding to each of one or more minor nucleic acid substances to the amount of extension product corresponding to the major nucleic acid substance; and

[0030] (d) Based on the proportion of (c), and based on the concentration of one or more chain terminators specific to the primary nucleic acid substance relative to the concentration of the primary nucleic acid substance, the amount of secondary nucleic acid substance is quantified relative to the amount of the primary nucleic acid substance.

[0031] In some embodiments of this method, the nucleic acid group comprises multiple minor nucleic acid substances, which are variants of a single major nucleic acid substance, and these multiple minor nucleic acid substances are identified in a single multiplexing reaction. In some embodiments, part (b) of the method is carried out in a set of at least two reaction vessels or compartments, wherein:

[0032] The first container or compartment includes extended conditions containing a chain terminator specific to the primary nucleic acid substance and free from chain terminators specific to one or more secondary nucleic acid substances; and

[0033] Each remaining container or compartment includes an extension condition containing a single chain terminator specific to and common to one or more minor nucleic acid substances, and not containing a chain terminator specific to the major nucleic acid substance or specific to the minor nucleic acid substance but not common to the single chain terminator.

[0034] In some embodiments of the methods provided herein, the sequences of the minor and major nucleic acid substances differ by a single base, and primers extend to or pass through this different single base. In some embodiments, the sequence of the minor nucleic acid substance includes insertions or deletions relative to the sequence of the major nucleic acid substance. In some embodiments, one or more minor nucleic acid substances are single nucleotide polymorphism (SNP) variants of the major nucleic acid substance. In some embodiments, the minor and major nucleic acid substances are mutants and wild-type alleles of the same gene, respectively.

[0035] In some embodiments of the methods provided herein, the primary nucleic acid material is derived from a host object, and the secondary nucleic acid material is derived from an object other than the host. In some embodiments, one or more secondary nucleic acid materials are present at approximately 10% of the copy number of the primary nucleic acid material. In some embodiments, one or more secondary nucleic acid materials are present at approximately 1% to less than 10% of the copy number of the primary nucleic acid material.

[0036] In some embodiments, one or more minor nucleic acid substances are present at approximately 2% to less than 10% of the copy number of the major nucleic acid substance.

[0037] In some embodiments of the methods provided herein, the concentration of the chain terminator specific to the major nucleic acid substance is from about 1% to about 20% of the concentration of the chain terminator specific to each minor nucleic acid substance. In some embodiments of the methods provided herein, the concentration of the chain terminator specific to the major nucleic acid substance is from about 0.1% to about 10% of the concentration of the chain terminator specific to each minor nucleic acid substance. In some embodiments of the methods provided herein, the concentration of the chain terminator specific to the major nucleic acid substance is from about 0.01% to about 5% of the concentration of the chain terminator specific to each minor nucleic acid substance. In some embodiments, the chain terminator is a chain-terminating nucleotide. In embodiments, the chain-terminating nucleotide is independently selected from ddATP, ddGTP, ddCTP, ddTTP, and ddUTP.

[0038] In some embodiments of the methods provided herein, the chain-terminating nucleotide specific to one or more minor nucleic acid substances consists of one chain-terminating nucleotide. In some embodiments, the chain-terminating nucleotide specific to one or more minor nucleic acid substances consists of two chain-terminating nucleotides. In some embodiments, the chain-terminating nucleotide specific to one or more minor nucleic acid substances consists of three chain-terminating nucleotides.

[0039] In some embodiments of the methods provided herein, the chain terminator comprises one or more acyclic terminators. In embodiments of the methods provided herein, the amplification in part (a) comprises about 30 to about 45 PCR amplification cycles using amplification primers. In some embodiments, the extension conditions in (b) comprise about 20 to about 300 cycles of extending the extension primers. In some embodiments, the extension conditions in (b) comprise at least 50 extension cycles.

[0040] In embodiments of the methods provided herein, one or more chain terminators and / or extension primers include detectable markers. In some embodiments, the markers are fluorescent markers or dyes. In some embodiments, the markers are mass markers. In embodiments, the markers can be detected, thereby identifying or quantifying one or more minor nucleic acid substances. In some embodiments, the markers are mass markers and are detected by mass spectrometry.

[0041] The methods provided herein, in some embodiments, include amplification of minor and / or major nucleic acid material prior to extension (using a chain terminator). In some embodiments, the amplification reaction conditions in (a) include water, genomic DNA, buffer, dNTPs, primer pairs, MgCl2, and polymerase, wherein the concentration of MgCl2 to the concentration of each dNTP is selected from ≤10:1, ≤9:1, ≤8:1, ≤7:1, ≤6:1, or ≤5:1. In some embodiments, the polymerase is a Taq polymerase at a concentration of at least about 0.03 units / μl. In embodiments of the methods provided herein, the amplification reaction conditions in (a) comprise about 400-700 μM of each dNTP, about 100 nM of primer pairs, and about 1.6 to about 4.8 mM of MgCl2. In some embodiments, a sequence tag is attached to one or more primers in the amplification primer pair. In some embodiments, free MgCl2 is used. 2+ The concentration is between 1.0 and 2.0 mM.

[0042] In some embodiments, specific MgCl2 concentrations for amplification performed in the methods described herein have been identified, allowing for high levels (e.g., 7-fold to 50-fold or more) of multiplexed PCR and primer extension reactions, as well as successful analysis of the amplification products, e.g., by detecting fluorescence signals or by mass spectrometry. When selecting the concentrations of dNTPs and MgCl2 used in the PCR amplification reaction (followed by mass extension and subsequent mass spectrometry analysis), consideration is given to the free MgCl2 content of the PCR reaction mixture. 2+ The concentration should be kept within a specific range, high enough to allow for coarse PCR amplification, and low enough not to adversely affect subsequent mass extension reactions and mass spectrometry analysis.

[0043] Therefore, this paper provides a multiplexing method for genotyping multiple polymorphic sites or other variants of minor nucleic acid substances by simultaneously amplifying multiple nucleic acid target regions of minor substances and corresponding regions of major nucleic acid substances under amplification conditions, thereby amplifying at least 60% of seven or more nucleic acid target regions by seven or more primer pairs to produce an amplified mixture of nucleic acid-target regions containing polymorphic sites or other variants. The amplified mixture of nucleic acid target regions is contacted with seven or more genotyping primers (i.e., extension primers or UEPs) under primer extension conditions in the presence of at least one chain terminator specific to the major nucleic acid substance and a chain terminator specific to the minor nucleic acid substance, thereby extending the primers to or through the corresponding polymorphic sites. A genotyping primer is present for each polymorphic site within the nucleic acid-target molecule, and the extended genotyping primers are detected by quality or other markers such as fluorescence or electrochemical labeling, wherein at least 60% of the genotypes of the seven or more attempted nucleic acid target regions are determined. In some implementations, the number of amplification primer pairs may be selected from 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or 13 or more.

[0044] In some embodiments of each of the methods described above, the sequence tag is attached to the 5′ end of one or both amplification primers of each primer pair. In other embodiments, a method for multiplexed detection of multiple sequence variants is used, employing conditions that allow determination (i.e., judgment) of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, up to 100%, of the attempt minor nucleic acid material (such as the amplification reaction conditions and / or primer quality extension reaction conditions provided herein). The conditions provided herein are applicable to multiple multiplexed reactions using multiple amplification primer pairs and 7- or more amplification reactions from multiple target nucleic acids. Furthermore, as described herein, all optimized amplification and / or primer quality extension genotyping reactions are applicable to multiplexed assays ranging from 2-dopairs to 6-dopairs and beyond.

[0045] In a particular embodiment, the sequence tag is linked to multiple primary and secondary amplification primer pairs selected from 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 or more primary and secondary primer pairs. The sequence tag can be linked to one or both of the primary and secondary primers in each pair. Typically, the sequence tag is linked to both the primary and secondary primers in each pair. The sequence tags used herein can be 5 to 20, 5 to 30, 5 to 40, or 5 to 50 nucleotides in length; decameric length sequence tags are particularly useful in the methods provided herein. In multiplexed amplification reactions, the sequence tag need not be the same sequence for each primer pair, nor need it be the same sequence for the primary and secondary primers in a particular amplification pair. In certain embodiments, the sequence tag is identical for each primer in the multiplexed amplification reaction. For example, in some embodiments, the sequence tag is a decameric unit and is attached to the 5′ end of each primary and secondary primer. In specific embodiments of the methods provided herein, only a single primer pair is used to amplify each specific nucleic acid target (minor nucleic acid material) region.

[0046] In embodiments of the method provided herein, the amplification reaction conditions of the above method include water, a mixture of minor and major nucleic acid substances, buffer, dNTPs, primary and secondary primer pairs, MgCl2, and polymerase, wherein the ratio of MgCl2 concentration to the concentration of each dNTP is selected from ≤10:1, ≤9:1, ≤8:1, ≤7:1, ≤6:1, or ≤5:1. In one specific embodiment, the ratio of MgCl2 concentration to the concentration of each dNTP is ≤7:1. In other embodiments, the amplification reaction conditions comprise approximately 400-700 μM, approximately 500-600 μM, or approximately 500 μM of each dNTP, and approximately 50-250 nM of primer pairs. In these embodiments, the total MgCl2 concentration can be between approximately 2.6 mM and approximately 4.8 mM MgCl2, between approximately 3.0 and approximately 4.5 mM MgCl2, and between approximately 3.5 mM and approximately 4.0 mM MgCl2. In the implementation method, when the concentrations of dNTPs and MgCl2 are selected, the free Mg 2+ The concentration is between approximately 1-2 mM. As used in this paper, free Mg... 2+ Concentration = Total Mg 2+ Concentration (e.g., total [MgCl2]) - Total dNTP concentration of all four dNTPs (e.g., 200 μM each dNTP = 800 μM total [dNTP]). In some embodiments, free Mg 2+The concentrations are between 1.1-1.9 mM, between 1.2-1.8 mM, between 1.3-1.7 mM, and between 1.4-1.6 mM. In one specific embodiment, free Mg... 2+ The concentration is approximately 1.5 mM. For each of these methods, the variety of amplification thermal cycles can be approximately 30 to approximately 35, 40, or 45 cycles. In one specific embodiment, the amplification reaction conditions comprise approximately 500 μM of each dNTP, approximately 100 nM of primer pairs, and approximately 3.5 mM MgCl2. For each of these methods, the polymerase can be Taq polymerase at a concentration of 0.03 units / μl (e.g., purchased from...). of In specific embodiments of the method provided herein, the amplification reaction conditions do not include the addition of one or any combination of additives selected from the following: BSA (bovine serum albumin), glycerol, DMSO (dimethyl sulfoxide), urea, or

[0047] The extension primer (UEP) hybridizes adjacent to the sequence variation, and the mixture also contains a preselected combination of dNTPs and ddNTPs or other chain terminators, such as reagents including acyclic terminators. In the preselected combination of dNTPs and ddNTPs, when ddNTPs are present in the mixture, no identical dNTPs are present. For these methods, the multiplicity of the primer mass extension thermal cycling is selected from at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, or more cycles. In embodiments of this method, the primer mass extension reaction conditions include about 20 μM to about 300 μM ddNTPs or other chain terminators specific to each minor nucleic acid substance (C[Mut]), typically about 20 μM to about 200 μM ddNTPs, about 40 μM to about 200 μM ddNTPs, about 100 μM to about 200 μM ddNTPs, or about 50 μM ddNTPs. The concentration of ddNTPs, a chain terminator specific to the major nucleic acid substance C[WT], is less than 20% of the concentration of a chain terminator specific to the minor nucleic acid substance (C[Mut]). C[WT] is typically between about 0.5% and less than about 20% of C[Mut], about 0.5% to less than about 15% of C[Mut], about 1% to about 15% of C[Mut], about 1% to about 10% of C[Mut], about 2% to about 10% of C[Mut], or about 1% to about 2% of C[Mut], and about 1 μM of UEP primers. In some embodiments, C[WT] is about 0.1% to about 10% of C[Mut] or about 0.01% to about 10% of C[Mut]. In some embodiments of these primer extension reactions, the extension reaction conditions also include about 0.05 to about 0.5 units of DNA polymerase per μL. In other embodiments, the primer extension reaction conditions further include about 0.1 to about 0.3 units of DNA polymerase per microliter. In other embodiments, the primer extension reaction conditions further include about 0.14 to about 0.2 units of DNA polymerase per microliter. In one embodiment, the primer extension reaction conditions further include about 0.14 units of DNA polymerase per microliter.

[0048] In some embodiments, the primer extension reaction has a primary chain terminator present in an amount of about 0.1% to about 10% of the secondary chain terminator. In other embodiments, the percentage of the primary chain terminator is adjusted individually for each transformation (i.e., A to C, A to G, A to T, C to A, C to G, C to T, G to A, G to C, G to T, T to A, T to C, and T to G) to account for differences in transformation-specific incorporation of the chain terminator. For example, the criterion for optimizing the percentage of the primary chain terminator relative to the secondary chain terminator may be the relative peak height of the secondary peak to the primary peak at a specific secondary frequency (e.g., less than about 10%, less than about 5%, less than about 2.5%, or less than about 1%). In some embodiments, for example, depending on a specific transformation, the adjusted percentage of the primary chain terminator relative to the secondary chain terminator concentration may be about 0.1% to about 10%, about 0.1% to about 5%, about 1% to about 4%, or about 0.1%, 0.2%, 0.3%, 0.4%. 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%. 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9.0%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8% or 9.9%.

[0049] In some implementations, for example, depending on a particular conversion, the adjusted percentage of the primary chain terminator relative to the secondary chain terminator concentration may be from about 0.01% to about 5%, from about 0.01% to about 4%, or from about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, or 0.4%. 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9% or 5.0%.

[0050] In some implementations, for at least one of a plurality of amplification primer pairs, the concentration of one primer is lower than that of another primer. Alternatively, for each of a plurality of primer pairs, the concentration of one primer is lower than that of another primer. In such implementations, the lower concentration primer for a specific nucleic acid target region (a region in the minor nucleic acid material) may have the same orientation as the extension (UEP) primer for that nucleic acid target region. Furthermore, the amplification product of the amplification step may be a single-stranded nucleic acid molecule.

[0051] A method for multiplexing minor and major nucleic acid material prior to extension is also provided, the method comprising: a) designing seven or more pairs of primary and secondary primers, wherein each primer pair amplifies a specific nucleic acid target region on the minor nucleic acid material; b) forming a mixture containing multiple primer pairs and one or more minor and major nucleic acid materials to amplify multiple nucleic acid target regions; and c) subjecting the mixture of step b) to multiple thermal cycles under amplification reaction conditions that allow amplification of more than 60% of the seven or more nucleic acid target regions, wherein the amplification reaction conditions include dNTPs and MgCl2, and wherein free MgCl2 is present. 2+ The concentration is between 1.0 and 2.0 mM (e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9). In other embodiments, the number of primary and secondary primer pairs may be selected from 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or 13 or more.

[0052] In some embodiments of each of the methods described above, a sequence tag is attached to the 5′ end of one or both amplification primers of each primer pair. In some embodiments, the amplification reaction conditions provided herein allow amplification of a certain percentage of seven or more nucleic acid target regions, said percentage being selected from greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, up to 100%, of the seven or more nucleic acid target regions. The conditions provided herein are applicable to multiplexed reactions of seven or more amplification reactions using multiple amplification primer pairs and mixtures from multiple major and minor nucleic acid substances.

[0053] Certain embodiments are further described in the following description, claims and drawings. Brief description of the attached figures

[0054] The accompanying drawings illustrate some non-limiting embodiments of the technology and are not necessarily drawn to scale.

[0055] Figure 1 This demonstrates the detection of minor alleles present at a frequency of 5% relative to the major allele using the methods described herein.

[0056] Figure 2 The results show that over time, the ddNTP chain terminator with wild-type specific ddNTP (C[WT]) will... ddNTP Concentration and mutant-specific ddNTPs (C[Mut]) ddNTP Different concentrations of the compound were incorporated into the single-base extension products corresponding to wild-type or mutant alleles.

[0057] Figure 3 The method described herein was used to detect minor alleles that were present at frequencies of 5%, 2.5%, or 1.25% relative to the major allele.

[0058] Figure 4 The detection sensitivity (y-axis) for each conversion (individual plot) for each dilution series sample type (0%, 1%, 2.5%, and 5%) is shown.

[0059] Figure 5 The graph shows the observed chimpanzee frequencies (y-axis) for the 5% chimpanzee (chimp) dilution series. The graph represents unique transformations, and the data points represent the chimpanzee frequencies observed in the trials within these transformations.

[0060] Figure 6 A block diagram showing the experimental elongation (y-axis) under different elongation reaction conditions (x-axis). Detailed Implementation

[0061] This article provides a method for identifying the presence or absence of one or more minor nucleic acid species in a sample containing a mixture of one or more minor nucleic acid substances and one or more major nucleic acid substances. A method for quantifying minor nucleic acid substances (e.g., frequency or copy number) relative to the amount of major nucleic acid substance in the sample is also provided.

[0062] The methods provided herein can be used to analyze minor nucleic acid substances present at a frequency or copy number of about 0.25% to at most about 50% relative to the major nucleic acid substance, with specific ranges including but not limited to about 0.25% to about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9.5%, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, or 1%. In some embodiments, minor nucleic acid substances are present at a frequency or copy number of about 1% or about 2% relative to the major nucleic acid substance to about 10%, 9.5%, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, or 2.5%. In other embodiments, minor nucleic acid substances are present at a frequency of about 1% or about 2% to less than about 10% relative to the major nucleic acid substance. In this implementation, the minor nucleic acid substance is present at a frequency of approximately 5% relative to the frequency or copy number of the major nucleic acid substance.

[0063] The method described in this paper provides detection sensitivity (limit of detection) for minor nucleic acids relative to the major nucleic acid substance, while allowing reliable identification and / or quantification relative to the presence and / or amount of the major nucleic acid substance.

[0064] For example, in iPLEX TM or homogeneous In the (hME) method (see, for example, U.S. Patent Publication No. 2013 / 0237428 A1, U.S. Patent Nos. 8,349,566 and 8,003,317, the contents of which are incorporated herein by reference in their entirety), a mixture of minor nucleic acid material (e.g., mutant alleles) and major nucleic acid material (e.g., wild-type alleles) is amplified by polymerase chain reaction (PCR) using a set of amplification primers, polymerase, and deoxynucleotides (dNTPs) to produce amplicons of wild-type and mutant material. After treatment with shrimp alkaline phosphatase (SAP) to dephosphorylate unincorporated dNTPs, an amplicon mixture is amplified using an extension primer (unextended primers or UEP), polymerase, and a termination mixture comprising a chain terminator (e.g., dideoxynucleotides or ddNTPs). UEPs hybridize with amplicones and extend to the differential sites between mutant and wild-type material (i.e., extension stops at the mutation sites where the bases differ between mutant and wild-type material) to produce single-base extension products or SBEs, such as iPLEX. TM The result can be extended by processing the resulting extended product (e.g., by desalting before mass spectrometry) or by analyzing the difference in detection signal (e.g., mass) relative to the wild-type allele.

[0065] The iPLEX mentioned above TM and homogeneity The (hME) method uses an equimolar mixture of ddNTPs in the extension step to produce extension products corresponding to wild-type and mutant materials. Therefore, in iPLEX... TM and homogeneity In the (hME) method, all other factors are equal except that the major nucleic acid substance is present in a large excess relative to the minor nucleic acid substance. Most UEPs are associated with the major nucleic acid substance and are extended using chain terminators specific to the major nucleic acid substance. Fewer UEP molecules are available for hybridization and extension of the minor nucleic acid substance. This weakens the magnitude of the detection signal corresponding to the minor nucleic acid substance, which is masked by the major detection signal from the major nucleic acid substance and may be included by background noise.

[0066] In contrast, the method presented herein uses a limited concentration of a chain terminator specific to wild-type or major nucleic acid material for the extension step, relative to chain terminators specific to mutants or minor nucleic acid material. When a chain terminator specific to the major nucleic acid material is added at a concentration lower than that of a chain terminator specific to one or more minor nucleic acid materials, a smaller number of UEPs are extended using the major nucleic acid material as a template, leaving a larger number of UEPs available for extension using the minor nucleic acid material as a template. Therefore, in a series of extension cycles, more extension products corresponding to the minor nucleic acid material accumulate, thereby increasing the detection signal corresponding to the minor nucleic acid material and improving the detection sensitivity (limit). The method presented herein, in which the concentration of the chain terminator is tilted to favor the extension of amplicones generated by the minor nucleic acid material, can be used to analyze any minor nucleic acid material present at a frequency lower than that of the major nucleic acid material in the sample of interest. In an embodiment, the method is advantageous for detecting minor nucleic acid substances present at a copy number or frequency less than 10% of that of the major nucleic acid substance, for example, at about 0.25% to less than 10%, about 0.5% to less than 10%, about 1% to less than 10%, or about 2% to less than 10% of the copy number or frequency of the major nucleic acid substance.

[0067] Figure 1 This illustrates an implementation of the method provided herein. A sample containing a mixture of minor nucleic acid material (few alleles) and major nucleic acid material (most alleles) is processed according to iPLEX. TM The method, or the method described herein, involves amplification and extension reactions to provide extension products for detection and analysis, in which a minority alleles are present at a frequency of 5% relative to the majority alleles. The leftmost figure shows the use of iPLEX. TM The analytical results of the method, using equimolar concentrations of chain terminators, are shown in the first figure. As the first figure shows, the signal peak on the left, corresponding to the extension product from the majority allele, dominates, causing the minority peak on the right to be reduced to background noise and undetectable. The middle figure shows the results obtained when the concentration of the chain terminator specific to the majority allele is 20% (1 / 5) of that of the chain terminator specific to the minority allele. As the middle figure shows, the detection signal intensity from the minority allele extension product (right peak) is now higher and more visible than the detection signal from the majority allele extension product (left peak). However, the minority allele signal remains small and close to background noise. The rightmost figure shows the results obtained when the concentration of the chain terminator specific to the majority allele is approximately 6-7% (one-fifteenth) of that of the chain terminator specific to the minority allele. As the right figure shows, the signal from the minority allele extension product (right peak) is now comparable to the signal from the majority allele (left peak). Therefore, Figure 1This indicates that adjusting the concentration of chain terminators favors the skewing of minor nucleic acid substances, with minor nucleic acid substances (which cannot be skewed by chain terminators such as iPLEX) occurring at frequencies less than 10%. TM The method can be effectively detected by the method provided in this article.

[0068] The concentration of the chain terminator specific to the major nucleic acid substance (C[WT]) is adjusted relative to the concentration of the chain terminator specific to the minor nucleic acid substance (C[Mut]) such that the C[WT]:C[Mut] ratio is not as high as the ratio that makes the minor nucleic acid substance undetectable. In some embodiments, the proportion of each minor nucleic acid substance can be adjusted based on the copy number or frequency of each minor nucleic acid substance relative to the corresponding major nucleic acid substance. Typically, for minor nucleic acid substances present at a frequency of 10% or less relative to the major nucleic acid substance, the concentration C[WT] of the major nucleic acid substance-specific chain terminator is less than 20% of the concentration of the chain terminator specific to the minor nucleic acid substance (C[Mut]). C[WT] is typically between about 0.5% and less than about 20% of C[Mut], about 0.5% to less than about 15% of C[Mut], about 1% to about 15% of C[Mut], about 1% to about 10% of C[Mut], about 2% to about 10% of C[Mut], or about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of C[Mut]. In some embodiments, C[WT] is about 0.1% to about 10% of C[Mut], about 0.01% to about 10% of C[Mut], or about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of C[Mut]. The ratio of the two concentrations should not be too low, i.e., C[WT] should not be much less than C[Mut]. When C[WT] is much lower than C[Mut], the amount of extended product obtained from the major nucleic acid substance will be lower, resulting in a low detection signal of the major nucleic acid substance. However, furthermore, since minor nucleic acid substances are typically present in amounts less than 10% of the major nucleic acid substances in the sample, there is a limitation on how many minor extension products can be generated. Even if C[Mut] is much higher than C[WT], the signal from the minor substance extension product is unlikely to be much higher than the signal from the major substance extension product. Therefore, when C[WT] is much lower than C[Mut], the signals from the major and minor nucleic acid substances are likely to be too low and too close to detectable background noise.

[0069] Figure 2The study described the specificity of ddNTP chain terminators (C[WT]) in various major substances. ddNTP ) and minor substance-specific ddNTP chain terminators (C[Mut)) ddNTP This study analyzed the detection signals from minor nucleic acid substances relative to those from major nucleic acid substances at various relative concentrations. After PCR amplification and SAP treatment, the amounts of amplicones from both minor and major nucleic acid substances in the sample were sufficient to serve as extension templates, for example, through single-base extension reactions using ddNTP chain-terminating nucleotides and amplification primers (UEPs). The molar concentration of the UEP primers was higher than that of the PCR amplification primers, allowing for repeated cycles of signal amplification during extension. Because the amplicones corresponding to the major nucleic acid substances were significantly in excess compared to the minor nucleic acid substances, most of the UEPs were terminated by chain-terminating nucleotides specific to the major nucleic acid substances ([WT]). ddNTP The extension is such that only a portion of the UEP will be terminated by a chain-terminating nucleotide ([Mut]) specific to the minor nucleic acid material. ddNTP Extension. After the first extension cycle, most UEP primers are extended using the major nucleic acid material as a template, reducing the number of UEP primers available for the next extension cycle. These extended UEP primers, i.e., the single-base extension products of the major nucleic acid material (SBE-WT), will also compete with the unextended primers (UEP) because they will bind to both the major and minor nucleic acid material amplicons (SBE-WT will compete with UEP for both major and minor nucleic acid material amplicons, thus reducing the amount of extension products generated using UEP). If the concentration of the major nucleic acid material-specific chain terminator ddNTP (C[WT]) is... ddNTP This is equal to the concentration of ddNTPs, the specific chain terminator for minor nucleic acid substances (C[Mut)). ddNTP Furthermore, if minor nucleic acid substances are present at a frequency or copy number lower than 10% of the major nucleic acid substances in the sample, the signal corresponding to the minor nucleic acid substances may be so low relative to the signal corresponding to the minor nucleic acid substances that it becomes undetectable in the background noise. This is in Figure 2 The top image (from left to right: signal from unextended primers, signal from extension products of major nucleic acid material, signal from extension products of minor nucleic acid material; horizontal dashed lines represent the background, i.e., the detection limit cannot be distinguished from noise) shows the signal.

[0070] On the other hand, if C[WT] ddNTP Less than C[Mut] ddNTP This results in lower consumption of UEP primers for the larger amounts of major nucleic acid material in the sample, allowing more UEP primers to be used for extension from amplicon corresponding to minor nucleic acid material in subsequent cycles. Because of C[Mut] ddNTP Significantly higher than C[WT] ddNTPThe reaction is completed during each cycle, therefore exceeding the total number of cycles for the single-base extension reaction, resulting in a much higher number of UEPs for ddNTP extension specific to minor nucleic acid substances, thus producing a higher signal relative to the signal corresponding to the major nucleic acid substance and distinct from background noise. This is in Figure 2 The second and third figures at the top (from left to right: signal from the unextended primer, signal from the extension product of the major nucleic acid material, signal from the extension product of the minor nucleic acid material; the horizontal dashed line represents the background, i.e., the detection limit cannot be distinguished from noise) are shown. Therefore, the method presented in this paper allows for the use of [Mut]... ddNTP Lower concentrations of [WT] ddNTP This detects minor nucleic acid substances present at a frequency of less than 10% relative to the major nucleic acid substances. However, if too little [WT] is added to the extension reaction... ddNTP When the amplicon of the major nucleic acid substance is significantly suppressed, low-frequency signals for both the major and minor nucleic acid substances can be generated when combined with small amounts of low-frequency nucleic acid substances. In this method, the signal from the major nucleic acid substance can be used as a positive control to ensure the integrity of the method. Therefore, if the signal from the major nucleic acid substance is too low, it can no longer be detected as a positive control, and additionally, the signal from the minor nucleic acid substance may also be too low for accurate identification. This is in... Figure 2 The background image (from left to right: signal from unextended primers, signal from the extension product of major nucleic acid material, signal from the extension product of minor nucleic acid material; the horizontal dashed line represents the background, i.e., the detection limit cannot be distinguished from noise) is shown.

[0071] like Figure 3 As shown, the method presented in this paper can be used to detect minor nucleic acid substances present at a frequency of less than 2%, down to 1.25%, relative to the major nucleic acid substances in a sample. Figure 3 The results are shown as follows: when samples were amplified and subsequently extended under three conditions: (A) concentration of ddNTPs, chain terminators specific to the major nucleic acid components (wild-type alleles); (C) [WT] ddNTP This is equal to the concentration of ddNTPs, chain terminators specific to minor nucleic acid substances (mutated alleles; C[Mut]). ddNTP (B)C[WT] ddNTP Less than C[Mut] ddNTP Approximately 20% of the concentration (i.e., C[WT]). ddNTP ∶C[Mut] ddNTP The ratio is 0.2); and (C)C[WT] ddNTP Less than C[Mut] ddNTP Approximately 5% of the concentration (i.e., C[WT]). ddNTP∶C[Mut] ddNTP The ratio is 0.05). Figure 3 As shown, when C[WT] ddNTP ∶C[Mut] ddNTP The ratio is 1, meaning the concentrations of the two chain terminators are equal (e.g., in iPLEX). TM When used in the method, the frequencies of mutant alleles were 5%, 2.5%, and 1.25% below the limit of detection (LOD). When C[WT] ddNTP ∶C[Mut] ddNTP When the proportion was reduced to 0.2, a 5% mutant allele frequency could be detected, while the frequencies of 2.5% and 1.25% mutant alleles remained below the detection limit. When C[WT] ddNTP ∶C[Mut] ddNTP When the proportion was further reduced to 0.05, the allele frequencies of all three mutants (5%, 2.5%, and 1.25%) could be detected.

[0072] The method provided herein can also be used to quantify one or more minor nucleic acid substances (e.g., frequency or copy number) relative to the amount of major nucleic acid substance in a sample. For example, if the relative concentrations of the specific chain terminators for the major and minor nucleic acid substances are known (i.e., C[WT] and C[Mut] are known), the minor and major nucleic acid substances can be obtained from the C[WT]:C[Mut] ratio using normalization coefficients. In embodiments of this method, the extension reaction is linearly amplified in each extension cycle, and the relative amounts of minor and major nucleic acid substances are inversely proportional to the C[WT]:C[Mut] ratio. Therefore, the presence of a detectable amount of signal corresponding to the major nucleic acid substance provides a positive control for the integrity of the method and can also be used to quantify the amount (e.g., frequency or copy number) of one or more minor nucleic acid substances relative to the amount of major nucleic acid substance in a sample.

[0073] The method presented herein can be applied to multiplexing forms. For example, when analyzing multiple minor nucleic acids that are variants of multiple major nucleic acids (e.g., mutants, alleles, polymorphisms, deletion variants, insertion variants, etc.), multiplexing can be performed in a series of reaction vessels, compartments, or containers, each containing an extension reaction mixture containing only one chain terminator (e.g., ddATP, ddCTP, ddUTP, ddGTP, or ddTTP). All major nucleic acids having the same specific chain terminator (e.g., all single-base extension products of these major nucleic acids terminate at ddATP) can be extended in one container. Similarly, all minor nucleic acids having the same specific chain terminator can be extended in another container. Using a series of containers, signals from multiple target minor nucleic acids can be analyzed, with each container having a higher concentration of chain terminator for each group of minor nucleic acids, sharing a common specific chain terminator, relative to the signal generated by the container amplifying the amplicons of the major nucleic acids. In one embodiment of the multiplexing method, multiple minor nucleic acids, all of which are variants of the same major nucleic acid, are analyzed.

[0074] The extension products corresponding to the major and minor nucleic acid substances obtained by the methods provided herein can be detected by a variety of methods. For example, the extension primers (UEPs) and / or chain terminators can be labeled with any type of chemical group or part that allows for the detection and / or quantification of signals, including but not limited to, mass markers, radioactive molecules, fluorescent molecules, antibodies, antibody fragments, haptens, carbohydrates, biotin, biotin derivatives, phosphorescent parts, luminescent parts, electrochemiluminescent parts, parts that generate electrochemical signals upon oxidation or reduction, such as complexes of iron, ruthenium, or osmium (see, for example, eSensor technology used by Genmark Diagnostics, Inc., as described in Pierce et al., J. Clin. Micribiol., 50(11): 3458-3465 (2012)), chromatic parts, and parts having detectable electronic spin resonance, capacitance, dielectric constant, or conductivity, or any combination thereof.

[0075] The extended products corresponding to the labels of minor and major nucleic acid substances can be analyzed by a variety of methods, including but not limited to: mass spectrometry, MALDI-TOF mass spectrometry, fluorescence detection, DNA sequencing gels, capillary electrophoresis on automated DNA sequencers, microchannel electrophoresis and other sequencing methods, mass spectrometry, time-of-flight mass spectrometry, quadrupole mass spectrometry, sector magnetic field mass spectrometry, electron mass spectrometry, infrared spectroscopy, ultraviolet spectroscopy, palentiostatic current analysis, current / electrochemical signal measurement, or by DNA hybridization techniques, including Southern blotting, slot blotting, dot blotting and DNA microarrays, where DNA fragments can be used as "probes" and "targets", ELISA, fluorescence assays, fluorescence resonance energy transfer (FRET), SNP-IT, GeneChips, HuSNP, BeadArray, TaqMan assay, Invader assay. or method.

[0076] The methods provided herein can be used, for example: (a) rapidly determining the presence of a specific target sequence (e.g., a target sequence containing genetic variation) in a sample; (b) performing mixture analysis, such as identifying mixtures and / or combinations thereof or determining the frequency of target sequences in a mixture (e.g., mixed communities, quasi-species); (c) detecting sequence variations (e.g., mutations, single nucleotide polymorphisms) in a sample; (d) performing haplotype tests; (e) performing microbial (e.g., pathogen) typing; (f) detecting the presence or absence of microbial target sequences in a sample; (g) identifying disease biomarkers; (h) detecting microsatellites; (i) identifying short tandem repeats; (j) identifying... (k) Detecting allelic variations; (l) Determining allele frequencies; (m) Determining methylation patterns; (n) Performing epigenetic experiments; (o) Re-sequencing regions of biomolecules; (p) Analyzing in human clinical research and medicine (e.g., detection of cancer biomarkers, detection of sequence variations; detection of sequence features that are favorable or unfavorable to specific drug administration); (q) Performing HLA typing; (r) Performing forensic analysis; (s) Performing vaccine quality control analysis; (t) Monitoring treatment; (u) Performing vector characterization; (v) Performing quality control of vaccines or production strains; and (w) Testing strain characteristics (x) Plants. These methods can also be used, for example, in various fields, including but not limited to commerce, education, medicine, agriculture, environment, disease surveillance, military defense, and forensic fields.

[0077] Nucleic acid

[0078] As used herein, the term "nucleic acid" refers to oligonucleotides or polynucleotides, including, but not limited to, native nucleic acids (e.g., deoxyribonucleic acid (DNA), ribonucleic acid (RNA)), synthetic nucleic acids, non-native nucleic acids (e.g., peptide nucleic acids (PNA)), unmodified nucleic acids, and modified nucleic acids (e.g., methylated DNA or RNA, labeled DNA or RNA, DNA or RNA having one or more modified nucleotides). The term "polynucleotide" refers to two or more nucleotides or nucleotide analogs linked by covalent bonds. Nucleic acids can be any type of nucleic acid suitable for the methods described herein. In some embodiments, nucleic acids can be DNA (e.g., complementary DNA (cDNA), genomic DNA (gDNA), plasmid and vector DNA, etc.), RNA (e.g., viral RNA, message RNA (mRNA), short repressive RNA (siRNA), ribosomal RNA (rRNA), tRNA, etc.) and / or DNA or RNA analogs (e.g., containing base analogs, sugar analogs, and / or non-native backbones, etc.). Nucleic acids can be in any form suitable for carrying out the methods described herein (e.g., linear, circular, supercoiled, single-stranded, double-stranded, etc.). In some embodiments, nucleic acids can be or may be derived from plasmids, bacteriophages, autonomously replicating sequences (ARS), centromeres, artificial chromosomes, chromosomes, cells, cell nuclei, or cell cytoplasm. In some embodiments, nucleic acids are derived from a single chromosome (e.g., a nucleic acid sample may be derived from a chromosome sampled from a diploid organism). In the case of fetal nucleic acids, the nucleic acids may be derived from paternal alleles, maternal alleles, or both maternal and paternal alleles.

[0079] As used in this article with respect to target nucleic acids, amplicones, primers, sequence tags, polynucleotides, or oligonucleotides, the term "minor nucleic acid substance" refers to a nucleic acid that, when its nucleotide sequence is aligned, differs from that of a "major nucleic acid substance" from another nucleic acid. Therefore, when the sequences of two substances differ by one or more nucleotides (e.g., approximately 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or greater than 100 nucleotides), the first nucleic acid substance is different from the second nucleic acid substance. In some embodiments, the nucleic acid material (e.g., minor or major nucleic acid material) obtained by PCR amplification of minor or major nucleic acid material or extended oligonucleotide material, the number of amplicon materials includes, but is not limited to, about 2 to about 10,000 nucleic acid materials, about 2 to about 1,000 nucleic acid materials, about 2 to about 500 nucleic acid materials, or sometimes about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 kinds of nucleic acid substances.

[0080] As used herein, the terms “minor nucleic acid substance” or “target nucleic acid substance” can refer to any nucleic acid substance of interest in a sample. Minor nucleic acid substances may include, but are not limited to, (i) a specific minor allele in two or more possible alleles, and (ii) nucleic acids having or not having a specific mutation, nucleotide substitution, sequence variation, repetitive sequence, marker, or differentiating sequence. As used herein, the term “major nucleic acid substance” refers to a nucleic acid substance that differs from a minor nucleic acid substance by one or more characteristics and is present in the sample at a higher frequency or copy number than the minor nucleic acid substance. As used herein, the term “genetic variation” refers to a nucleic acid substance that differs by one or more characteristics. As used herein, the term “variation” refers to a nucleic acid substance that differs by one or more characteristics. Characteristics include, but are not limited to, one or more methyl or methylated states, one or more phosphate groups, one or more acetyl groups, and one or more deletions, additions, or substitutions of one or more nucleotides. Examples of one or more deletions, additions, or substitutions of one or more nucleotides include, but are not limited to, the presence or absence of a specific mutation, the presence or absence of nucleotide substitutions (e.g., single nucleotide polymorphisms (SNPs)), the presence or absence of repetitive sequences (e.g., di, tri, tetra, and pentanucleotide repeats), the presence or absence of markers (e.g., microsatellites), and the presence or absence of distinguishing sequences (e.g., sequences that distinguish one organism from another (e.g., sequences that distinguish one viral strain from other viral strains)). Different target nucleic acids can be distinguished by any known method, such as by quality, binding, distinguishable tags, etc., as described herein.

[0081] In the methods provided herein, a sample may contain a mixture of one or more “minor nucleic acid” substances and one or more “major nucleic acid” substances, or a mixture may be generated by combining more than one sample containing minor nucleic acid substances and major nucleic acid substances. Minor nucleic acid substances include variants of major nucleic acid substances and may include, but are not limited to, mutants of wild-type (major nucleic acid) alleles (minor nucleic acids), variants of genes found in more than one host (e.g., viral oncogenes (minor nucleic acids) that are variants of normal healthy genes (major nucleic acids)), polymorphisms, including single nucleotide polymorphisms (SNPs), insertions, deletions, or other forms of mutations in major nucleic acid substances. The minor nucleic acid substance is present in the sample (or sample combination) at a frequency or copy number of about 0.25% to at most about 50%, with specific ranges including but not limited to about 0.25% to about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9.5%, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, or 1%. In some embodiments, the minor nucleic acid substance is present at a frequency or copy number of about 1% or about 2% to about 10%, 9.5%, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, or 2.5% relative to the major nucleic acid substance. In other embodiments, the minor nucleic acid substance is present at a frequency of about 1% or about 2% to less than about 10% relative to the major nucleic acid substance.

[0082] As used herein, the terms "multiple" target nucleic acids, minor nucleic acid substances, and / or major nucleic acid substances refer to more than one target nucleic acid substance, minor nucleic acid substance, and / or major nucleic acid substance. "Multiple" can be approximately 2 to approximately 10,000 nucleic acid substances, approximately 2 to approximately 1,000 nucleic acid substances, approximately 2 to approximately 500 nucleic acid substances, or sometimes approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 80, 85, 90, 95. In some embodiments, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 nucleic acid substances are used. The detection or identification of nucleic acids leads to the detection of minor nucleic acid substances and can indicate the presence or absence of specific mutations, sequence variations (mutations or polymorphisms), or genetic variations (e.g., sequence variations, sequence differences, or polymorphisms). The detection or identification of nucleic acids also typically leads to the detection or identification of major nucleic acid substances, which can serve as a basis for positive controls and / or quantification of minor nucleic acid substances. Within a variety of minor and major nucleic acid substances, it is possible to detect and / or quantify substances that are the same or different; to detect and / or quantify minor nucleic acid substances that are all of the same major nucleic acid substance or multiple minor nucleic acid substances that are variants of multiple major nucleic acid substances.

[0083] In some embodiments, the oligonucleotide material hybridizes with a nucleic acid template (e.g., an amplicon) to form a double-stranded nucleic acid, and the oligonucleotide material hybridizing with the template is referred to herein as a hybridized oligonucleotide material. In some embodiments, the hybridized oligonucleotide material may contain one or more nucleotides that do not hybridize with the template. For example, the hybridized oligonucleotide material may contain one or more mismatched nucleotides (e.g., non-complementary nucleotides), and sometimes may contain the 5′ and / or 3′ regions of the non-hybridized nucleotides. In some embodiments, the hybridized oligonucleotide material includes a tag (e.g., a quality-distinguished tag, a sequence tag, a luminescent tag, or a radioactive tag). In some embodiments, the hybridized oligonucleotide material includes a trapping agent (e.g., biotin or any member of a binding pair). In some embodiments, the hybridized oligonucleotide material includes a termination nucleotide.

[0084] The term "chain terminator" is used interchangeably with "chain terminator reagent" or "chain terminator" as used herein, and refers to a molecule that stops the extension reaction when an extension primer is added. Chain terminators can include nucleotide analogs that, when present in a polynucleotide chain, prevent further chain extension. Exemplary chain terminators as chain-terminating nucleotides include dideoxynucleotides, such as ddA (dideoxyadenine), ddT (dideoxythymidine), ddC (dideoxycytosine), ddG (dideoxyguanine), and ddU (dideoxyuracil).

[0085] As used herein, the term "nucleotide" refers to both natural and non-natural nucleotides. Nucleotides include, but are not limited to, naturally occurring nucleoside mono-, di-, and triphosphates: deoxyadenosine mono-, di-, and triphosphates; deoxyguanosine mono-, di-, and triphosphates; deoxythymidine mono-, di-, and triphosphates; deoxycytidine mono-, di-, and triphosphates; deoxyuridine mono-, di-, and triphosphates; and deinosine mono-, di-, and triphosphates (referred to herein as dA, dG, dT, dC, dU, and dI, or A, G, T, C, U, and I, respectively). Nucleotides also include, but are not limited to, modified nucleotides and nucleotide analogues. Modified nucleotides and nucleotide analogues include, but are not limited to, deazapurine nucleotides such as 7-deaza-deoxyguanosine (7-deaza-DG) and 7-deaza-deoxyadenosine (7-deazaadenosine) mono-, di-, and triphosphates, deuterated-deoxythymidine (deuterated-dT) mono-, di-, and triphosphates, and methylated nucleotides such as 5-methyldeoxycytidine triphosphate. 13 C / 15 N-labeled nucleotides and deoxyinosine mono-, di-, and triphosphates. Modified nucleotides, isotopically enriched nucleotides, depleted nucleotides, tagged and labeled nucleotides, and nucleotide analogs can be obtained using a variety of functional and attachment site combinations.

[0086] The term "composition" used herein with respect to nucleic acids refers to a tangible article comprising one or more nucleic acids. A composition is sometimes a sample extracted from a source, and is a composition of all samples at the source, and sometimes is a source of one or more nucleic acids. A composition may contain nucleic acids. In some embodiments, a composition may contain genomic DNA. In some embodiments, a composition may contain maternal DNA, fetal DNA, or a mixture of maternal and fetal DNA. In some embodiments, a composition may contain fragments of genomic DNA. In some embodiments, a composition may contain nucleic acids derived from viruses, bacteria, yeast, fungi, mammals, or mixtures thereof.

[0087] Nucleic acid samples can be derived from one or more sources and can contain a mixture of minor and major nucleic acid substances. Samples can also be combined to produce a mixture that includes both minor and major nucleic acid substances. For example, samples can be collected from biological, mineral, or geological sites (e.g., soil, rocks, mineral sediments, fossils) or forensic sites (e.g., crime scenes, contraband, or suspected contraband). Thus, the source can be environmental, such as geological, agricultural, war zone, or soil-based. The source can also be any type of organism, such as any plant, fungus, protist, prokaryote, virus, or animal, including but not limited to humans, non-humans, mammals, reptiles, cattle, cats, dogs, goats, pigs, monkeys, apes, gorillas, bulls, cattle, bears, horses, sheep, poultry, mice, rats, fish, dolphins, whales, and sharks, or any animal or organism that may have detectable nucleic acids. The source can also refer to different parts of an organism, such as internal, external, living or non-living cells, tissues, fluids, etc. Therefore, a sample can be a "biological sample," which refers to any material obtained from a living or previously living source, such as animals like humans or other mammals, plants, bacteria, fungi, protozoa, or viruses. The source can be in any form, including but not limited to solid materials such as tissues, cells, cell pellets, cell extracts, or biopsies, or biological fluids such as urine, blood, saliva, amniotic fluid, exudate from infected or inflamed areas, or oral wash containing buccal cells, hair, cerebrospinal fluid, and synovial fluid, as well as organs. Samples can also be obtained at different time points than another sample, where the samples originate from the same or different sources. Nucleic acids can be derived from nucleic acid libraries, such as cDNA or RNA libraries. Nucleic acids can be products of nucleic acid purification or isolation and / or amplification of nucleic acid molecules in the sample. The nucleic acids provided for use in the sequence analysis methods described herein may comprise nucleic acids from one sample or two or more samples (e.g., from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900 or more samples).

[0088] Nucleic acids can be processed in various ways during, before, or after the methods provided herein. For example, nucleic acids can be reduced in size (e.g., cleavage, digestion by nucleases or restriction enzymes, dephosphorylation, demethylation), increased in size (e.g., phosphorylation, reaction with methylation-specific reagents, attachment to detectable tags), treated with nucleic acid cleavage inhibitors, etc.

[0089] In some embodiments, nucleic acids can be analyzed according to the methods provided herein without processing. In some embodiments, nucleic acids are provided for processing following the methods described herein. For example, nucleic acids can be extracted, isolated, purified, or amplified from a sample. As used herein, the term "isolation" means removing nucleic acids from their original environment (e.g., the natural environment in which nucleic acids occur naturally or the host cell from which exogenous nucleic acids are expressed), thus altering the nucleic acids from their original environment "artificially." Isolated nucleic acids generally contain fewer non-nucleic acid components (e.g., proteins, lipids) compared to the component content in the source sample. Compositions containing isolated nucleic acids can be substantially isolated (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% free of non-nucleic acid components). As used herein, the term "purified" means that the provided nucleic acid contains fewer types of nucleic acids compared to the sample source from which it is derived. The composition containing nucleic acids can be substantially purified (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% free of other nucleic acid types).

[0090] Nucleic acids can be processed, in some embodiments, by methods of generating nucleic acid fragments prior to providing nucleic acids for the methods described herein. In some embodiments, the fragmented or cleaved nucleic acids may have a nominal, average, or geometric mean length of about 5 to about 10,000 base pairs, about 100 to about 1.00 base pairs, about 100 to 500 base pairs, or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 base pairs. Fragments can be generated by any suitable method known in the art, and the average, geometric mean, or nominal length of the nucleic acid fragments can be controlled by selecting an appropriate fragment generation method. In some embodiments, shorter nucleic acids can be used to analyze sequences that contain almost no sequence variations and / or contain a large amount of known nucleotide sequence information. In some embodiments, longer nucleic acids can be used to analyze sequences that contain more sequence variations and / or contain a smaller amount of unknown nucleotide sequence information.

[0091] Amplification and extension

[0092] In embodiments of the methods provided herein, nucleic acid material (e.g., minor and / or major nucleic acid material) may be amplified in some embodiments. As used herein, the term "amplification" and its grammatical variations refer to a method for generating copies of a template nucleic acid. For example, a nucleic acid template may be linearly or exponentially generated into two or more nucleic acid amplicon (copies) having the same or substantially the same nucleotide sequence as the template or a portion thereof. In some embodiments, nucleic acid amplification is typically specific (e.g., the amplicon has the same or substantially the same sequence) and can sometimes be nonspecific (e.g., the amplicon has a different sequence). Nucleic acid amplification is sometimes beneficial when the amount of the target sequence present in the sample is low. By amplifying the target sequence and detecting the synthesized amplicon, the sensitivity of the assay can be improved because less target sequence is required at the start of the assay for detecting the target nucleic acid. In some embodiments, the nucleic acid material (minor or major nucleic acid material) is sometimes not amplified prior to hybridization extension oligonucleotides (primers, i.e., UEPs).

[0093] Amplification conditions are known and can be selected for the specific nucleic acid to be amplified. Amplification conditions include certain reagents, some of which may include, but are not limited to, nucleotides (e.g., nucleotide triphosphates), modified nucleotides, oligonucleotides (e.g., primer oligonucleotides for polymerase-based amplification and oligonucleotide building blocks for ligase-based amplification), one or more salts (e.g., magnesium-containing salts), one or more buffers, one or more polymerizing agents (e.g., ligases, polymerases), one or more cleavage enzymes (e.g., enzymes that cleave one strand of a double-stranded nucleic acid), and one or more nucleases (e.g., exonucleases, endonucleases, RNases). Any polymerase suitable for amplification can be used, such as polymerases with or without exonuclease activity, DNA polymerases, and RNA polymerases, such as mutant forms of these enzymes. Any ligase suitable for ligating the 5′ end of one oligonucleotide to the 3′ end of another oligonucleotide can be used. Amplification conditions may also include certain reaction conditions, such as isothermal or temperature cycling conditions. Methods for cycling temperatures during amplification are known, for example, by using a thermal cycling apparatus. The term "cycling" refers to amplification (e.g., an amplification reaction or extension reaction) using a single primer or multiple primers, wherein temperature cycling is employed. In some embodiments, amplification conditions may also include emulsions (e.g., oils), which may be used to form multiple reaction chambers in which single nucleic acid molecules can be amplified. Amplification is sometimes an exponential product-generating process and sometimes a linear product-generating process.

[0094] It can amplify the strand of a single-stranded nucleic acid target, and can amplify one or both strands of a double-stranded nucleic acid target. In some embodiments, the amplification product (amplifier) ​​is about 10 nucleotides to about 10,000 nucleotides in length, about 10 to about 1,000 nucleotides in length, about 10 to about 500 nucleotides in length, about 10 to about 100 nucleotides in length, and sometimes about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, or 1,000 nucleotides in length.

[0095] Any suitable amplification technique and conditions can be chosen for amplifying a specific nucleic acid. Known amplification methods include, but are not limited to, polymerase chain reaction (PCR), extension and ligation, ligation amplification (or ligase chain reaction (LCR)), and amplification methods based on the use of Q-β replicase or template-dependent polymerase (see US Patent Publication No. US20050287592). Chain displacement amplification (SDA), thermophilic SDA, nucleic acid sequence-based amplification (3SR or NASBA), and transcription-associated amplification (TAA) can also be used. The reagents, apparatus, and hardware used to perform the amplification methods are commercially available, and the amplification conditions are known, and the target nucleic acid can be selected.

[0096] In some implementations, polymerase-based amplification can be achieved using universal primers. In these processes, the hybridization region that hybridizes with one or more universal primers is incorporated into the template nucleic acid. Such a hybridization region can be incorporated into (i) a primer that hybridizes with and extends the target nucleic acid, and / or (ii) an oligonucleotide ligated to, for example, the target nucleic acid or the product of (i) (e.g., using a ligase). Amplification methods involving universal primers can provide the advantage of amplifying multiple target nucleic acids, for example, using only one or two amplification primers.

[0097] In some embodiments, certain minor and major nucleic acid substances may be extended before or after amplification. The term "extension" as used herein, and its grammatical variations, refers to the elongation of one strand of a nucleic acid. For example, in some embodiments, oligonucleotides hybridizing with minor or major nucleic acid substances or amplicon generated from minor or major nucleic acid substances may be extended. The extension reaction is carried out under extension conditions, and various such conditions are known and selected for specific applications. Extension conditions may include certain reagents, including but not limited to one or more oligonucleotides, extending nucleotides (e.g., nucleotide triphosphates (dNTPs)), chain terminators or nucleotides (e.g., one or more dideoxynucleotide triphosphates (ddNTPs) or acyclic terminators), one or more salts (e.g., magnesium-containing salts), one or more buffers (e.g., using β-NAD, Triton X-100), and one or more polymerizing agents (e.g., DNA polymerase, RNA polymerase). The concentration C[WT] of the chain terminator specific to the major nucleic acid substance is typically less than about 20% of the concentration (C[Mut]) of the chain terminator specific to the minor nucleic acid substance. C[WT] is typically about 0.5% to less than about 20% of C[Mut], about 0.5% to less than about 15% of C[Mut], about 1% to about 15% of C[Mut], about 1% to about 10% of C[Mut], about 2% to about 10% of C[Mut], or about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of C[Mut]. In some embodiments, C[WT] is about 0.1% to about 10% of C[Mut], about 0.01% to about 10% of C[Mut], or about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of C[Mut].

[0098] In some implementations, the extension can be carried out under isothermal or non-isothermal conditions (e.g., thermal cycling conditions). One or more nucleic acid substances can be extended in the extension reaction, and one or more molecules of each nucleic acid substance can be extended. Nucleic acids can be extended with one or more nucleotides, and in some embodiments, the length of the extended product is about 10 nucleotides to about 10,000 nucleotides, about 10 to about 1,000 nucleotides, about 10 to about 500 nucleotides, about 10 to about 100 nucleotides, and sometimes about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, or 1,000 nucleotides. The length of oligonucleotide extension can be determined by combining the termination nucleotide (e.g., ddNTP), hybridization position, or other factors. In some implementations, the amplification and extension processes are performed in the same detection process.

[0099] In some embodiments, the extension reaction includes repeating multiple temperature cycles to amplify the amount of extension product in the reaction. In some embodiments, the extension reaction is cycled two or more times. In some embodiments, the extension reaction is cycled 10 or more times. In some embodiments, the extension reaction is cycled about 10, 15, 20, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, or 600 or more times. In some embodiments, the extension reaction is cycled 20-50 times. In some embodiments, the extension reaction is cycled 20-100 times. In some embodiments, the extension reaction is cycled 20-300 times. In some embodiments, the extension reaction is cycled 200-300 times. In some embodiments, the extension reaction is cycled at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times.

[0100] In some embodiments, minor or major nucleic acid material is extended in the presence of an extending composition, wherein the nucleic acid material is extended by one nucleotide. The extending composition may contain one or more buffers, salts, enzymes (e.g., polymerases, Klenow, etc.), water, templates (e.g., DNA, RNA, amplicon, etc.), primers (e.g., oligonucleotides), nucleotide triphosphates, glycerol, macromolecular exclusion molecules, and any other additives used in the art. The extending composition may include terminating nucleotides (e.g., dideoxynucleotides (e.g., ddNTPs)) or other chain terminators, non-terminating or extending nucleotides (e.g., dNTPs), or mixtures of terminating and non-terminating nucleotides. An extending composition consisting essentially of one or more specific terminating nucleotides may contain any other components of the extending composition (e.g., buffers, salts, templates, primers, etc.), but not any other terminating nucleotides or nucleotide triphosphates (e.g., dNTPs) other than those described above. For example, an extending composition consisting essentially of ddTTP and ddCTP does not contain ddATP, ddGTP, or any other dNTPs. In some embodiments, the nucleotides in the extension composition are only the terminating nucleotides, and the target nucleic acid is extended by one nucleotide (i.e., the extension composition sometimes does not contain an extension nucleotide). In some embodiments, the extension composition consists essentially of a terminating nucleotide (e.g., ddNTP). In embodiments of the methods provided herein, the concentration C[WT] of the chain terminator specific to the major nucleic acid substance is less than 20% of the concentration (C[Mut]) of the chain terminator specific to the minor nucleic acid substance, and C[WT] is typically from about 0.5% to less than about 20% of C[Mut], from about 0.5% to less than about 15% of C[Mut], from about 1% to about 15% of C[Mut], from about 1% to about 10% of C[Mut], from about 2% to about 10% of C[Mut], or from about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of C[Mut]. In some embodiments, C[WT] is about 0.1% to about 10% of C[Mut], about 0.01% to about 10% of C[Mut], or about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of C[Mut]. In some embodiments, the minor or major nucleic acid material is extended in the presence of the extending composition, wherein the nucleic acid material is extended by one nucleotide.The extension composition may contain one or more buffers, salts, enzymes (e.g., polymerases, Klenow, etc.), water, templates (e.g., DNA, RNA, amplicon, etc.), primers (e.g., oligonucleotides), nucleotide triphosphates, glycerol, macromolecular exclusion molecules, and any other additives used in the art. The extension composition may include terminating nucleotides (e.g., dideoxynucleotides (e.g., ddNTPs)) or other chain terminators, non-terminating or extending nucleotides (e.g., dNTPs), or mixtures of terminating and non-terminating nucleotides. An extension composition consisting essentially of one or more specific terminating nucleotides may contain any other components of the extension composition (e.g., buffers, salts, templates, primers, etc.), but not any other terminating nucleotides or nucleotide triphosphates (e.g., dNTPs) besides those described above. For example, an extension composition consisting essentially of ddTTP and ddCTP does not contain ddATP, ddGTP, or any other dNTPs. In some embodiments, the nucleotides in the extension composition are only terminating nucleotides, and the target nucleic acid is extended by one nucleotide (i.e., the extending nucleotide is sometimes absent from the extension composition). In some embodiments, the extension composition consists essentially of terminating nucleotides (e.g., ddNTPs). In embodiments of the methods provided herein, the concentration C[WT] of the chain terminator specific to the major nucleic acid substance is less than 20% of the concentration (C[Mut]) of the chain terminator specific to the minor nucleic acid substance. C[WT] is typically from about 0.5% to less than about 20% of C[Mut], from about 0.5% to less than about 15% of C[Mut], from about 1% to about 15% of C[Mut], from about 1% to about 10% of C[Mut], from about 2% to about 10% of C[Mut], or from about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of C[Mut]. In some embodiments, C[WT] is about 0.1% to about 10% of C[Mut], about 0.01% to about 10% of C[Mut], or about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of C[Mut].

[0101] In some embodiments, the chain terminator or chain-terminating nucleotide comprises one or more detectable markers. Detectable markers include, but are not limited to, mass markers, radioactive molecules, fluorescent molecules, antibodies, antibody fragments, haptens, carbohydrates, biotin, biotin derivatives, phosphorescent portions, luminescent portions, electrochemiluminescent portions, portions that generate an electrochemical signal upon oxidation or reduction, such as complexes of iron, ruthenium, or osmium (see, for example, eSensor technology used by Genmark Diagnostics, Inc., as described in Pierce et al., J. Clin. Micribiol., 50(11): 3458-3465(2012)), chromatic portions, and portions having detectable electronic spin resonance, capacitance, dielectric constant, or conductivity, or any combination thereof. In some embodiments, the chain terminator or chain-terminating nucleotide comprises a single detectable marker. In some embodiments, the first chain terminator or chain-terminating nucleotide comprises a detectable marker different from the detectable marker of the second chain terminator or chain-terminating nucleotide. In some embodiments, the extension composition includes one or more chain terminators or chain-terminating nucleotides, wherein each chain terminator or chain-terminating nucleotide includes a different detectable marker. In some embodiments, the extension composition comprises one or more chain terminators or chain-terminating nucleotides, wherein each contains the same detectable marker. In some embodiments, the extension composition comprises a chain terminator or chain-terminating nucleotide and an extension nucleotide (e.g., dNTP), and one or more of the nucleotides (e.g., the terminating nucleotide and / or the extension nucleotide) include a detectable marker.

[0102] Any suitable extension reaction can be selected and utilized. Extension reactions can be employed, for example, by incorporating a deoxynucleotide and / or dideoxynucleotide into an extending oligonucleotide that hybridizes to a region in the target nucleic acid adjacent to the SNP site to distinguish SNP alleles. Primers are typically extended using polymerase. In some embodiments, the oligonucleotide extends only one deoxynucleotide or dideoxynucleotide complementary to the SNP site. In some embodiments, the oligonucleotide can be extended by incorporation of a dNTP and terminated by a ddNTP, or in some embodiments terminated by ddNTP incorporation but without dNTP extension. In some embodiments, extension can be performed using: unmodified extending oligonucleotides and unmodified dideoxynucleotides, unmodified extending oligonucleotides and biotinylated dideoxynucleotides, extending oligonucleotides containing deoxyinosine and unmodified dideoxynucleotides, extending oligonucleotides containing deoxyinosine and biotinylated dideoxynucleotides, extension by biotinylated dideoxynucleotides, or extension by biotinylated deoxynucleotides and / or unmodified dideoxynucleotides.

[0103] In some embodiments, the oligonucleotide material can hybridize with a template (e.g., a minor or major nucleic acid material) of a neighboring genetic variant or variant under hybridization conditions (e.g., the 3′ end of the oligonucleotide material may be located at the 5′ end of the genetic variant site, and may be 0 to 10 nucleotides away from the 5′ end of the genetic variant site). Several variants may be present at the genetic variant site of the target nucleic acid. The genetic variant is sometimes a single nucleotide polymorphism (SNP) or a single nucleotide variant. Several single nucleotide variants may be present at a single base position on the template target located at the 3′ of the hybridized oligonucleotide. Several single nucleotide variants may differ due to the single base at the 3′ position of the template target on the hybridized oligonucleotide material. In some embodiments, the oligonucleotide material is extended by one nucleotide at the variant position. In some embodiments, depending on the number of variants present, the oligonucleotide may be extended by any of the five termination nucleotides (e.g., ddATP, ddUTP, ddTTP, ddGTP, ddCTP). The major nucleic acid material and its minor nucleic acid material variants or corresponding amplicons can serve as templates, and it is partially possible to determine which termination nucleotide is added to the oligonucleotide during the extension reaction. The primary nucleic acid substance may have two or more variants of the secondary nucleic acid substance. In some embodiments, the primary nucleic acid substance has two, three, or four variants of the secondary nucleic acid substance.

[0104] In the multiplexed forms of the methods provided herein, a primary nucleic acid substance and one or more variants of secondary nucleic acids can be analyzed in a single reaction vessel, with the chain terminator specific to the primary nucleic acid substance differing from the chain terminator specific to one or more secondary nucleic acids. In some embodiments, all secondary nucleic acids are terminated by the same chain terminator, different from the chain terminator specific to the primary nucleic acid substance. In some embodiments, at least one secondary nucleic acid substance has a chain terminator different from the chain terminators of the other secondary nucleic acids. In some embodiments, the chain terminator for each secondary nucleic acid substance in the reaction is different from each other and also different from the chain terminator specific to the primary nucleic acid substance.

[0105] In some multiplexed forms of the methods provided herein, mixtures of more than one major nucleic acid substance and more than one minor nucleic acid substance can be analyzed in a series of reaction vessels. All major nucleic acid substances having the same specific chain terminator are extended in the same reaction vessel (to produce, for example, a single-base extension product using the chain terminator), and in embodiments, the only chain terminator in the vessel is a chain terminator specific (and shared) to the major nucleic acid substance in the vessel. Similarly, all minor nucleic acid substances having the same specific chain terminator are extended in the same reaction vessel, and in embodiments, the only chain terminator in the vessel is a chain terminator specific (and shared) to the minor nucleic acid substance in the vessel.

[0106] In the methods provided herein, including embodiments of multiple forms of the methods, the concentration of the chain terminator specific to the major nucleic acid substance (C[WT]) may be less than 20% of the concentration of the chain terminator specific to the minor nucleic acid substance (C[Mut]), C[WT] typically being from about 0.5% to less than about 20% of C[Mut], from about 0.5% to less than about 15% of C[Mut], from about 1% to about 15% of C[Mut], from about 1% to about 10% of C[Mut], from about 2% to about 10% of C[Mut], or from about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of C[Mut]. In some embodiments, C[WT] is about 0.1% to about 10% of C[Mut], about 0.01% to about 10% of C[Mut], or about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of C[Mut].

[0107] The resulting extension product can be identified by a detection label that may be present on one or more extension primers (UEPs), a chain terminator, and one or more nucleotides (if present in the extension reaction). Labels may include, but are not limited to, mass markers, radioactive molecules, fluorescent molecules, antibodies, antibody fragments, haptens, carbohydrates, biotin, biotin derivatives, phosphorescent moieties, luminescent moieties, electrochemiluminescent moieties, moieties that generate an electrochemical signal upon oxidation or reduction, such as complexes of iron, ruthenium, or osmium (see, for example, eSensor technology used by Genmark Diagnostics, Inc., as described in Pierce et al., J. Clin. Micribiol., 50(11): 3458-3465(2012)), chromatic moieties, and moieties having detectable electronic spin resonance, capacitance, dielectric constant, or conductivity, or any combination thereof.

[0108] The extended products corresponding to the labels of minor and major nucleic acid substances can be analyzed by a variety of methods, including but not limited to: mass spectrometry, MALDI-TOF mass spectrometry, fluorescence detection, DNA sequencing gels, capillary electrophoresis on automated DNA sequencers, microchannel electrophoresis and other sequencing methods, mass spectrometry, time-of-flight mass spectrometry, quadrupole mass spectrometry, sector magnetic field mass spectrometry, electron mass spectrometry, infrared spectroscopy, ultraviolet spectroscopy, palentiostatic current analysis, current / electrochemical signal measurement, or by DNA hybridization techniques, including Southern blotting, slot blotting, dot blotting and DNA microarrays, where DNA fragments can be used as "probes" and "targets", ELISA, fluorescence assays, fluorescence resonance energy transfer (FRET), SNP-IT, GeneChips, HuSNP, BeadArray, TaqMan assay, Invader assay. or method.

[0109] In some embodiments, the relative amount (e.g., frequency or copy number) of minor nucleic acid material relative to major nucleic acid material can be determined using a normalization coefficient as a ratio of its detection signal to the concentration of the major nucleic acid material-specific chain terminator to the concentration of the minor nucleic acid material-specific chain terminator. In embodiments of this method, the amplification of major and minor nucleic acids during extension is linear and proportional to the relative amounts (ratios) of the molecules of minor and major nucleic acids and their chain terminators in the assay. In some embodiments, the amount (e.g., copy number, concentration, percentage) of minor nucleic acid material is quantified by normalizing the ratio of the signal of minor nucleic acid material to the signal of major nucleic acid material using a coefficient. This coefficient is inversely proportional to the fraction of the concentration of the chain terminator or nucleotide specific to major nucleic acid material relative to the concentration of the chain terminator or nucleotide specific to minor nucleic acid material (i.e., the lower the fraction of the chain terminator specific to major nucleic acid material relative to the chain terminator specific to minor nucleic acid material, the larger the coefficient). In embodiments of the method provided herein, a chain terminator specific to the major nucleic acid substance is added in an amount sufficient to generate a detectable signal. This can be used as a positive control to identify the presence of minor nucleic acid substances and can also serve as a basis for quantifying the relative amount (e.g., frequency or copy number) of minor nucleic acid substances in a sample.

[0110] In some embodiments, a minor nucleic acid substance may be present in greater abundance than other minor nucleic acid substances. In some embodiments, the relative concentrations of each specific chain terminator among the various minor nucleic acid substances can be adjusted to optimize the magnitude of the detection signal corresponding to each minor nucleic acid substance. In some embodiments, the primary nucleic acid substance includes first, second, and third variant minor nucleic acid substances, wherein the second variant is present in greater abundance than the first and third variants. In some embodiments, the primary nucleic acid substance includes first, second, third, and fourth variant minor nucleic acid substances, wherein the second variant is present in greater abundance than the first, third, and fourth variants. Variants expressed as greater abundance are generally present at higher concentrations or are represented by a greater number of molecules (e.g., copies) when compared to another variant. Higher concentrations can be more than 2-fold. In some embodiments, higher concentrations are 10-fold or more. In some embodiments, higher concentrations are 100-fold, 1000-fold, or 10000-fold or more. In some embodiments, the primary nucleic acid substance is present at a concentration 100-fold or higher than that of the minor nucleic acid substances. In some embodiments, minor nucleic acid substances represent less than 30%, 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.8%, 0.75%, 0.5%, 0.1%, 0.05%, 0.01%, or less of the major nucleic acid substances. In some embodiments, minor nucleic acid substances constitute about 5% to about 0.75% of the major nucleic acid substances. In some embodiments, minor nucleic acid substances represent less than 30%, 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.8%, 0.75%, 0.5%, 0.1%, 0.05%, 0.01%, or less of the total nucleic acid substances in the composition.

[0111] In some embodiments, the terminating nucleotide present in the extension composition (or absent in some embodiments) determines which terminating nucleotide to add to the oligonucleotide. In some embodiments, the extension composition contains one or more terminating nucleotides (e.g., ddNTPs). In some embodiments, the extension composition contains one or more terminating nucleotides and one or more non-terminating nucleotides (e.g., dNTPs). In some embodiments, the extension composition contains only terminating nucleotides corresponding to a specific minor or major nucleic acid substance, thus allowing extension only of that substance. In some embodiments, in samples containing multiple minor nucleic acid substances, the extension composition includes only those terminating nucleotides corresponding to the minor nucleic acid substance (variant) to be detected.

[0112] As used herein, the term "signal-to-noise ratio" refers to a quantitative measure of signal quality by quantifying the ratio of the intensity of a signal to the intensity of noise when using a detection process (e.g., mass spectrometry). In some embodiments, an intensity peak on one spectrum has a greater signal-to-noise ratio than a low-intensity peak produced by the same analyte (e.g., an extended oligonucleotide) on another spectrum. In some embodiments, an extended oligonucleotide derived from a major nucleic acid substance (e.g., a wild-type allele, a second variant, a wild-type variant) generates noise. In some embodiments, the signal produced by an extended oligonucleotide derived from a minor nucleic acid substance (e.g., a mutant variant, a mutant allele, a SNP) is masked by noise generated by a more abundant minor or major extended oligonucleotide. As used herein, the term "signal" in the context of "signal-to-noise ratio" refers to the intensity of the signal peak of the extended oligonucleotide. In some embodiments, the term "signal" in the context of "signal-to-noise ratio" generally refers to the intensity of the signal peak of an extended oligonucleotide derived from a minor nucleic acid substance (e.g., a mutant variant, a mutant allele, a SNP). In some implementations, a termination nucleotide that allows for the extension of the major nucleic acid substance (e.g., wild-type allele) is added at a concentration that allows for enhanced signal generation from the extension of the minor nucleic acid substance, while still providing a signal from the major nucleic acid substance as a control and as a basis for quantifying the minor nucleic acid substance.

[0113] As used herein, the term "sensitivity" refers to the amount of analyte that can be detected at a given signal-to-noise ratio when using a detection process (e.g., mass spectrometry). In some embodiments, sensitivity can be improved by reducing the background or noise level. In some embodiments, extended oligonucleotides derived from a major nucleic acid substance (e.g., wild-type allele, wild-type variant) generate noise. In some embodiments, sensitivity increases when the signal generated from the extended oligonucleotides derived from the major nucleic acid substance is reduced.

[0114] Any suitable type of nucleotide can be incorporated into the amplification or extension product. In some embodiments, the nucleotide can be a naturally occurring nucleotide, a terminator nucleotide, or a non-naturally occurring nucleotide (e.g., a nucleotide analog or derivative). In some embodiments, certain nucleotides may contain members of a detectable label and / or a binding pair or fluorescent labeling pair for FRET detection (e.g., one member of the pair may be on the terminator nucleotide incorporated into the UEP by extension, while the other member of the pair may be at another position on the extension product oligonucleotide).

[0115] The solution containing amplicons generated by the amplification method or the solution containing extension products generated by the extension method can be further processed. For example, the solution can be contacted with a reagent to remove the phosphate moiety from free nucleotides that have never been incorporated into the amplicon or extension product. An example of such a reagent is a phosphatase (e.g., alkaline phosphatase, such as shrimp alkaline phosphatase). The amplicon and extension product can also be bound to a solid, washed, contacted with a reagent to remove the terminal phosphate (e.g., exposed to a phosphatase), contacted with a reagent to remove the terminal nucleotides (e.g., an exonuclease), contacted with a cleaving reagent (e.g., an endonuclease, a ribonuclease), etc.

[0116] As used herein, the term "oligonucleotide" refers to two or more nucleotides or nucleotide analogs linked by covalent bonds. Oligonucleotides have any convenient length, and in some embodiments, the length is about 5 to about 200 nucleotides, about 5 to about 150 nucleotides, about 5 to about 100 nucleotides, about 5 to about 75 nucleotides, or about 5 to about 50 nucleotides, and sometimes the length is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 80, 85, 90, 95, 100, 125, 150, 175, or 200 nucleotides. Oligonucleotides may include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), naturally occurring and / or non-naturally occurring nucleotides or combinations thereof, and any chemical or enzymatic modifications thereof (e.g., methylated DNA, DNA with modified nucleotides). Oligonucleotides are sometimes shorter than the length of the amplicon or target nucleic acid, but not necessarily shorter than the primers or polynucleotides used for amplification. Oligonucleotides typically contain nucleotide subsequences or hybridization sequences that are complementary or substantially complementary to the amplicon, target nucleic acid, or their complementary sequences (e.g., approximately 95%, 96%, 97%, 98%, 99%, or more than 99% identical to the complementary sequences of the amplicon or target nucleic acid at alignment). Oligonucleotides may also contain nucleotide subsequences that are not complementary or substantially non-complementary to the amplicon, target nucleic acid, or their complementary sequences (e.g., at the 3′ or 5′ end of the nucleotide subsequence in primers that are complementary or substantially complementary to the amplicon). In some embodiments, the oligonucleotide may contain a detectable molecule (e.g., a tag, a fluorophore, a radioisotope, a colorimetric agent, a particle, an enzyme, etc.) and / or (in some embodiments) a binding pair member (e.g., biotin / avidin, biotin / streptavidin).

[0117] As used herein, the term "in solution" refers to a liquid, such as a liquid containing one or more nucleic acids. Nucleic acids and other components in solution may be dispersed throughout the solution, and the solution typically contains water (e.g., an aqueous solution). The solution may contain any convenient amount of oligonucleotides, and typically contains at least the same amount of oligonucleotides as the amplicon material or target nucleic acid material to be detected.

[0118] As used herein, the term "hybridization sequence" refers to a nucleotide sequence in an oligonucleotide capable of specifically hybridizing with an amplicon, target nucleic acid, or its complementary sequence. Hybridization sequences are easy to design and select, and their length is suitable for hybridization with an amplicon, target sequence, or its complementary sequence in solution as described herein. In some embodiments, the length of the hybridization sequence in each oligonucleotide is about 5 to about 200 nucleotides (e.g., about 5 to 10, about 10 to 15, about 15 to 20, about 20 to 25, about 25 to 30, about 30 to 35, about 35 to 40, about 40 to 45, or about 45 to 50, about 50 to 70, about 80 to 90, about 90 to 110, about 100 to 120, about 110 to 130, about 120 to 140, about 130 to 150, about 140 to 160, about 150 to 170, about 160 to 180, about 170 to 190, about 180 to 200 nucleotides).

[0119] As used herein, the term "hybridization conditions" refers to conditions under which two nucleic acids with complementary nucleotide sequences can interact with each other. Hybridization conditions can be highly stringent, moderately stringent, or low stringent, and these different levels of stringency are known. Hybridization conditions that allow for amplification and / or extension are typically selected based on the application of interest.

[0120] As used herein, the term "specific hybridization with a single amplicon or target nucleic acid" means hybridization substantially with one amplicon or target nucleic acid substance and substantially without hybridization with other amplicon or target nucleic acid substances in solution. Specific hybridization excludes mismatches, allowing, for example, the design of oligonucleotides to specifically hybridize with a particular allele, and only with that allele. Oligonucleotides that are uniformly matched or complementary to the allele will hybridize specifically with that allele, whereas hybridization will not occur if one or more base mismatches are present.

[0121] As used herein, the term "hybridization site" refers to a specific location on the amplicon or target nucleic acid of another nucleic acid hybridization. In some embodiments, the end of an oligonucleotide is adjacent to or substantially adjacent to a site on the amplicon or target nucleic acid material that has a sequence different from that of the other amplicon or target nucleic acid material. When there are no nucleotides between the site and the oligonucleotide end, the oligonucleotide end is "adjacent" to the site. In some embodiments, when there are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides between the site and the oligonucleotide end, the oligonucleotide end is "substantially adjacent" to the site.

[0122] Can distinguish between marking and releasing

[0123] As used herein, the terms "distinguishing marker" and "distinguishing tag" refer to the types of markers or tags that can be distinguished from each other and used to identify the nucleic acid to which the tag is attached. The multiplexing methods described herein can select and use a variety of types of markers and tags. For example, oligonucleotides, amino acids, small organic molecules, luminescent molecules, light-absorbing molecules, light-scattering molecules, isotopes, enzymes, etc., can be used as distinguishing markers or tags. In some embodiments, oligonucleotides, amino acids, and / or small organic molecules with different lengths, different mass-to-charge ratios, different electrophoretic mobilities (e.g., capillary electrophoretic mobilities), and / or different masses can also be used as distinguishing markers or tags. Therefore, fluorophores, radioisotopes, colorimetric agents, luminescent agents, chemiluminescent agents, light-scattering agents, etc., can be used as markers. The choice of marker depends on the required sensitivity, ease of coupling with the nucleic acid, stability requirements, and available instruments. The term "distinguishing feature" used herein with respect to distinguishing markers and tags refers to any feature of a marker or tag that can be distinguished from another marker or tag (e.g., the mass and others described herein). In some implementations, a marker composition that distinguishes between markers and labels can be selected and / or designed to produce optimal flight behavior in the mass spectrometer and allow for differentiation between markers and labels at a high level of multiplexing.

[0124] For the methods used herein, specific target (major or minor) nucleic acid substances, amplicon substances, and / or extended oligonucleotide substances are typically paired with distinguishable detectable markers, such that the detection of a specific marker or tag directly identifies and / or quantifies the presence of a specific target minor or nucleic acid substance, amplicon substance, and / or extended oligonucleotide substance in a specific composition. Therefore, a distinguishable feature of a marker, for example, can be used to identify a target nucleic acid substance in a composition because that specific distinguishable feature corresponds to a specific target nucleic acid. Markers and tags can be attached to nucleic acids (e.g., oligonucleotides) by any known method and at any location (e.g., at the 5′ of an oligonucleotide). Therefore, as used herein, for each specific marker, for each specific target nucleic acid substance, "specific correspondence" means a marker that pairs with a target substance. When the presence of a marker is detected, the presence of the target nucleic acid substance bound to that marker is detected and / or, in some embodiments, quantified.

[0125] The term "species" used in this document to refer to distinguishable labels or markings (collectively, "markers") means a mark that is detectably distinguishable from another mark. In some embodiments, the number of marking substances includes, but is not limited to, about 2 to about 10,000 marking substances, about 2 to about 500,000 marking substances, about 2 to about 100,000, about 2 to about 50,000, about 2 to about 10,000, and about 2 to about 500 marking substances, or sometimes about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 80, 85, 90, 95, 100, 125, 150 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, or 500000 types of labeled substances.

[0126] As used herein, the term "quality-distinguished marker" refers to a marker distinguishable by quality. A variety of quality-distinguished markers can be selected and used, such as complexes, amino acids, and / or polynucleotides. Different lengths and / or compositions of nucleotide strings (e.g., nucleic acids, complexes), amino acid strings (e.g., peptides, polypeptides, complexes), and / or polynucleotides can be distinguished by quality and used as markers. Any number of units can be used in quality-distinguished markers, and the upper and lower limits of these units depend in part on the resolution and quality window of the system used to detect and distinguish these markers. Therefore, the length and composition of quality-distinguished markers can be selected in part based on the resolution and quality window of the detector used to detect and distinguish the markers.

[0127] As used herein, the term "complex" refers to a set of monomeric units, not a specific order of those units. For nucleic acids, the term "complex" refers to a base composition of nucleic acids where bases are monomeric units. The number of each type of base can be determined by B... n Indicates (i.e., A) a C c G g T t Where A0C0G0T0 represents an “empty” complex or a complex without bases. A natural complex is a complex in which all component monomer units (e.g., bases for nucleic acids and amino acids for polypeptides) are greater than or equal to zero. In some embodiments, at least one of A, C, G, or T is equal to 1 or greater (e.g., A0C0G1T0, A1C0G1T0, A2C1G1T2, A3C2G1T5). To compare sequences to determine sequence variations, in the methods provided herein, “non-natural” complexes containing negative monomer units can be generated by an algorithm used to process the data. For polypeptides, a complex refers to the amino acid composition of a polypeptide fragment, where similarly, the number of each type of amino acid is represented. A complex substance may correspond to multiple sequences. For example, polymer A2G3 corresponds to the sequences AGGAG, GGGAA, AAGGG, GGAGA, etc. Typically, there is a unique complex corresponding to a sequence, but more than one sequence may correspond to the same complex. In some embodiments, a complex substance pairs with (e.g., corresponding to) a target nucleic acid substance, an amplicon substance, and / or an oligonucleotide substance. In the embodiments described herein (e.g., A0C0G5T0 and A0C5G0T0 are different and mass-distinguished complex substances), the different complex substances have different base compositions and distinguishable masses. In some embodiments, a group of complex substances has different base compositions and the same length. In some embodiments, a group of complex substances has both different base compositions and lengths.

[0128] Nucleotide complexes used as quality-distinguishing markers can have any length, wherein all complex substances are detectably distinguishable, for example, lengths of about 1 to 15, 5 to 20, 1 to 30, 5 to 35, 10 to 30, 15 to 30, 20 to 35, 25 to 35, 30 to 40, 35 to 45, 40 to 50, or 25 to 50, or sometimes about 55, 60, 65, 70, 75, 80, 85, 90, 85, or 100 nucleotides. Peptide or polypeptide complexes used as quality-distinguishing markers can have any length, wherein all complex substances are detectably distinguishable, for example, lengths of about 1 to 20, 10 to 30, 20 to 40, 30 to 50, 40 to 60, 50 to 70, 60 to 80, 70 to 90, or 80 to 100 amino acids. As mentioned above, the limitation on the number of units in a complex is usually limited by the resolution and quality window of the detection methods used to distinguish multicomponent substances.

[0129] The terms "multiplex" and "tandem complex" are used synonymously herein (collectively referred to as "multiplex") and refer to a molecule comprising two or more units connected to each other (e.g., typically continuous; sometimes branched in some embodiments). In some embodiments, multiplexes are sometimes nucleic acids and / or artificial polymers. In some embodiments, multiplexes may comprise units of the same type (e.g., homomultiplexes), and sometimes multiplexes may comprise units of different types (e.g., heteromultiplexes). Multiplexes may comprise any type of unit, including nucleotide units, amino acid units, small organic molecular units (e.g., triphenylmethyl), specific nucleotide sequence units, specific amino acid sequence units, etc. In one embodiment, the homomultiplex of three specific sequence units ABC is ABCABCABC. Multiplexes may comprise any number of units, as long as each multiplex can be distinguished from other substances. For example, in some embodiments, the triphenylmethyl polymer may comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, or 1000 triphenylmethyl units.

[0130] In some embodiments, a distinguishable tag can be released from a nucleic acid product (e.g., an extended oligonucleotide). The link between the distinguishable tag and the nucleic acid can be transcribed and cleaved, allowing detection of one or more released tags to identify and / or quantify any type of nucleic acid product (e.g., U.S. Patent Application Publication No. US20050287533A1, entitled “Target-Specific Complex and Method of Use” by Ehrich et al.). Methods for such linking and cleaving (“cleavage conditions”) are known. In some embodiments, the tag can be separated from other parts of the molecule to which it is linked. In some embodiments, the tag (e.g., a polymer) is cleaved from a larger nucleotide string (e.g., an extended oligonucleotide). Non-limiting examples of links include links that can be cleaved by nucleases (e.g., ribonucleases, endonucleases); links that can be cleaved by chemicals; links that can be cleaved by physical treatment; and photocleavable linkers that can be photocleaved (e.g., o-nitrobenzyl, 6-nitrovinylaminooxycarbonyl, 2-nitrobenzyl). When using emission light detection systems (e.g., matrix-assisted laser desorption / ionization (MALDI) mass spectrometry involving laser emission light), optically cleavable connectors offer advantages because cleaving and detection are combined and performed in a single step.

[0131] In some embodiments, the marker may be part of a larger unit and can be separated from that unit prior to detection. For example, in some embodiments, the marker is a group of consecutive nucleotides in a larger nucleotide sequence, and the marker is cleaved from the larger nucleotide sequence. In such embodiments, the marker is typically located at an end of the nucleotide sequence or nucleic acid in which it resides. In some embodiments, the marker or its precursor is located in a transcription cassette, which includes a promoter sequence operatively linked to a precursor sequence encoding the marker. In a later embodiment, the promoter is sometimes an RNA polymerase recruitment promoter that produces RNA containing or composed of the marker. The marker-containing RNA can be cleaved prior to detection to release the marker (e.g., with RNase).

[0132] In some embodiments, the distinguishable marker or tag is not cleaved from the extended oligonucleotide, and in some embodiments, the distinguishable marker or tag contains a trapping agent. In some embodiments, detecting the distinguishable feature includes detecting the presence or absence of the extended oligonucleotide, and in some embodiments, the extended oligonucleotide contains a trapping agent.

[0133] Detection and multiplication degree

[0134] As used herein, the term “detection” refers to the identification of labeled substances. Any suitable detection device can be used to distinguish labeled substances in a sample. Detection devices suitable for detecting a large number of distinguishable labels include, but are not limited to, certain mass spectrometry and gel electrophoresis devices. Examples of mass spectrometry formats include, but are not limited to, matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), MALDI orthogonal TOF MS (OTOF MS; two-dimensional), laser desorption / ionization mass spectrometry (LDMS), electrospray (ES) MS, ion cyclotron resonance (ICR) MS, and Fourier transform MS. The methods described herein can be readily applied to mass spectrometry formats in which the analyte is volatilized and ionized (“ionized MS”, e.g., MALDI-TOF MS, LDMS, ESMS, linear TOF, OTOF). Orthogonal ion extraction MALDI-TOF and axial MALDI-TOF can produce relatively high resolution, and thus relatively high levels of multiplexing. Detection devices suitable for detecting luminescent, light-absorbing, and / or light-scattering labels include, but are not limited to, certain photodetectors and optical detectors (e.g., for fluorescent, chemiluminescent, absorption, and / or light-scattering labels).

[0135] The methods provided herein allow for high-throughput detection or discovery of multiple minor nucleic acid substances present in samples or combinations of samples containing one or more major nucleic acid substances. Multiplexing offers the advantage of identifying multiple minor nucleic acid substances (e.g., some with different sequence variations) in as few as a single mass spectrometer or other detection system (such as fluorescence or electrical signals), compared to individual minor nucleic acid substances requiring separate mass spectrometry or other analyses. In some embodiments, the methods provided herein are suitable for high-throughput, highly automated methods for analyzing sequence variations with high speed and accuracy. In some embodiments, the methods provided herein can be multiplexed at a high level in a single reaction. Multiplexing can be applied when the genotype at the polymorphic locus is unknown, and in some embodiments, when the genotype at the locus is known.

[0136] In some implementations, the number of multiplexed target (minor and / or major) nucleic acid substances includes, but is not limited to, about 2-1000, and sometimes about 1-3, 3-5, 5-7, 7-9, 9-11, 11-13, 13-15, 15-17, 17-19, 19-21, 21-23, 23-25, 25-27, 27-29, 29-31, 31-33, 33-35, 35-37, 37-39, 39-41, 41-43, 43-45, 45-47, 47-49, 49-51, 51-53, 53-55, 55-57, 57-59, 59-61, 61-63, 63-65, 65-67, 67-69, 69-71. 71-73, 73-75, 75-77, 77-79, 79-81, 81-83, 83-85, 85-87, 87-89, 89-91, 91-93, 93-95, 95-97, 97-101, 101-103, 103-105, 105-107, 107-109, 109-111 111-113, 113-115, 115-117, 117-119, 121-123, 123-125, 125-127, 127-129, 129-131, 131-133, 133-135, 135-137, 137-139, 139-141, 141-143, 143- 145, 145-147, 147-149, 149-151, 151-153, 153-155, 155-157, 157-159, 159-161, 161-163, 163-165, 165-167, 167-169, 169-171, 171-173, 173-175, 1 75-177, 177-179, 179-181, 181-183, 183-185, 185-187, 187-189, 189-191, 191-193, 193-195, 195-197, 197-199, 199-201, 201-203, 203-205, 205-20 7, 207-209, 209-211, 211-213, 213-215, 215-217, 217-219, 219-221, 221-223, 223-225, 225-227, 227-229, 229-231, 231-233, 233-235, 235-237, 237 -239, 239-241, 241-243, 243-245, 245-247, 247-249, 249-251, 251-253, 253-255, 255-257, 257-259, 259-261, 261-263, 263-265, 265-267, 267-269269-271, 271-273, 273-275, 275-277, 277-279, 279-281, 281-283, 283-285, 285-287, 287-289, 289-291, 291-293, 293-295, 295-297, 297-299, 299-301, 301-303, 303-305, 305-307, 307-309, 309-311, 311-313, 313-315, 315-317, 317-319, 319-321, 321-323, 323-325, 325-3 27, 327-329, 329-331, 331-333, 333-335, 335-337, 337-339, 339-341, 341-343, 343-345, 345-347, 347-349, 349-351, 351-353, 353-355, 355-357, 357-359, 359-361, 361-363, 363-365, 365-367, 367-369, 369-371, 371-373, 373-375, 375-377, 377-379, 379-381, 381-383, 383 -385, 385-387, 387-389, 389-391, 391-393, 393-395, 395-397, 397-401, 401-403, 403-405, 405-407, 407-409, 409-411, 411-413, 413-415, 415-417, 417-419, 419-421, 421-423, 423-425, 425-427, 427-429, 429-431, 431-433, 433-435, 435-437, 437-439, 439-441, 441-443, 443-445, 445-447, 447-449, 449-451, 451-453, 453-455, 455-457, 457-459, 459-461, 461-463, 463-465, 465-467, 467-469, 469-471, 471-473, 473-475, 475-477, 477-479, 479-481, 481-483, 483-485, 485-487, 487-489, 489-491, 491-493, 493-495, 495-497, 497-501 kinds of substances or more.

[0137] The design methods for resolving mass spectrometry through multiplexing experiments can include primer and oligonucleotide design methods, relative concentrations of reagents such as chain terminators, selection of detection markers, and other reaction design methods. Regarding primer and oligonucleotide design in multi-pathway experiments, the same general primer design guidelines are used for single-pathway reactions, such as avoiding spurious initiations and primer dimers; only the multi-pathway reactions involve more primers. Furthermore, for mass spectrometry analysis, the analyte peak in a given mass spectrum is fully resolved from the products of any experiment, which is a multi-pathway experiment including a pausing peak and any other byproduct peaks. Additionally, the analyte peak ideally falls within a user-specified mass window, for example, in the range of 5,000–8,500 Da. In some embodiments, extended oligonucleotides can be designed relative to the target sequence of a given SNP chain. In such embodiments, the length is typically within, for example, user-specified limits (e.g., 17 to 24 bases or 17–26 bases) and typically does not include indeterminate bases in the target sequence. Hybridization intensity is sometimes determined by calculating the sequence-dependent melting (or hybridization / dissociation) temperature T. m To measure. Due to their hairpin potential, pseudo-initiation potential, primer dimer potential, low-complexity regions, and problematic subsequences (such as GGGG), certain primer choices may be unacceptable or disadvantageous relative to other primer choices. Methods and software for designing extending oligonucleotides (e.g., according to these standards) are known and include, for example, SpectroDESIGNER (Sequenom).

[0138] As used herein, the term "detection rate" or "judgment rate" refers to the number of detections obtained relative to the number of attempts to obtain (e.g., identified genotypes or mutants). In other words, for a 12-fold reaction, if 10 genotypes are ultimately determined by the methods provided herein, 10 detections have been obtained, and the detection rate is 10 / 12. Different events can cause a particular attempt to fail, resulting in a detection rate below 100%. Occasionally, in cases where a mixture of dNTPs and ddNTPs is used for termination, for example, after the introduction of a non-terminating nucleotide (i.e., dNTP), unsuitable extension products can occur by pausing the polymerase. The quality difference between such incorrect termination and correct termination primer extension reactions at polymorphic sites is sometimes too small to be consistently resolved and can lead to misjudgments if an inappropriate termination mixture is used. The quality difference between correct and incorrect termination (i.e., caused by pausing) and between correct termination and salt adducts and correct termination and nonspecific incorporation are generally maximized to reduce the number of misjudgments.

[0139] The accuracy of a multiplex test can be determined by evaluating the number of detections obtained (e.g., correct or accurate assessment) and / or the number of false positive and / or false negative events in one or more tests. Accuracy can also be assessed by comparing the accuracy of the corresponding single test for the target being assessed in each multiplex test. In some embodiments, one or more methods can be used to determine the decision rate. For example, a manual method can be used in combination with automated or computerized methods for making detections, and in some embodiments, the detection rates of each method can be summed to calculate the overall detection rate. In some embodiments, when multiplexing two or more target nucleic acids (minor and / or major nucleic acid substances) (e.g., 50 or more target nucleic acids), the accuracy of the detection rate can be about 99% or greater, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 87-88%, 85-86%, 83-84%, 81-82%, 80%, 78%-79%, or 76-77%. In some implementations, the detection rate of each target substance in a multiplexing assay comprising about 2 to 200 target substances is greater than or equal to 80% or more (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more).

[0140] In some implementations, the error rate may be determined based on the detection rate or accuracy rate. For example, the error rate might be the number of false detections. In some implementations, for example, the error rate may be less than 100% of the detection rate or accuracy rate. The error rate may also be referred to as the "failure rate." Identifying false positives and / or false negatives can readjust the detection rate and error rate. In some implementations, running more tests can also help identify false positives and / or false negatives, thereby adjusting the detection rate and / or error rate. In some implementations, when multiplexing two or more target nucleic acids as primary and / or secondary nucleic acid substances (e.g., fifty or more target nucleic acid substances), the error rate may be about 1% or less, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%.

[0141] application

[0142] The following are examples of non-restrictive applications of the reuse technology described in this article.

[0143] 1. Detect sequence variations (e.g., genetic variants).

[0144] Improved methods are provided for identifying the genomic basis of diseases and their biomarkers. Candidates of sequence variations (e.g., genetic variants) that can be identified by the methods provided herein include sequences containing sequence variations as polymorphisms. Polymorphisms include naturally occurring, somatic sequence variations, and variations caused by mutations. Polymorphisms include, but are not limited to: sequence microvariants in which one or more nucleotides in a local region vary from individual to individual; insertions and deletions ranging in size from one nucleotide to millions of bases; and microsatellites or nucleotide repeats with varying numbers of repeats. Nucleotide repeat sequences include homologous repeat sequences, such as dinucleotide, trinucleotide, tetranucleotide, or larger repeat sequences, identical sequences repeated multiple times, and heterologous repeat sequences where sequence motif repetitions are found. For a given locus, the number of nucleotide repeats can vary from individual to individual.

[0145] Polymorphic markers or sites are differentially expressed gene loci. Such sites can be as small as a single base pair (SNP). Polymorphic markers include, but are not limited to, restriction fragment length polymorphisms (RFLPs), variable-number tandem repeats (VNTRs), hypervariable regions, microsatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats and other repeat patterns, simple sequence repeats, and insertion elements such as Alu. Polymorphism also manifests as different Mendelian alleles in genes. Polymorphism can be observed through differences in protein expression, protein modifications, RNA expression modifications, DNA and RNA methylation, alterations in regulators of gene expression and DNA replication, and any other alterations in genomic or organelle nucleic acids.

[0146] Furthermore, many genes possess polymorphic regions. Since an individual possesses any of several allelic variants of a polymorphic region, individuals can be identified based on the type of allelic variant of that polymorphic region. This can be used, for example, for forensic purposes. In other cases, understanding the types of allelic variants an individual possesses is crucial. For example, allelic differences in certain genes, such as the major histocompatibility complex (MHC) gene, are involved in graft rejection or graft-versus-host disease during bone marrow transport. Therefore, there is a strong need to develop rapid, sensitive, and accurate methods for determining the types of allelic variants of polymorphic regions of genes or genetic disorders. The methods or kits provided herein can be used to genotype an individual by determining the types of one or more allelic variants of one or more polymorphic regions in one or more genes or chromosomes. Genotyping an individual using the methods provided herein can be used for forensic or species testing purposes, and the polymorphic region may be present in mitochondrial genes or may be a short tandem repeat.

[0147] Single nucleotide polymorphisms (SNPs) are typically diploid systems, meaning an individual can possess two alleles for any given marker. This implies that each SNP marker has relatively low information content compared to microsatellite markers, and may have more than 10 alleles. SNPs also tend to be highly population-specific; a marker that is polymorphic in one population may not be highly polymorphic in another. SNPs found at approximately 1,000 bases per kilobase (see Wang et al., (1998) Science 280: 1077-1082) offer the potential to generate very high-density genetic maps, which is very useful for developing haplotype systems for genes or regions of interest, and due to the nature of SNPs, they may actually be polymorphisms associated with the phenotype of the disease being studied. The low mutation rate of SNPs also makes them good markers for studying complex genetic traits.

[0148] A significant focus of genomics is the identification of SNPs, which is important for a variety of reasons. They allow for both indirect testing (haploid association) and direct testing (functional variants). They are among the most abundant and stable genetic markers. Common diseases are best explained by common genetic alterations, and natural variations in populations help in understanding the interactions between disease, treatment, and the environment.

[0149] Sensitive detection of somatic mutations is particularly valuable to the cancer research community, which is interested in identifying the genetic determinants of tumor initiation and proliferation. Information obtained from sensitive methods can also be used to analyze mutations to predict patient outcomes and inform relevant treatment options. In some implementations, sensitive detection methods are required that can detect genetic variants less than or equal to 5% of their corresponding wild-type sequence. In some implementations, methods capable of detecting less than or equal to 1% of the wild-type variant are implemented. In some implementations, methods can be implemented to detect less than or equal to 5%, 4%, 3%, 2%, 1%, 0.8%, 0.75%, 0.5%, 0.1%, 0.05%, or 0.01% of the wild-type variant. Furthermore, in prenatal diagnosis, this type of method can elucidate paternally derived mutations in utero.

[0150] In some embodiments, allelic analysis can be performed by generating extended oligonucleotides from nucleic acid targets carrying one or more somatic mutations of interest (e.g., SNPs, disease markers, etc., and combinations thereof). In some embodiments, detecting the presence or absence of extended oligonucleotides representing the release of alleles carrying somatic mutations can be used as a rapid method for screening the presence or absence of specific mutations in a target population. In some embodiments involving the generation of extended oligonucleotides from mutated alleles, the extended oligonucleotides can be detected when the appropriate mutated allele generates an extended oligonucleotide product.

[0151] 2. Identification of disease markers

[0152] This article provides a method for the rapid and accurate identification of sequence variations as genetic markers of disease, which can be used to diagnose or determine the prognosis of disease. Diseases characterized by genetic markers can include, but are not limited to, atherosclerosis, obesity, diabetes, autoimmune diseases, and cancer. All diseases in organisms have a genetic component, whether inherited or generated by the body's response to environmental stresses, such as viruses and toxins. The ultimate goal of ongoing genomic research is to use this information to develop new methods for identifying, treating, and potentially curing these diseases. The first step is to screen diseased tissues and identify genomic variations at the individual sample level. The identification of these “disease” markers depends on the ability to detect genomic marker variations to identify erroneous genes or sequence variants. Genomic markers (all genetic loci, including single nucleotide polymorphisms (SNPs), microsatellites and other non-coding genomic regions, tandem repeat sequences, introns, and exons) can be used to identify all organisms, including humans. These markers provide a method that not only identifies populations but also allows for population stratification based on their responses to factors such as disease, drug treatment, and resistance to environmental factors. In some embodiments, the disease marker is sometimes a mutation, and may be a relatively rare allele, such as a somatic mutation against a wild-type allele background (e.g., cancerous tissue versus normal tissue, mutant viral type versus normal viral type (e.g., HIV)). In some embodiments, a rare allele or mutation represents less than 5%, 4%, 3%, 2%, 1%, 0.8%, 0.75%, 0.5%, 0.1%, 0.05%, or 0.01% of the wild type. In some embodiments, a rare allele or mutation may represent less than 1% of the wild type.

[0153] 3. Microbial identification

[0154] This document provides procedures or methods for identifying the genus, species, strain, clone, or subtype of microorganisms and viruses. Microorganisms and viruses are selected from a variety of organisms, including but not limited to bacteria, fungi, protozoa, ciliates, and viruses. Microorganisms are not limited to a specific genus, species, strain, subtype, or serotype or any other classification. Microorganisms and viruses can be identified by determining sequence variations in the target microorganism sequence relative to one or more reference sequences or samples. Reference sequences can be obtained, for example, from the same or different genera, species, strains, or serotypes or any other classification, or from other microorganisms from a host prokaryotic or eukaryotic organism or any mixed population.

[0155] The identification and typing of pathogens (e.g., bacteria or viruses) are crucial in the clinical management of infectious diseases. Precise identification of the microorganism is not only used to differentiate disease states from healthy states, but also forms the basis for determining the source and spread of infection, and whether antibiotics or other antimicrobial therapies are most appropriate. Furthermore, it allows for monitoring of treatment. Traditional pathogen typing methods use a variety of phenotypic characteristics, including growth characteristics, color, cell or colony morphology, antibiotic sensitivity, staining, odor, serotyping, biochemical typing, and reactivity with specific antibodies, to identify microorganisms (e.g., bacteria). All of these methods require the culture of suspected pathogens, which has several significant drawbacks, including high material and labor costs, risks of worker exposure, false positives due to mishandling, and false negatives due to low numbers of viable cells, or the stringent culture requirements for many pathogens. Moreover, culture methods require considerable time to achieve diagnosis, and due to the potentially life-threatening nature of such infections, antimicrobial therapy is often initiated before results are obtained. Some organisms cannot be maintained in cultures or exhibit very slow growth rates (e.g., Mycobacterium tuberculosis takes 6–8 weeks).

[0156] In many cases, pathogens exist in small quantities and / or are very similar to organisms that make up the normal flora, and may not be distinguishable from harmless strains by the methods described above. In these cases, determining the presence of pathogenic strains may require the higher resolution provided by the molecular typing methods presented herein.

[0157] 4. Detect the presence of viral or bacterial nucleic acid sequences indicating infection.

[0158] The method described herein can be used to determine the presence of a viral or bacterial nucleic acid sequence indicating infection by identifying sequence variations present in the viral or bacterial nucleic acid sequence relative to one or more reference sequences. Reference sequences may include, but are not limited to, sequences obtained from an infectious organism, an associated non-infectious organism, or a sequence derived from a host organism.

[0159] Viruses, bacteria, fungi, and other infectious organisms contain different nucleic acid sequences, including sequence variants, that differ from those contained in host cells. Target DNA sequences can be part of foreign genetic sequences, such as the genome of invading microorganisms, including, for example, bacteria and their bacteriophages, viruses, fungi, protozoa, etc. The methods presented herein are particularly suitable for distinguishing different variants or strains of microorganisms (e.g., pathogenicity, less pathogenicity, drug resistance, and non-resistance, etc.) in order to, for example, select appropriate therapeutic interventions.Examples of pathogenic viruses that infect humans and animals and can be detected by publicly available methods include, but are not limited to, retroviridae (e.g., human immunodeficiency viruses such as HIV-1 (also known as HTLV-III, LAV, or HTLV-III / LAV; Ratner et al., Nature, 313:227-284 (1985); Wain Hobson et al., Cell, 40:9-17 (1985), HIV-2 (Guyader et al., Nature, 328:662-669 (1987); European Patent Publication No. 0269 520; Chakrabarti et al., Nature, 328:543-547 (1987); European Patent Application No. 0 655 501) and other isolates such as HIV-LP (International Publication No. WO 94 / 00562); Picornaviridae (e.g., poliovirus, hepatitis A virus (Gust et al., Intervirology, 20:1-7 (1983)); Enteroviruses, human Coxsackievirus, rhinovirus, Echovirus); Caliciviridae (e.g., strains causing gastroenteritis); Clonorviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bucciaviridae (e.g., Hantavirus, Burgavirus, sandfly virus, and Nairobi virus); Azopsis Viriviruses (hemorrhagic fever viruses); Reoviridae (e.g., reovirus, rotavirus, and rotavirus); Dinucleotide Viridae; Hepatitis Viridae (hepatitis B virus); Parvoviridae (parvovirus); Parvoviridae (most adenoviruses); Papillomaviruses (papillomavirus, polyomavirus); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus type 1 (HSV-1) and HSV-2, varicella-zoster virus, cytomegalovirus, herpesviruses); Poxviridae (smallpox virus, vaccinia virus, poxvirus); Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., pathogens of spongiform encephalopathy, vectors of delta hepatitis (considered a defective satellite of hepatitis B virus), vectors of non-A, non-B hepatitis (Category 1 = internal transmission; Category 2 = external transmission, i.e., hepatitis C); Norwalk and related viruses, and astroviruses.

[0160] Examples of infectious bacteria include, but are not limited to, Helicobacter pylori, Borrelia burgdorferi, Legionella pneumophila, and mycobacteria (e.g., Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, and Mycobacterium gordonii).gordonae), Salmonella, Staphylococcus aureus, Neisseriagonorrheae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A), Streptococcus agalactiae (Group B), Streptococcus spp. (green streptococci), Streptococcus faecalis, Streptococcus bovis, Streptococcus spp. (anaerobic species), Streptococcus pneumoniae, Campylobacter spp., Enterococcus spp., Haemophilus influenzae, Bacillus anthracis Corynebacterium diphtheriae, Corynebacterium spp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides spp., Facterobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira spp., and Actinomyces israelli, as well as any variants, including antibiotic-resistant variants.

[0161] Examples of infectious fungi include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans. Other infectious organisms include protozoa such as Plasmodium falciparum and Toxoplasma gondii.

[0162] 5. Overview of Antibiotics

[0163] The methods presented in this paper can improve the speed and accuracy of detecting nucleotide changes involved in drug resistance, including antibiotic resistance. Genotypes involved in resistance to isoniazid, rifampin, streptomycin, fluoroquinolones, and ethionamide have been identified [Heym et al., Lancet 344:293 (1994) and Morris et al., J. Infect. Dis. 171:954 (1995)]. Combinations of isoniazid (inh) and rifampin (rif), along with pyrazinamide and ethambutol or streptomycin, are routinely used as the first-line attack for confirmed cases of Mycobacterium tuberculosis [Banerjee et al., Science 263:227 (1994)]. The increasing emergence of such resistant strains necessitates the development of rapid assays to detect them, thereby reducing the cost and community health hazards associated with following ineffective and potentially harmful treatments. Identification of several genotypes involved in drug resistance facilitates rapid screening for nucleotide changes leading to resistance using mutation detection techniques. Furthermore, this technique aids in treatment monitoring and tracking of microbial community structure, as well as surveillance monitoring during treatment. In addition, correlation and monitoring of mixed populations can be conducted.

[0164] 6. Unit-based classification

[0165] The method presented in this article can be used to detect haplotyping. In any diploid cell, on any gene or other chromosomal segment, there are two haplotyps, each containing at least one distinct difference. In many well-studied genetic systems, haplotyping is more strongly associated with phenotypes than single nucleotide variants. Therefore, haplotyping is valuable for understanding the genetic basis of various phenotypes, including disease predisposition or susceptibility, responses to therapeutic interventions, and other phenotypes of interest in medicine, animal husbandry, and agriculture.

[0166] The haplotype approach presented in this paper allows selection of a portion of a sequence from one of an individual's two homologous chromosomes, and selection of genotype-linked SNPs on that sequence portion. Direct haplotype resolution can yield increased information content, improving the diagnosis of any relevant disease genes or the identification of associations with those diseases.

[0167] 7. Microsatellites

[0168] The method presented in this paper allows for rapid and definitive detection of microsatellite sequence variations. Microsatellites (sometimes called variable-number tandem repeats or VNTRs) are short tandem repeat nucleotide units of 1 to 7 or more bases, with dinucleotide, trinucleotide, and tetranucleotide repeats being the most prominent. Microsatellites are present in every 100,000 bp of genomic DNA (JL Weber and PE Can, Am. J. Hum. Genet. 44, 388 (1989); J. Weissenbach et al., Nature 359, 794 (1992)). For example, CA dinucleotide repeats account for approximately 0.5% of the human extramitochondrial genome; CT and AG repeats together account for approximately 0.2%. CG repeats are rare and are most likely due to regulatory functions of CpG islands. Microsatellites are highly polymorphic relative to their length, widely distributed throughout the genome, primarily enriched in noncoding sequences, and their functions within the genome are unknown. Microsatellites may be important in forensic applications because a population will maintain a variety of microsatellites that are unique to that population and will be different from other mismatched populations.

[0169] Many changes within microsatellites may be silent, but some can lead to significant alterations in gene products or expression levels. For example, in some tumors, trinucleotide repeat sequences found in gene coding regions are affected (CT Caskey et al., Science 256, 784 (1992)), and microsatellite alterations can lead to genetic instability that contributes to cancer predisposition (PJ McKinnen, Hum. Genet. 175, 1997 (1987); J. German et al., Clin. Genet. 35, 57 (1989)).

[0170] 8. Short series repetition

[0171] The methods presented herein can be used to identify short tandem repeat (STR) regions in certain target sequences of the human genome relative to, for example, reference sequences in the human genome that do not contain STR regions. STR regions are polymorphic regions unrelated to any disease or condition. Many loci in the human genome contain polymorphic short tandem repeat (STR) regions. STR loci contain short repeat sequence elements of 3 to 7 base pairs in length. An estimated 200,000 expected trimeric and tetrameric STRs are frequently present in the human genome, occurring once every 15 kb (see, for example, International PCT application WO 9213969A1, Edwards et al., Nucl. Acids Res. 19:4791 (1991); Beckmann et al., (1992) Genomics 12:627-631). Nearly half of these STR loci are polymorphic, providing a rich source of genetic markers. Variations in the number of repeat units at specific loci were observed in variable nucleotide tandem repeat (VNTR) genes (Nakamura et al., (1987) Science 235: 1616-1622); and microsatellite loci (Jeffreys et al., (1985) Nature 314: 67-73), which contain longer repeat units, and microsatellite or dinucleotide repeat loci (Luty et al., (1991) Nucleic Acids Res. 19: 4308; Litt et al., (1990) Nucleic Acids Res. 18: 4301; Litt et al., (1990) Nucleic Acids Res. 18: 5921; Luty et al., (1990) Am. J. Hum. Genet. 46: 776-783; Tautz (1989) Nucl. Acids Sequence variations (Res. 17: 6463-6471; Weber et al., (1989) Am. J. Hum. Genet. 44: 388-396; Beckmann et al., (1992) Genomics 12: 627-631). VNTR typing is a well-established tool in microbial typing, for example, Mycobacterium tuberculosis (MIRU typing).

[0172] Examples of STR loci include, but are not limited to, the pentanucleotide repeat sequence in the human CD4 locus (Edwards et al., Nucl. Acids Res. 19: 4791 (1991)); the tetranucleotide repeat sequence in the human aromatase cytochrome P-450 gene (CYP19; Polymeropoulos et al., Nucl. Acids Res. 19: 195 (1991)); the tetranucleotide repeat sequence in the human coagulation factor XIIIA subunit gene (F13A1; Polymeropoulos et al., Nucl. Acids Res. 19: 4306 (1991)); the tetranucleotide repeat sequence in the F13B locus (Nishimura et al., Nucl. Acids Res. 20: 1167 (1992)); and the human c-les / fps proto-oncogene (FES; Polymeropoulos et al., Nucl. Acids Res. 20: 1167 (1992)); and the human c-les / fps proto-oncogene (FES; Polymeropoulos et al., Nucl. Acids Res. 20: 1167 (1992)). Tetranucleotide repeat sequences in Res. 19: 4018 (1991); tetranucleotide repeat sequences in the LFL gene (Zuliani et al., Nucl. Acids Res. 18: 4958 (1990)); trinucleotide repeat sequence variations in the human pancreatic phospholipase A-2 gene (PLA2; Polymeropoulos et al., Nucl. Acids Res. 18: 7468 (1990)); tetranucleotide repeat sequence variations in the VWF gene (Ploos et al., Nucl. Acids Res. 18: 4957 (1990)); and tetranucleotide repeat sequences in the human thyroid peroxidase (hTPO) locus (Anker et al., Hum. Mol. Genet. 1: 137 (1992)).

[0173] 9. Biological identification

[0174] STR loci and other polymorphic regions of genes are sequence variations and are extremely useful markers for human identification, paternal and maternal lineage testing, genetic mapping, transplantation and genetic disputes, twin conjugation testing, detection of human inbreeding, quality control of human cultured cells, identification of human remains, and forensic detection of semen samples, bloodstains, microorganisms, and other substances. These loci are also useful markers in commercial animal breeding and pedigree analysis, as well as in commercial plant breeding. Linkage analysis using polymorphic DNA markers can identify economically important traits in plant crops and animals. This article provides efficient and accurate methods for identifying species of such loci.

[0175] 10. Detection of allele variations

[0176] The method presented in this paper allows for high-throughput, rapid, and accurate detection of allelic variants. The study of allelic variants involves not only the detection of specific sequences in complex backgrounds but also the differentiation between sequences with few or even single nucleotide differences. One approach to detecting allelic-specific variants via PCR is based on the fact that Taq polymerase struggles to synthesize DNA strands when a mismatch exists between the template strand and the 3′ end of the primer. Allelic-specific variants can be detected by using primers that perfectly match only one possible allele; mismatches with other alleles prevent primer extension, thus inhibiting the amplification of the sequence. The method presented in this paper is also applicable to association studies, copy number variation, detection of disease markers, and SNP genotyping for typing.

[0177] 11. Determine allele frequencies

[0178] The methods described herein are valuable for identifying one or more genetic markers whose frequency varies within a population as a function of age, ethnicity, sex, or some other criterion. For example, the age-dependent distribution of ApoE genotypes is known in the art (see, for example, Schechter et al., (1994) Nature Genetics 6: 29–32). The frequency of sequence variations known to be associated with disease to some extent can also be used to detect or monitor the progression of disease states. For example, the N291S polymorphism (N291S) in the lipoprotein lipase gene results in a serine substitution of asparagine at amino acid codon 291, leading to decreased levels of high-density lipoprotein cholesterol (HDL-C) associated with an increased risk of atherosclerosis in men, particularly myocardial infarction (see, Reymer et al., (1995) Nature Genetics 10: 28–34). Furthermore, determining changes in allele frequencies can allow for the identification of previously unknown sequence variations and ultimately the identification of genes or pathways involved in disease onset and progression.

[0179] 12. Epigenetics

[0180] The methods provided herein can be used to study changes in a target nucleic acid or protein relative to a reference nucleic acid or protein that are not based on sequence, such as the type of bases or amino acids, which are naturally occurring monomeric units of nucleic acids or proteins. For example, the methods provided herein can be used to identify sequence-independent features such as methylation patterns, the presence of modified bases or amino acids, or differences in higher-order structures between the target molecule and the reference molecule to produce fragments that are cleaved at sequence-independent sites. Epigenetics is the study of genetic information based on differences in gene expression rather than differences in gene sequence. Epigenetic alterations are heritable changes in gene function or higher-order nucleic acid structure that cannot be explained by changes in nucleic acid sequence, occurring during mitosis and / or meiosis. Examples of features subject to epigenetic variation or alteration include, but are not limited to, DNA methylation patterns, histone modifications, and Polycomb-trithorax (Pc-G / tx) protein complexes in animals (see, for example, Bird, A., Genes Dev., 16:6-21 (2002)).

[0181] Epigenetic changes typically (though not always) lead to changes in gene expression, and are typically (though not always) heritable. For example, as discussed further below, changes in methylation patterns are an early event in the development and progression of cancer and other diseases. In many cancers, certain genes are inappropriately switched off or on due to aberrant methylation. The ability of methylation patterns to suppress or activate transcription can be heritable. Pc-G / trx protein complexes, such as methylation, can suppress transcription in a heritable manner. Pc-G / trx polyprotein assemblies target specific regions of the genome, effectively freezing the embryonic gene expression state, whether the gene is active or inactive, and stably propagating that state through development. The ability of Pc-G / trx histoproteins to target and bind to the genome affects only the expression levels of genes contained within the genome, without affecting the nature of the gene products. The methods presented herein can be used with specific cleavage reagents or specific extension reactions that identify changes in the target sequence relative to a reference sequence based on sequence-independent changes, such as epigenetic changes.

[0182] 13. Methylation pattern

[0183] The method presented in this paper can be used to detect sequence variations as epigenetic changes in target sequences, such as changes in methylation patterns within target sequences. Cellular methylation analysis is an emerging research discipline. Covalent addition of methyl groups to cytosine is primarily found on CpG dinucleotides (microsatellites). While the function of CpG islands not located in promoter regions remains to be explored, CpG islands in promoter regions are of particular interest because their methylation status regulates the transcription and expression of related genes. Methylation in promoter regions leads to silencing of gene expression. This silencing is permanent and persists through mitosis. Due to its important role in gene expression, DNA methylation influences developmental processes, imprinting and X chromosome inactivation, as well as tumorigenesis, senescence, and the suppression of parasitic DNA. Methylation is thought to be involved in the carcinogenesis of many broad-spectrum cancers, such as lung, breast, and colon cancers, as well as leukemia. There are also associations between methylation and protein dysfunction (long QT syndrome) or metabolic diseases (transient neonatal diabetes mellitus, type 2 diabetes mellitus).

[0184] Bisulfite treatment of genomic DNA can be used to analyze the location of methylated cytosine residues within the DNA. Bisulfite treatment of nucleic acids deamidates cytosine residues to uracil residues, while methylated cytosine remains unchanged. Therefore, by comparing the sequence of a target nucleic acid that has not been treated with bisulfite with the sequence of a nucleic acid treated with bisulfite using the method presented herein, the degree of methylation in the methylated nucleic acid and the location of cytosine methylation can be inferred.

[0185] Methylation analysis via restriction endonuclease reactions is possible by using restriction enzymes with methylation-specific recognition sites such as HpaII and MSPI. The basic principle is that certain enzymes are blocked by methylated cytosine in the recognition sequence. Once this differentiation is complete, the resulting fragments can be further analyzed using the methods presented in this paper.

[0186] These methods can be coupled with bisulfite restriction assay (COBRA). Bisulfite treatment causes the loss of the BstUI recognition site in amplified PCR products, resulting in the appearance of novel detectable fragments in the analysis compared to untreated samples. The methods presented in this article can be combined with specific cleavage of methylation sites to provide rapid and reliable information on methylation patterns in target nucleic acid sequences.

[0187] 14. Resequencing

[0188] The significant increase in the amount of available genomic sequence information from a wide variety of organisms has increased the need for technologies that allow large-scale comparative sequence analysis to correlate sequence information with function, phenotype, or species. The applications of these technologies for comparative sequence analysis can be broad, including SNP discovery and sequence-specific identification of pathogens. Therefore, resequencing and high-throughput mutation screening technologies are crucial for identifying mutations behind diseases and the genetic variability that may lead to differential drug responses.

[0189] To meet these needs, several methods have been developed. Current technologies for high-throughput DNA sequencing include DNA sequencers using electrophoresis and laser-induced fluorescence detection. Electrophoresis-based sequencing methods have inherent limitations in detecting heterozygotes and are affected by GC compression. Therefore, DNA sequencing platforms that generate digital data without using electrophoresis can overcome these problems. Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) measures nucleic acid fragments and outputs digital data. The method presented in this paper allows for high-throughput, high-speed, and high-precision detection of sequence identity and sequence variations relative to a reference sequence. This method enables the routine use of MALDI-TOF MS sequencing for accurate mutation detection, such as screening for founder mutations in BRCA1 and BRCA2 associated with breast cancer development.

[0190] 15. Disease outbreak surveillance

[0191] In an era of global transportation and travel, close monitoring of pathogen outbreaks is essential to prevent their global spread and achieve control. DNA-based genotyping using high-throughput technologies enables rapid sample throughput in relatively short timeframes, as required by outbreak scenarios (e.g., surveillance in hospital settings, early warning systems). Monitoring relies on the regions of microbial markers used but can conveniently monitor genus, species, strain, or subtype-specific levels. These methods can be used for biocontrol, clinical and drug surveillance, and metagenomic applications (e.g., gut microbiota analysis). Such monitoring of treatment progression or failure is described in U.S. Patent Nos. 7,255,992, 7,217,510, 7,226,739, and 7,108,974, which are incorporated herein by reference.

[0192] 16. Vaccine quality control and production cloning quality control

[0193] The methods provided in this article can be used to control the types of recombinant production clones (not limited to vaccines), which can be vaccines or, for example, insulin or any other production clone or biological or medical product.

[0194] 17. Microbial monitoring for production control and quality in pharmaceuticals

[0195] The methods described herein can be used to control the quality of pharmaceutical products, for example, by detecting the presence or absence of certain microbial target nucleic acids in these products.

[0196] Reagent test kit

[0197] In some embodiments, a kit is provided for carrying out the methods described herein. The kit often comprises one or more containers containing one or more of the components described herein. The kit contains one or more components in any number of individual containers, pouches, tubes, vials, multiwell plates, etc., or the components may be combined in different combinations within such containers. For example, one or more of the following components may be included in the kit: (i) one or more nucleotides (e.g., terminated nucleotides and / or non-terminated nucleotides); wherein one or more may include detection markers; (ii) one or more nucleotides containing a capture agent; (iii) one or more oligonucleotides, wherein one or more may include detection markers (e.g., amplification primers, one or more extension primers (UEP), tagged oligonucleotides, oligonucleotides containing a capture agent); (iv) one or more enzymes (e.g., polymerases, endonucleases, restriction enzymes, etc.); (v) control components (e.g., control genomic DNA, primers, synthesis templates, target nucleic acids, etc.); (vi) one or more buffers; and (vii) printing materials (e.g., instructions, labels, etc.). In the implementation of the kit, the relative amount of the terminating nucleotide is present in the solution or in a relative amount such that, when dissolved according to the provided instructions, the concentration of the chain-terminating nucleotide specific to the major nucleic acid substance (C[WT]) is less than the concentration of the chain-terminating nucleotide specific to the minor nucleic acid substance (C[Mut]). In embodiments, the concentration C[WT] of the chain terminator specific to the major nucleic acid substance is less than 20% of the concentration (C[Mut]) of the chain terminator specific to the minor nucleic acid substance. C[WT] is typically from about 0.5% to less than about 20% of C[Mut], from about 0.5% to less than about 15% of C[Mut], from about 1% to about 15% of C[Mut], from about 1% to about 10% of C[Mut], from about 2% to about 10% of C[Mut], or from about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of C[Mut]. In some embodiments, C[WT] is about 0.1% to about 10% of C[Mut], about 0.01% to about 10% of C[Mut], or about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of C[Mut].

[0198] The kit is sometimes used in conjunction with a treatment and may contain instructions for performing one or more methods and / or instructions for performing one or more compositions. The kit can be used to perform the methods described herein. The instructions and / or instructions may be in tangible form (e.g., paper) or electronic form (e.g., computer-readable documents on tangible media such as a compressed disk) and may be included in a kit insert. The kit may also include a written description of the internet location providing such instructions or descriptions.

[0199] Example

[0200] The following examples illustrate, but do not limit, this technology.

[0201] Example 1 - PCR Amplification

[0202] In embodiments of the methods provided herein, PCR amplification is performed on samples or combinations of samples containing a mixture of one or more major nucleic acid substances and one or more minor nucleic acid substances using suitable amplification primers. Exemplary amplification conditions are shown below, for example, in U.S. Patent Application Publication No. 2013 / 0237428 A1, U.S. Patent No. 8,349,566 and U.S. Patent No. 8,003,317, and Oeth et al. Application specification document number 8876-006, R04, published on November 10, 2006, is incorporated herein by reference in its entirety. For example, amplification conditions can be set as follows:

[0203] Table 1 - PCR Mixtures

[0204]

[0205] Mix the components from Table 1 and gently vortex. Exemplary conditions for PCR cycling are as follows: 94°C for 15 minutes; 45 cycles, 94°C for 20 seconds / 56°C for 30 seconds / 72°C for 1 minute; 72°C for 3 minutes; cool to 4°C.

[0206] Example 2 - Treatment with shrimp alkaline phosphatase (SAP)

[0207] Following amplification, unincorporated dNTPs were dephosphorylated by SAP treatment. The "SAP mixture" was prepared using the reagent combinations shown in Table 2:

[0208] Table 2 - SAP Mixtures

[0209]

[0210] The SAP mixture (total volume: 2 μl) prepared according to Table 2 was added to the 5 μl PCR reaction from Example 1. Each 5 μl PCR reaction was gently mixed or vortexed in this manner. The treated samples were incubated in a standard thermal cycler as follows: 37 °C for 40 min; 85 °C for 5 min; cooled to 4 °C.

[0211] Example 3 - UEP Primer Extension

[0212] 2 μl of the extension mixture was added to each SAP-treated sample from Example 2, one of which was prepared as shown in Table 3 below:

[0213] Table 3 - Extended Mixtures

[0214]

[0215] The components in Table 1 are mixed and gently vortexed. Exemplary conditions for extended cycles can be as follows: a 2-step 200 short-cycle program is performed in a standard thermal cycler at 94°C for 30 seconds; 40 cycles of 94°C for 5 seconds / (52°C for 5 seconds / 80°C for 5 seconds – 5 cycles, for a total of 200 short cycles); 72°C for 3 minutes; cooling to 4°C. The 200 short-cycle program uses two cycle periods, one of 5 cycles within the 40-cycle period, thus producing a 200-cycle program.

[0216] Example 4 - MALDI-TOF Analysis

[0217] Desalting of the extended product formed according to Example 3 was achieved by adding 6 mg of CLEAN resin (Sequinam). Using a nanodistributor, 15 nmol of the pyrolysis reaction was automatically distributed to a pre-loaded matrix. The sample was placed on a silicon chip. Mass spectra were acquired using a MassARRAY Compact Analyzer (MALDI-TOF mass spectrometer). The sample was transferred to a 384-well plate. ( On (San Diego, California). The whole Microchip transfer to BRUKER / The mass spectrometer allows for automated sample measurement. It analyzes positive ions and accumulates approximately 100 single-shot spectra (e.g., 5 grating positions x 20 shots / position). All samples are analyzed in linear time-of-flight mode using delayed ion extraction and a total accelerating voltage of 20 kV. For further details, please refer to [link to documentation]. (San Diego, California) The Nanodispenser User Guide outlines the process of "allocating primer mass extension reaction products to..." Dispensing Primer Mass Extension Reaction Products onto Chapter 1. The Typer system (Typer version 3.0) is used to... Obtaining spectra, such as (San Diego, California) As described in the "SPECTROACQUIRE" chapter of the "Typer User Guide".

[0218] Example 5 - Exemplary scheme and results using tilted ddNTP concentrations

[0219] As described above, the method presented herein combines the detection sensitivity of minor nucleic acid substances (such as minor alleles) in a sample with the ability to quantify their frequency, quantity, or copy number relative to major nucleic acid substances (such as major alleles). This is achieved by adjusting the concentration range of a chain terminator (e.g., ddNTP) specific to the major nucleic acid substance in the extension reaction such that it is lower than the concentration of a chain terminator specific to the minor nucleic acid substance, thereby increasing the detection limit for signals from the minor nucleic acid substance, but not so low that the signal of the major nucleic acid substance is reduced to the background noise level, thus excluding its use as a positive control (to ensure the integrity of the method, i.e., to ensure that the observed results are true when the signal corresponding to the minor nucleic acid substance is detected or not) or as the basis for quantifying the relative amount (e.g., frequency, copy number) of the minor nucleic acid substance.

[0220] An example solution is as follows:

[0221] Experimental Design

[0222] Each assay consists of three primers: two PCR primers and one single-base extension primer. The amplicon (the nucleic acid length can vary, but is generally recommended to be less than 150 bp to ensure successful amplification of samples such as circulating cell-free DNA and degraded DNA isolated from FFPE (formalin-fixed paraffin-embedded tissue)). A quality tag is added to the 5' end of the primers to remove unincorporated PCR primers from the analysis quality window or to allow for validation using next-generation sequencing. Several requirements also apply to extension probe design. First, the quality of the extension products must be adequately spaced by quality differences to ensure no conflicts between assays (e.g., signal overlap). Second, multiplexing is a design-based reaction where each reaction vessel is limited to nucleic acid material that binds the same specific chain-terminating nucleotide. For example, in an "A" multiplexing reaction, all nucleic acid material in the reaction uses ddA as the chain-terminating nucleotide, and all assays using this nucleotide can be combined. Additional design freedom is gained by allowing reverse design; that is, in this case, probing the sequence on the opposite strand can study "T" because the probe will target "A" on the opposite strand.

[0223] PCR amplification

[0224] PCR was performed with a total volume of 20 μL, supplemented with 10 μL of DNA template and 10 μL of master mixture, which consisted of 1 mM MgCl2, 125 μM dNTPs, 0.125 U uracil-DNA glycosylase (New England Biolabs, Ipswich, MA), 4 U Taq polymerase, and 100 nM of each PCR primer. The reaction was initially incubated at 30 °C for 10 min, followed by incubation at 94 °C for 2 min. 45 cycles of PCR were performed at 94 °C for 30 sec, 56 °C for 30 sec, and 72 °C for 1 min. A final incubation at 72 °C for 5 min completed the PCR. 5 μL of amplified product was adjusted by adding 2 μL of 0.5 U SAP in 0.24x shrimp alkaline phosphatase (SAP) buffer (total volume 7 μL), incubating at 37 °C for 40 min, and then denaturing the SAP enzyme at 85 °C for 10 min. Unless otherwise stated, all reagents used were from Agena Bioscience, Inc.

[0225] Single base extension

[0226] By adding 2 μL of 0.2x extension buffer, 200 μM of minor allele variant nucleotide amplicon and different concentrations of major allele variant nucleotide amplicon ranging from 0.25 μM to 20 μM, different concentrations of extension primers, and 0.14 U... The master mixture of Pro enzyme was used. Single-base extension reactions were performed in a total volume of 9 μL. Reaction parameters included an initial incubation of 30 seconds at 94 °C, followed by five nested cycles of 5 seconds at 94 °C, 5 seconds at 52 °C, and then 5 seconds at 80 °C for 40 cycles. Single-base extension was completed by incubation at 72 °C for 3 minutes.

[0227] Capture and data acquisition

[0228] Prior to nanodispensing, the product was conditioned with 5 μL (3 mg) of anion exchange resin slurry for desalting. Finally, the analytes were dispensed using an RS1000 nanodispenser. II. On a solid support. Use 4. Data were obtained by the instrument using MALDI-TOF mass spectrometry.

[0229] Exemplary results of the analysis based on the method provided in this paper are shown in Figure 1-3 .like Figure 1 As shown, samples containing a mixture of minor nucleic acid material (few alleles) and major nucleic acid material (most alleles) are processed according to iPLEX. TM The method, or the method described herein, involves amplification and extension reactions in which a minority alleles are present at a frequency of 5% relative to the majority alleles. The leftmost figure shows the results using iPLEX. TM The analytical results of the method, using equimolar concentrations of chain terminator. As shown in the first figure, the signal peak corresponding to the left-hand extension product from the majority allele is so dominant that the minority peak on the right is reduced to background noise and is undetectable. The middle figure shows the results obtained when the concentration of the majority allele-specific chain terminator is 20% (1 / 5) of the concentration of the minority allele-specific chain terminator. As shown in the middle figure, the detection signal intensity from the minority allele extension product (right peak) is now higher and more visible than the detection signal from the majority allele extension product (left peak). However, the minority allele signal remains small and close to the level of background noise. The rightmost figure shows the results obtained when the concentration of the majority allele-specific chain terminator is approximately 6-7% (one-fifteenth) of the concentration of the minority allele-specific chain terminator. As shown in the right figure, the signal from the minority allele extension product (right peak) is now comparable to the signal from the majority allele (left peak). Therefore, Figure 1 This indicates that adjusting the concentration of chain terminators favors the skewing of minor nucleic acid substances, with minor nucleic acid substances (which cannot be skewed by chain terminators such as iPLEX) occurring at frequencies less than 10%. TM The method can be effectively detected by the method provided in this article.

[0230] In addition, such as Figure 3As shown, the method presented in this paper can be used to detect minor nucleic acid substances present at a frequency of less than 2%, down to 1.25%, relative to the major nucleic acid substances in a sample. Figure 3 The results are shown as follows: when samples were amplified and subsequently extended under three conditions: (A) concentration of ddNTPs, chain terminators specific to the major nucleic acid components (wild-type alleles); (C) [WT] ddNTP This is equal to the concentration of ddNTPs, chain terminators specific to minor nucleic acid substances (mutated alleles; C[Mut]). ddNTP (B)C[WT] ddNTP Less than C[Mut] ddNTP Approximately 20% of the concentration (i.e., C[WT]). ddNTP ∶C[Mut] ddNTP The ratio is 0.2); and (C)C[WT] ddNTP Less than C[Mut] ddNTP Approximately 5% of the concentration (i.e., C[WT]). ddNTP ∶C[Mut] ddNTP The ratio is 0.05). Figure 3 As shown, when C[WT] ddNTP ∶C[Mut] ddNTP The ratio is 1, meaning the concentrations of the two chain terminators are equal (e.g., in iPLEX). TM When used in the method, the frequencies of mutant alleles were 5%, 2.5%, and 1.25% below the limit of detection (LOD). When C[WT] ddNTP ∶C[Mut] ddNTP When the proportion was reduced to 0.2, a 5% mutant allele frequency could be detected, while the frequencies of 2.5% and 1.25% mutant alleles remained below the detection limit. When C[WT] ddNTP ∶C[Mut] ddNTP When the proportion was further reduced to 0.05, the allele frequencies of all three mutants (5%, 2.5%, and 1.25%) could be detected.

[0231] Example 6 - iPLEX+ Feasibility Study

[0232] iPLEX chemistry, implemented on the MassARRAY system, provides accurate and sensitive detection of single nucleotide polymorphisms (SNPs), somatic mutations, and copy number variations (CNVs). Unbound by theory, iPLEX chemistry offers a sensitivity of over 10% for detecting minor variants of somatic mutations. The 10% minor allele frequency threshold is a practical limitation of the iPLEX method stemming from the properties of the MassARRAY platform. The MassARRAY is a MALDI-TOF mass spectrometer characterized by an 80cm linear TOF analyzer with a maximum dynamic range of 50X (minimum to peak in the spectrum). This 50X dynamic range is further reduced by ion signal intensity and noise levels. For example, the iPLEX somatic mutation application has employed a conservative 10X dynamic range threshold (10% minor variant frequency). iPLEX+ chemistry aims to improve the sensitivity of existing iPLEX chemistry to levels below 10% minor variants.

[0233] In somatic mutation applications, mutation frequency is determined as the ratio of the intensity of the minor mutation peak to the intensity of the dominant wild-type (WT) peak, or the percentage of the minor mutation peak. In either case, the intensity of the WT peak is used as the background for measuring the mutation peak. For example, considering the theoretical 50X dynamic range and the actual 10X dynamic range of the MassARRAY instrument, the hypothetical 5% of minor alleles will be detected at levels below the limit of detection (LOD) threshold and therefore will not be reported as high-confidence mutations.

[0234] The change in the dynamic range limitation of conventional iPLEX was investigated by consuming the amount of WT ion peaks compared to conventional iPLEX. This modified version of iPLEX is called iPLEX+.

[0235] During the iPLEX reaction, the unextended primer nucleotides (UEP) are converted into the product by incorporating a template-specific single-terminant nucleotide (acyclic nucleotide) over 200 extension reaction cycles. The extension reaction is a bimolecular reaction in which the amount of product (the observed peak) is proportional to the product of the concentration of the DNA template at the WT or minor peak and the amount of the terminant nucleotide:

[0236] [WT 产物 ]~[DNA WT ]×[WT 终止剂 ],

[0237] [secondary 产物 [DNA secondary] × [Secondary] 终止剂 ].

[0238] Given the high concentration of the WT DNA template and the same concentration of WT and minor terminators in regular iPLEX, the relative intensity of the detected peaks will be proportional to the amount of DNA template. One way to reduce the amount of the WT peak and shift the equilibrium “biased” towards the minor product is to reduce the amount of WT terminator nucleotides compared to the minor terminator nucleotides. iPLEX+ is the WT-depleted form of regular iPLEX, where a previous equimolar mixture of the four terminator nucleotides has now been depleted for the WT alleles (e.g., depleted to less than 1% of the total). By reducing the amount of the WT-specific terminator, the equilibrium of the extension reaction shifts towards the formation of the minor product. The enrichment of the minor peak will vary depending on the amount of WT terminator available for the reaction.

[0239] A feasibility study was conducted to determine whether the WT-consuming iPLEX method (a mixture of WT-consuming terminator nucleotides), termed iPLEX+, would allow for the detection of less than 10% of minor alleles. The performance of iPLEX+ was investigated using a comprehensive experimental cohort covering all potential WT-mutation scenarios (transformations) and a model system (where 0%, 1%, 2.5%, and 5% were generated from a dilution series of human-chimpanzee DNA) was used. The model system was validated using an orthogonal technique (Ion Torrent PGM). Three rounds of comprehensive studies were conducted, involving inquiries into the iPLEX+ cohort for over 300 trials, reflecting multiple techniques for replicating the entire dilution series. Several extension reaction parameters were evaluated after each feasibility round to improve the overall cohort's performance. Other objectives included determining the sensitivity of the iPLEX+ method, determining whether iPLEX+ is quantifiable, and establishing initial biochemical parameters (terminator nucleotide mixture, amount of terminator nucleotides, and extension reaction parameters). This study shows that the iPLEX+ method allows for the detection of 5% mutants with nearly 100% sensitivity, over 90% sensitivity for 2.5% mutants, and an average sensitivity of 35-61% for 1% mutants (50% is the average sensitivity, depending on the method conditions).

[0240] Model System

[0241] The model system used to simulate low-frequency mutations and query all possible WT-mutant allele combinations is a mixture of human and chimpanzee (Pan Troglodytes) genomic DNA. Whole-genome sequence alignment was performed between the human hg19 reference assembly and the chimpanzee 2013 assembly to select short, upright homologous sequences with a single stable nucleotide mismatch between humans and chimpanzees. These regions became candidates for the iPLEX+ assay. Another set of criteria applied to orthologous regions was that they must map to human exons within 5 exons or larger ORFs, and the distance between regions on the same chromosome must be greater than 5000 bp. The final number of qualified assay candidates was 5,822.

[0242] Experimental Design

[0243] Agena Bioscience Experiment Design Suite 2.0 was used to design iPLEX+ experiments using candidates from human-chimpanzee models. The experimental design was implemented using a “somatic mutation” setting with a maximum multiplexing level of 15. iPLEX+ presents multiplexing constraints that are not part of the general iPLEX experimental design. The WT-consuming composition of the termination mixture produces one of four nucleotides for cases where all experiments in the “channel” are WT. All multiplex experiments with the WT major allele are referred to as A-channels or A-conversions. An A-conversion has the depleted A nucleotide extending all major WT products, and the C, G, and T nucleotides extending minor products. The experimental design was performed prior to design with input sequences having specific experimental orientation to eliminate reverse experimental designs with complementary nucleotides (a common feature of experimental designers). Experiments were designed separately for the four channels (WT-A, C, G, T), and multiplexing was incorporated into the final design. The final design has all the functions of 12 possible WT-minor conversions, with three conversions for each WT terminator (e.g., the A-channel has three WT-minor conversions a / C, a / G, a / T). The combined experimental design has 334 experiments in 24 multiplexings and has all possible transformations represented by at least 20 experiments.

[0244] biological samples

[0245] Biological samples were generated using a pooled human genome HapMap01 (Coriell) DNA sample and chimpanzee DNA from a single individual, Max (Coriell). In the all-human-chimpanzee mixture, human DNA was the major component providing the WT alleles, while chimpanzee DNA served as a minor component. The total amount of DNA in each sample (pure or mixture) was set at approximately 3300 copies of genomic DNA, corresponding to 10 ng of DNA per sample.

[0246] Verification using iPLEX

[0247] Using 100% human samples, 100% chimpanzee samples, and 50% human-chimpanzee mixtures, 334 assays were validated using standard iPLEX chemistry. Preliminary validation using standard iPLEX was performed to identify poorly performing assays. Poorly performing assays were those with low elongation (typically below 80%) and poor template specificity, manifested as assay bias (non-specific elongation of non-template alleles). Assay elongation was calculated as the fraction of the assay product peak across all assay peaks, including UEP.

[0248]

[0249] iPLEX validation was performed to identify experiments that failed due to technical or biological reasons. These reasons included poor design (poor primer specificity, poor annealing properties, poor multiplexing, etc.), biological (sequence variations or mutant SNPs within the amplicon at the PCR or extension primer site, non-specific annealing of the test primers), or technical reasons (test located at the salt adduct site, poor quality of the test primers). Of the 334 designed experiments, 15 experiments had extension rates below 80% and were therefore marked as poor. In addition to extension rates, routine iPLEX data were used to identify experiments with skewed or non-specific amplification. Since the experiments query stable regions in human and chimpanzee DNA, it was expected that 100% of human and 100% of chimpanzee samples would be homozygous. However, at heterozygous allele sites, 41 experiments had minor allele frequencies of 10% or higher. For example, an experiment that was heterozygous in 100% chimpanzee samples showed non-template extension of the G allele (human) and resulted in heterozygous detection, where only the expected homozygous T allele was present. Therefore, a total of 49 unique trials were found to have poor extension rates or nonspecific extension. The design-to-panel success rate was 85%, which is within the ADS 2.0 acceptance criterion of a design trial success rate >80%. Of the 49 poor trials, 24 failed trials (very poor extension rates <0.5 and non-template allele extension) were removed from further analysis.

[0250] iPLEX+ Round 1

[0251] The first round of the iPLEX+ experiments consisted of the following studies. Human / chimpanzee DNA was pooled into dilution series to represent 0%, 1%, 2.5%, and 5% chimpanzee contributions. The 0% chimpanzee sample served as a 100% human WT control. The WT terminator nucleotide fraction was set to 1% in all four terminator mixtures (a / CGT, c / AGT, g / CAT, and t / ACG). Each dilution series was queryed in 16 technical replicates. The following criteria were evaluated when determining method performance:

[0252] • Extension of the trial in 0% chimpanzee samples,

[0253] • Chimpanzee frequency in 0% chimpanzee sample

[0254] • Test sensitivity,

[0255] Conversion performance and sensitivity,

[0256] • Overall sensitivity.

[0257] The first two criteria were used as quality control measures to remove experiments that failed during the experimental process. To assess the sensitivity of individual experiments, it had to be extended in 0% chimpanzee (100% human WT DNA sample) to produce a minimum detectable WT peak (SNR of 5 or higher). Without a reliable WT signal in 0% chimpanzee, the performance of experiments in 1%, 2.5%, and 5% chimpanzee could not be assessed due to the lack of comparison with the baseline. The second criterion in the WT sample—observed minor chimpanzee alleles—was used to remove non-specific experiments that failed due to experimental design rather than the iPLEX+ method.

[0258] Given the WT-consuming nature of iPLEX+, extension rates for iPLEX+ assays at 100% WT were expected to be lower than those for 5% chimpanzee and conventional iPLEX. The average assay extension rate for conventional iPLEX was 0.95, while iPLEX+ for 0% chimpanzee samples had an extension rate of 0.40, and iPLEX+ for 5% chimpanzee samples had an extension rate of 0.78. The WT-consuming formulation of the nucleotide terminator mixture biases the balance towards minor allele products; therefore, 100% human WT samples without minor products showed significantly lower assay extension rates. One of the tasks of the feasibility study was to establish a WT nucleotide concentration that ensures assay amplification in 100% WT samples. After evaluating the method performance in the first round, 96 assays showed no extension in 100% human WT samples, and 36 assays showed nonspecific chimpanzee extension in WT samples. Therefore, only 57% of the assays were functional.

[0259] After removing poorly performing experiments, experimental performance and group sensitivity were estimated. The primary criterion used to estimate experimental performance was the chimpanzee frequencies observed in experiments at 1%, 2.5%, and 5% chimpanzee dilutions, distinguishable from 0% chimpanzee WT samples. Chimpanzee frequencies were derived from the allele ratio between two peaks: the human allele peak and the chimpanzee allele peak. The allele peak ratio was expressed as a numerical genotype as follows:

[0260]

[0261] Among them, strength 低质量 It is the peak intensity and intensity of low-quality alleles.高质量 This represents the peak intensity of high-quality alleles. The numeric genotype indicates the frequency of minor alleles in chimpanzees when their alleles are of low quality. When chimpanzee alleles are of high quality, the chimpanzee frequency is calculated as 1 - numeric genotype.

[0262] The performance of each trial was evaluated using 16 technical replicates. Data (observed chip frequencies) were plotted as a lattice diagram for each trial, with the y-axis representing the observed chimpanzee frequency and the x-axis representing the dilution series, with WT samples representing 0% chimpanzees. Data were evaluated based on whether a sample could be distinguished from 0% chimpanzees to a statistically significant degree. The statistics used were t-tests with multiple comparisons performed using Bonferroni correction, and a critical α value was set at 0.95. Trial chr2_AtoC_104 significantly distinguished 2.5% and 5% chimpanzee samples from 0% chimpanzee samples. The following two trials, chr20_AtoC_118 and chr4_AtoC_146, could only distinguish 5% from 0% chimpanzees, while trial chr1_AtoG_115 distinguished all dilutions from 0% chimpanzees. The sensitivity per conversion and overall sensitivity of iPLEX+ was calculated using the significance results of each trial. Figure 4 The sensitivity for each conversion of iPLEX+ is shown. The sensitivity for each dilution level (0%, 1%, 2.5%, and 5% chimpanzee) was calculated as the mean of trials that were statistically significantly different from that dilution level (0% chimpanzee). For example, Figure 4 The top left corner shows that the sensitivity for 1% chimpanzees is 0, for 2.5% chimpanzees it is 0.33, and for 5% chimpanzees it is 1.0, based on tests representing the A to C conversion. This means that in any test at this conversion, 1% cannot distinguish from 0, can distinguish from the 2.5% dilution 33% of the time, and can distinguish from the 5% dilution 100% of the time. The overall sensitivity for each dilution series is 0.61 for 1%, 0.88 for 2.5%, and 1.0 for the 5% chimpanzee frequency sample.

[0263] iPLEX+ sensitivity varies depending on the experiment and transformation. Significant experimental and transformation-specific biases were observed in the quantification of iPLEX+. Figure 5 The results show the chimpanzee frequencies observed in different transformations of chimpanzee DNA into humans using a 5% dilution.

[0264] In the first round of the feasibility study, the WT nucleotide depletion method of iPLEX+ was shown to be effective in reducing WT allele signal and improving assay sensitivity to below 10%. Based on this data, iPLEX+ can be used to detect minor variants below 10% with excellent sensitivity: 100% for 5% mutants, 88% for 2.5% mutants, and 61% for 1% mutants. Some drawbacks of the first-round method included poor assay extension and assay-specific nucleotide incorporation in WT samples. To overcome the low assay extension, reduce product strength, and mitigate some assay-specific incorporation bias, a round of optimization was performed, in which the nucleotide termination mixture (1% for WT nucleotides and equimolar for mutant alleles in the first round) was optimized for each transformation.

[0265] Nucleotide terminator optimization

[0266] Nucleotide terminator optimization was performed using a technique replicated at a 5% chimpanzee dilution, yielding 16 data points per experiment (14 replicates and 2 NTCs). The following terminator mixtures were evaluated: 2%, 4%, 8%, and 16% WT nucleotides. The goal of nucleotide terminator optimization was to determine the optimal ratio of WT nucleotide terminator for each transformation. The optimization criterion was the relative peak height of the 5% chimpanzee minor allele peak to the WT peak. The optimization objective was to produce terminator mixtures that resulted in equal peak heights for the WT and 5% chimpanzee peaks.

[0267] Different concentrations of WT nucleotides elicited different responses in chimpanzee minor alleles. Increasing the amount of WT terminator from 1% to 16% reduced the observed minor allele fraction across all transformations. Increasing the WT nucleotide fraction from 8% to 16% resulted in a 5% reduction in the signal of chimpanzee minor alleles, making iPLEX+ similar to conventional iPLEX. As with previous iPLEX+ experiments, the general diffusion of data points per WT percentage indicates the incorporation of transformation-specific alleles. However, a 2% WT nucleotide amount is the optimal amount for detecting an average of 5% chimpanzee minor alleles as a 0.5 chimpanzee allele frequency.

[0268] The adjusted WT nucleotide percentage (based on the average of that transformation) was calculated for each transformation to produce a 0.5 chimpanzee allele frequency. These data are shown in Table 4. The adjusted WT nucleotide percentage (bottom row) was selected for the next round of iPLEX+ feasibility.

[0269] Table 4

[0270]

[0271]

[0272] iPLEX+ Round 2

[0273] Except for the termination mixture, the second round of the iPLEX+ feasibility study was identical in layout and scope to the first round. The first round was conducted using 1% WT nucleotide terminator in its universal nucleotide mixture, while the second round used a transformation-specific WT nucleotide amount (Table 4). The second round of iPLEX+ feasibility studies consisted of the following: Human / chimpanzee DNA was pooled into dilution series to represent 0%, 1%, 2.5%, and 5% chimpanzee contributions. Each dilution series was queried in 16 technical replicates. As with the first round, the following criteria were evaluated in determining method performance:

[0274] • Extension of the trial in 0% chimpanzee samples,

[0275] • Chimpanzee frequency in 0% chimpanzee sample

[0276] • Test sensitivity,

[0277] Conversion performance and sensitivity,

[0278] • Overall sensitivity.

[0279] Similar to Round 1, poorly performing trials were identified in Round 2. 44 trials (compared to 96 in Round 1) failed to produce sufficient signal in 100% WT samples. This represents a 2.2-fold improvement over Round 1, entirely due to the increased WT nucleotide concentration in Round 2. In addition to the failed trials, 33 trials extended chimpanzee alleles in 100% WT samples (compared to 36 in Round 1). A total of 77 trials (25% of the groups) performed poorly in Round 2, compared to 136 trials (43% of the groups) in Round 1. These trials were removed from further analysis.

[0280] The effect of increased WT nucleotide contribution on elongation was investigated (0% chimpanzee, 5% chimpanzee, and conventional iPLEX). No significant change in elongation compared to round 1 was observed. The mean elongation of the 0% chimpanzee sample was 0.41 (compared to 0.40 in round 1), and the mean elongation of the 5% chimpanzee sample was 0.63 (compared to 0.78 in round 1).

[0281] Using the same methodology as in Round 1, the sensitivity of each transformation and the overall sensitivity of the group were examined. Moderate variations were observed at each transformation level; however, the overall performance of the Round 2 groups was similar to that of Round 1.

[0282] The overall sensitivity for the second-round group was 0.52 for 1% of chimpanzees (compared to 0.61 in the first round), 0.91 for 2.5% of chimpanzees (compared to 0.88 in the first round), and 0.98 for 5% of chimpanzees (compared to 1.0 in the first round). Furthermore, as in the first round, a significant degree of trial-specific extension was observed, resulting in high variability in the minor allele portions observed in each dilution series. Therefore, the main benefit of the transformation-specific WT nucleotide approach is that fewer trials at 0% were observed in the second round (44 trials) compared to the first round (96 trials), with no extension in the second round.

[0283] Orthogonal verification of model systems

[0284] Following the first round, nucleotide optimization and a second round of experiments were conducted to validate the human-chimpanzee model system used in the development of iPLEX+. Experiments (as described in the model system) were established to inquire about different target regions in the human and chimpanzee genomes, and dilution series samples were generated to represent the minor allele frequencies of 0%, 1%, 2.5%, and 5% chimpanzees against a major human allele background. A series of experiments were performed using an ion-torsional PGM (Life Technologies) to validate the target regions and dilutions. Libraries for sequencing were prepared by amplifying iPLEX(+) designed amplicons using iPLEX PCR mixtures and dNTPS. The recommended gDNA input in this PCR reaction was 10-20 ng copies / multiplexation. The amount of DNA in each multiplexation was measured using the BioAnalyzer DNA 1000 kit and merged into equimolar fractions. Targets were made for the ion-torsional input. Rapid DNA fragmentation and library preparation groups used multiplexing of amplicon libraries to 50 ng each. Ion-fluidized library preparation involved end repair, adapter connection of P1 and A adapters, nick translation, and small 6x cycle PCR. Cleanup after each step was performed using Ampure XP beads. Library QC and quantification were performed using the BioAnalyzer DNA 1000 kit, and the library was fed into the Ion-fluidized PGM OT2 200 kit at 26 pmol. Templated ISPs were enriched using the Ion-fluidized OT2 ES machine. These templated ISPs were sequenced on the PGM system using the Ion-fluidized 200 sequencing kit and a 240 nucleotide flow rate. Each model was sequenced using a single 318 or 318v2 chip. Data analysis was performed by aligning sequencing reads to a BED file containing genomic coordinates of the target amplicon designed with iPLEX(+). Target representation and minor variant frequencies were automatically calculated and validated using the Ion-fluidized coverage analysis and variant detector plugins.

[0285] Ion data for 278 targets were confirmed using a 5% chimpanzee dilution library, representing 310 targets (90% of the targets). The median frequency of minor chimpanzee alleles in the 5% chimpanzee library was 4.9%, with a median target coverage of 10,000-fold. The minimum coverage cutoff for the analysis was 5,000-fold. A 2.5% dilution series library resulted in the identification of 189 targets (61%), with a median minor allele frequency of 3.6% and a median target coverage of 8,200-fold.

[0286] Each dilution library contained less than 5% outliers with significantly higher chimpanzee allele frequencies than expected at 5% and 2.5%. Examination of the correlation between the targets in the 5% and 2.5% dilution series revealed that variant outliers found in both the 5% and 2.5% data belonged to the same target trial.

[0287] The failure to identify 1% of variants during the 1% ion PGM run was primarily due to poor chip loading, resulting in insufficient target coverage to identify 1% of chimpanzee variants. The 0% chimpanzee library did not produce any variants as expected.

[0288] PGM data confirmed that the overall quality of the human-chimpanzee library was good, with 90% of the experimental targets from a 5% chimpanzee dilution achieving a nearly identical median minor variant frequency of 4.9% as confirmed by PGM. Evidence that library outliers belong to the same experimental targets suggests that these artifacts are due to challenges in the experimental design rather than the iPLEX+ method.

[0289] Nucleotide amount adjustment

[0290] The second round of iPLEX+ feasibility work continued to improve the extension rate of iPLEX+ experiments in WT samples. Increasing the WT nucleotide concentration in a transformation-specific manner resulted in a 2.2% reduction in the number of experiments that failed due to poor extension in WT samples. Furthermore, in studies using WT samples to query the iPLEX+ group, the following parameters of the extension response were considered, and their effects on the experimental extension rate in WT samples were investigated:

[0291] • PCR annealing time - Increasing the annealing time during extension will allow the polymerase (thermosequenase) to "find" the consumed WT nucleotides more times.

[0292] • Total nucleotide concentration - Maintain the same WT nucleotide ratio as in round 2, but allow a higher total amount to increase WT incorporation rate.

[0293] • 300-cycle PCR - Increasing the number of cycles from 200 to 300 will produce more products and stronger allele peaks.

[0294] • Increasing the amount of polymerase - Increasing the polymerase concentration will increase the amount of product due to faster turnover and resulting in a stronger allele peak.

[0295] Figure 6 The box plot shows the test extension rate for each condition as described above.

[0296] The conditions that produced the highest experimental extension rates in 0% chimpanzee WT samples were #1, #2, #5, and #6 (from left to right on the x-axis). The constant component for these four conditions was the 2X nucleotide amount. The 2X nucleotide amount represents twice the total nucleotide concentration, and the WT conversion-specific ratio remained the same as in round 2.

[0297] iPLEX+ Round 3

[0298] The third and final round of the iPLEX+ feasibility study utilized previously determined transformation-specific WT nucleotide concentrations and 2X total nucleotide concentrations. Similar to rounds 1 and 2, the third round of the iPLEX+ feasibility study consisted of the following: Human / chimpanzee DNA was pooled into dilution series to represent 0%, 1%, 2.5%, and 5% chimpanzee contributions. Each dilution series was queried in 16 technical replicates. As in round 1, the following criteria were evaluated in determining method performance:

[0299] • Extension of the trial in 0% chimpanzee samples,

[0300] • Chimpanzee frequency in 0% chimpanzee sample

[0301] • Test sensitivity,

[0302] Conversion performance and sensitivity,

[0303] • Overall sensitivity.

[0304] 37 trials failed due to poor extension in the 0% chimpanzee WT samples and in 33 samples of nonspecific extension chimpanzees in the WT samples. Further improvement was observed compared to Round 2 (44 trials). The poor trial score obtained in Round 3 was 23% (compared to 25% in Round 2). A modest improvement in trial extension rate was also observed by using 2N total terminator nucleotides. The mean trial extension rates in the 0% and 5% chimpanzee samples were 0.53 and 0.72, respectively. This is an improvement compared to the results of Round 2 (0.41 for 0% chimpanzees and 0.63 for 5% chimpanzee samples). As in Rounds 1 and 2, the sensitivity for each transformation and the overall sensitivity of the group were determined. Moderate variations were observed at each transformation level; however, the overall performance of the Round 3 groups was again similar to the overall performance of the first two rounds, except for the sensitivity in the 1% chimpanzee samples.

[0305] The overall sensitivity for the third round groups was 0.35 for 1% chimpanzees (compared to 0.52 in round 2), 0.89 for 2.5% chimpanzees (compared to 0.91 in round 2), and 0.99 for 5% chimpanzees (compared to 0.98 in round 1). The sensitivity of the 1% chimpanzee sample decreased from 0.52 to 0.35 across the three rounds. Without being bound by theory, two possible explanations for the observed decrease in sensitivity (0.61 in round 1, 0.52 in round 2, and 0.35 in round 3) due to the increased concentration of the terminator nucleotide are as follows:

[0306] Increasing the percentage of WT nucleotides and the total nucleotide concentration leads to an increase in the peak intensity of the major WT alleles. This, in turn, causes the method to perform similarly to conventional iPLEX, where the 1% minor allele signal is a low-intensity noise peak.

[0307] Increasing the percentage of WT nucleotides and the total nucleotide concentration reduced the number of poor extension tests from 96 in round 1 to 44 in round 2 and 37 in round 3. In rounds 2 and 3, the “most recent worst” tests by QC showed almost no reduction in chimpanzee sensitivity by 1% due to their lower signal-to-noise ratio, but these were taken into account when calculating overall sensitivity.

[0308] Example 7 - Optimal Nucleotide Concentration

[0309] The optimal nucleotide concentrations for each possible transformation in the iPLEX+ reaction are shown in Table 5. The WT / Mut (major allele / minor allele) ratio is also shown.

[0310] Table 5

[0311]

[0312]

[0313] Example 8 - iPLEX+ Validation Study

[0314] A study was conducted to validate the iPlex+ assay. The function, sensitivity, and reproducibility of somatic mutation detection in NRAS and EGFR genes (models) were tested.

[0315] Test function

[0316] The experiment used 200 copies / ul of mutants for the 5% mutant population. All other minor variants were titrated from this starting value. The wild-type contribution was 4000 copies / ul.

[0317] • Assess the suitability of wild-type extension for the assay using a wild-type template and a standard iPLEX termination mixture. Evaluate non-specific interactions of assays that fail to extend to suitable wild-type nucleotides and, if appropriate, redesign or move to another assay.

[0318] • Evaluate the nonspecific extension of the trial to the non-template control and move it to another key.

[0319] • Assess appropriate extensions in the 5% mutant model using a custom nucleotide mixture. Observe all appropriate variant genotypes.

[0320] • The exonuclease activity and additional signals of the experiment were evaluated.

[0321] • Evaluation experiments were conducted using peak SNR (signal-to-noise ratio). During development, three experiments showed repetitive poor performance in peak SNR. The cause of each experiment was examined. The proposed cause was mitigated through redesign and / or reallocation.

[0322] Test sensitivity

[0323] Preliminary studies investigated the nucleotide ratios necessary for minor variant detection at 5% or less (initial nucleotide mixture). Triple runs were performed on samples and triplicate on WT samples, each with the same weight. The initial nucleotide mixture did not provide the required sensitivity. High wild-type signal resulted in a low proportion of mutants, which did not allow for differentiation from other mutation classes (data not shown).

[0324] To improve sensitivity to mutant alleles, the WT nucleotide contribution was reduced. This assessment used 5%, 2%, and 1% minor variants from WT and NTC samples. Each sample was run in triplicate. The reduction in wild type successfully increased the proportion of mutants to allow for greater minor variant identification; however, the SNR of the WT peak decreased below the cutoff value (SNR of 5) used for detection peaks in several trials (data not shown).

[0325] The concentration of WT nucleotides was slightly increased in the adjusted nucleotide mixture to increase the WT SNR. This evaluation used the same standard and minor variants as previous experiments to investigate the reduction in WT nucleotides. WT samples were run in duplicate, each weighing one NTC. Improving the WT contribution of the mixture to a concentration between the original mixture and the reduced WT combination successfully adjusted many assays for identifying poor WT signals or poor variant types (data not shown). Data evaluating all three nucleotide mixture assays suggest the use of a customized nucleotide ratio between WT and mutants.

[0326] The customized nucleotide mixtures were evaluated using the same twelve controls in quadruplicate as in the previous two experiments. WT and NTC were also run in quadruplicate per replicate. The customized nucleotide ratio mixtures successfully provided a differentiateable mutation ratio while maintaining sufficient WT SNR to allow for peak identification and detection by software (SNR values ​​greater than 5) (see Table 6). The customized nucleotide mixtures (Table 6) represent a further refinement of the optimal nucleotide concentration mixtures in Table 5.

[0327] Table 6

[0328]

[0329]

[0330] Reproducibility

[0331] The minor variant models (5%, 2.5%, 1%, and WT) used in the sensitivity experiments were evaluated in 24 replicates (96 replicates of WT). The minor variant types of each model produced statistically significant differences in distribution (data not shown).

[0332] Example 9 - Non-limiting example of the implementation method

[0333] The following provides non-limiting examples of some implementations of this technology.

[0334] A1. A multiplexing method for identifying the presence or absence of one or more minor nucleic acid substances in a nucleic acid group, comprising a mixture of the one or more minor nucleic acid substances and one or more major nucleic acid substances, wherein each minor nucleic acid substance is a variant of a corresponding major nucleic acid substance and has a copy number less than that of its corresponding major nucleic acid substance, wherein the method comprises:

[0335] (a) Amplify the target region of the mixture simultaneously with amplification primers under amplification conditions including dNTPs, thereby producing an amplified mixture of nucleic acids containing major and minor nucleic acid substances.

[0336] (b) The amplification mixture is contacted with the extension primers under extension conditions including a chain terminator, wherein:

[0337] (i) One or more major nucleic acid substances share a common chain terminator that is specific to the major nucleic acid substance but non-specific to the minor nucleic acid substance, and

[0338] (ii) Each of one or more minor nucleic acid substances has a chain terminator that is specific to the minor nucleic acid substance and non-specific to the major nucleic acid substance, wherein the chain terminator that is specific to the minor nucleic acid substance is: (A) unique to the specific minor nucleic acid substance in the amplification mixture and not shared by other minor nucleic acid substances in the amplification mixture, or (B) at least one of one or more minor nucleic acid substances shares a common chain terminator with at least one other minor nucleic acid substance in the amplification mixture.

[0339] The primer is extended to or through different nucleotide positions in the minor nucleic acid material relative to the major nucleic acid material, thereby producing extension products corresponding to chain terminations of the minor and major nucleic acid materials, respectively, wherein the concentration of the chain terminator specific to the major nucleic acid material is less than the concentration of one or more chain terminators specific to each of the minor nucleic acid materials; and

[0340] (c) Analyze the extended products of (b) to identify the presence or absence of one or more minor nucleic acid substances.

[0341] A2. The method as described in embodiment A1, wherein the nucleic acid group comprises a variety of minor nucleic acid substances, which are variants of a single major nucleic acid substance, and the variety of minor nucleic acid substances are identified in a single multiplexing reaction.

[0342] A3. The method as described in embodiment A1, wherein (b) is carried out in a group of at least two reaction vessels or compartments, wherein:

[0343] The first container or compartment includes extended conditions containing a chain terminator specific to the primary nucleic acid substance and free from chain terminators specific to one or more secondary nucleic acid substances; and

[0344] Each remaining container or compartment includes an extension condition containing a single chain terminator specific to and common to one or more minor nucleic acid substances, and not containing a chain terminator specific to the major nucleic acid substance or specific to the minor nucleic acid substance but not common to the single chain terminator.

[0345] A4. The method as described in any one of embodiments A1-A3, wherein the concentration of each chain terminator is known.

[0346] B1. A method for quantifying one or more minor nucleic acid substances in a nucleic acid group, said nucleic acid group comprising a mixture of said one or more minor nucleic acid substances and a major nucleic acid substance, wherein said minor nucleic acid substances are variants of the same major nucleic acid substance and each exists at a copy number lower than that of the major nucleic acid substance, the method comprising:

[0347] (a) Amplify the target region of the mixture simultaneously with amplification primers under amplification conditions including dNTPs, thereby producing an amplified mixture of nucleic acids containing major and minor nucleic acid substances.

[0348] (b) The amplification mixture is contacted with extension primers under extension conditions, said extension conditions including for (i) each of one or more minor nucleic acid substances, and (ii) a major nucleic acid substance-specific chain terminator, whereby the primers extend to or through different nucleotide positions in the minor nucleic acid substances relative to the major nucleic acid substances, thereby producing extension products corresponding to chain terminations of the minor nucleic acid substances and the major nucleic acid substances, respectively, wherein: (1) the concentration of each chain terminator is known; and (2) the concentration of the chain terminator specific to the major nucleic acid substance is less than the concentration of the chain terminator specific to one or more minor nucleic acid substances;

[0349] (c) Determine the ratio of the amount of extension product corresponding to each of one or more minor nucleic acid substances to the amount of extension product corresponding to the major nucleic acid substance; and

[0350] (d) Based on the proportion of (c), and based on the concentration of a chain terminator specific to one or more minor nucleic acid substances, the amount of minor nucleic acid substance is quantified relative to the amount of major nucleic acid substance, wherein the concentration of the chain terminator specific to one or more minor nucleic acid substances is relative to the concentration of the chain terminator specific to the major nucleic acid substance.

[0351] C1. A method for multiplexing one or more minor nucleic acid substances in a quantitative nucleic acid group, said nucleic acid group comprising a mixture of said one or more minor nucleic acid substances and a major nucleic acid substance, wherein each minor nucleic acid substance is a variant of the corresponding major nucleic acid substance and exists at a copy number lower than that of its corresponding major nucleic acid substance, said method comprising:

[0352] (a) Amplify the target region of the mixture simultaneously with amplification primers under amplification conditions including dNTPs, thereby producing an amplified mixture of nucleic acids containing major and minor nucleic acid substances.

[0353] (b) The amplification mixture is contacted with the extension primers under extension conditions including a chain terminator, wherein:

[0354] (i) One or more major nucleic acid substances share a common chain terminator that is specific to the major nucleic acid substance but non-specific to the minor nucleic acid substance, and

[0355] (ii) Each of one or more minor nucleic acid substances has a chain terminator that is specific to the minor nucleic acid substance and non-specific to the major nucleic acid substance, wherein the chain terminator that is specific to the minor nucleic acid substance is: (A) unique to the specific minor nucleic acid substance in the amplification mixture and not shared by other minor nucleic acid substances in the amplification mixture, or (B) at least one of one or more minor nucleic acid substances shares a common chain terminator with at least one other minor nucleic acid substance in the amplification mixture.

[0356] The primer is extended to or through different nucleotide positions in the minor nucleic acid material relative to the major nucleic acid material, thereby producing extension products corresponding to chain terminations of the minor and major nucleic acid materials, respectively, wherein: (1) the concentration of each chain terminator is known; and (2) the concentration of the chain terminator specific to the major nucleic acid material is less than the concentration of the chain terminator specific to one or more minor nucleic acid materials.

[0357] (c) Determine the ratio of the amount of extension product corresponding to each of one or more minor nucleic acid substances to the amount of extension product corresponding to the major nucleic acid substance; and

[0358] (d) Based on the proportion of (c), and based on the concentration of a chain terminator specific to one or more minor nucleic acid substances relative to the concentration of a chain terminator specific to the major nucleic acid substance, the amount of minor nucleic acid substance is quantified relative to the amount of major nucleic acid substance.

[0359] C2. The method as described in embodiment B1 or embodiment C1, wherein the nucleic acid group comprises a variety of minor nucleic acid substances, which are variants of a single major nucleic acid substance, and the variety of minor nucleic acid substances are identified in a single multiplexing reaction.

[0360] C3. The method as described in embodiment C1, wherein (b) is carried out in a group of at least two reaction vessels or compartments, wherein:

[0361] The first container or compartment includes extended conditions containing a chain terminator specific to the primary nucleic acid substance and free from chain terminators specific to one or more secondary nucleic acid substances; and

[0362] Each remaining container or compartment includes an extension condition containing a single chain terminator specific to and common to one or more minor nucleic acid substances, and not containing a chain terminator specific to the major nucleic acid substance or specific to the minor nucleic acid substance but not common to the single chain terminator.

[0363] D1. The method of any one of embodiments A1-A4, wherein the sequences of the minor and major nucleic acid substances differ by a single base, and the primers extend to or pass through different single bases.

[0364] D2. The method as described in any one of embodiments A1-A4, B1, C1-C3 and D1, wherein the sequence of the minor nucleic acid substance includes insertions or deletions relative to the sequence of the major nucleic acid substance.

[0365] D3. The method as described in any one of embodiments A1-A4, B1, C1-C3, D1 and D2, wherein one or more minor nucleic acid substances are single nucleotide polymorphism (SNP) variants of the major nucleic acid substance.

[0366] D4. The method as described in any one of embodiments A1-A4, B1, C1-C3 and D1-D3, wherein the minor and major nucleic acid substances are mutants and wild-type alleles of the same gene, respectively.

[0367] D5. The method as described in any one of embodiments A1-A4, B1, C1-C3 and D1-D4, wherein the primary nucleic acid substance is derived from a host object and the secondary nucleic acid substance is derived from an object other than the host, or the secondary nucleic acid substance is derived from a host object and the primary nucleic acid substance is derived from an object other than the host.

[0368] D6. The method as described in any one of embodiments A1-A4, B1, C1-C3 and D1-D5, wherein each of the one or more minor nucleic acid substances is present at a copy number less than about 10% of the copy number of the major nucleic acid substance.

[0369] D7. The method as described in embodiment D6, wherein each of the one or more minor nucleic acid substances is present at a copy number of about 1% to less than 10% of the copy number of the major nucleic acid substance.

[0370] D8. The method as described in embodiment D7, wherein each of the one or more minor nucleic acid substances is present at a copy number of about 2% to less than 10% of the copy number of the major nucleic acid substance.

[0371] D9. The method as described in embodiment D7, wherein each of the one or more minor nucleic acid substances is present at about 5% or less of the copy number of the major nucleic acid substance.

[0372] D10. The method as described in embodiment D7, wherein each of the one or more minor nucleic acid substances is present at about 2% or less of the copy number of the major nucleic acid substance.

[0373] D10.1. The method as described in embodiment D7, wherein each of the one or more minor nucleic acid substances is present at approximately 1% of the copy number of the major nucleic acid substance.

[0374] D11. The method as described in any one of embodiments A1-A4, B1, C1-C3 and D1-D10.1, wherein the concentration of the chain terminator specific to the major nucleic acid substance is about 1% to about 20% of the concentration of the chain terminator specific to each minor nucleic acid substance.

[0375] D12. The method as described in any one of embodiments A1-A4, B1, C1-C3 and D1-D10.1, wherein the concentration of the chain terminator specific to the major nucleic acid substance is from about 0.1% to about 10% of the concentration of the chain terminator specific to each minor nucleic acid substance.

[0376] D13. The method as described in embodiment D12, wherein the concentration of the chain terminator specific to the major nucleic acid substance is from about 0.1% to about 4% of the concentration of the chain terminator specific to each minor nucleic acid substance.

[0377] D13.1. The method as described in any one of embodiments A1-A4, B1, C1-C3 and D1-D10.1, wherein the concentration of the chain terminator specific to the major nucleic acid substance is from about 0.01% to about 10% of the concentration of the chain terminator specific to each minor nucleic acid substance.

[0378] D13.2. The method as described in embodiment D13.1, wherein the concentration of the chain terminator specific to the major nucleic acid substance is about 0.01% to about 4% of the concentration of the chain terminator specific to each minor nucleic acid substance.

[0379] D14. The method as described in any one of embodiments A1-A4, B1, C1-C3 and D1-D13.2, wherein the concentration of the chain terminator for the major nucleic acid substance varies based on a specific major / minor substance conversion.

[0380] D15. The method of any one of embodiments A1-A4, B1, C1-C3 and D1-D14, wherein the chain terminator is a chain-terminating nucleotide.

[0381] D16. The method as described in embodiment D15, wherein the chain-terminating nucleotide is independently selected from ddATP, ddGTP, ddCTP, ddTTP, and ddUTP.

[0382] D17. The method as described in embodiments D15 or D16, wherein the chain-terminating nucleotide specific to one or more minor nucleic acid substances consists of a single chain-terminating nucleotide.

[0383] D18. The method as described in embodiments D15 or D16, wherein the chain-terminating nucleotide specific to one or more minor nucleic acid substances consists of two chain-terminating nucleotides.

[0384] D19. The method as described in embodiments D15 or D16, wherein the chain-terminating nucleotide specific to one or more minor nucleic acid substances consists of three chain-terminating nucleotides.

[0385] D20. The method of any one of embodiments A1-A4, B1, C1-C3 and D1-D19, wherein the chain terminator comprises one or more acyclic terminators.

[0386] D21. The method as described in any one of embodiments A1-A4, B1, C1-C3 and D1-D20, comprising about 30 to about 45 PCR amplification cycles as in (a).

[0387] D22. The method as described in any one of embodiments A1-A4, B1, C1-C3 and D1-D21, wherein the extension condition in (b) comprises about 20 to about 300 cycles.

[0388] D23. The method as described in embodiment D22, wherein the extension condition in (b) includes at least 50 cycles.

[0389] D24. The method as described in any one of embodiments A1-A4, B1, C1-C3 and D1-D23, wherein one or more chain terminators include a detectable marker.

[0390] D25. The method as described in embodiment D24, wherein the marker is a fluorescent marker or a dye.

[0391] D26. The method as described in embodiment D24, wherein the mark is a quality mark.

[0392] D27. The method of any one of embodiments D24-D26 further includes detecting the marker to identify or quantify the one or more minor nucleic acid substances.

[0393] D28. The method as described in embodiment D27, wherein the marker is a mass marker and is detected by mass spectrometry.

[0394] D28.1. The method as described in embodiment D27, wherein the label is a fluorescent label or dye and is detected by electrophoresis or real-time PCR.

[0395] D29. The method as described in any one of embodiments A1-A4, B1, C1-C3 and D1-D28.1, wherein the amplification reaction conditions in (a) include water, genomic DNA, buffer, dNTPs, primer pairs, MgCl2 and polymerase, wherein the ratio of the concentration of MgCl2 to the concentration of each of the dNTPs is selected from ≤10∶1, ≤9∶1, ≤8∶1, ≤7∶1, ≤6∶1 or ≤5∶1.

[0396] D30. The method as described in embodiment D29, wherein the polymerase is a Taq polymerase at a concentration of at least about 0.03 units / μl.

[0397] D31. The method as described in any one of embodiments A1-A4, B1, C1-C3 and D1-D30, wherein the amplification reaction conditions in (a) comprise about 400-700 μM of each dNTP, about 100 nM of primer pairs and about 1.6 to about 4.8 mM MgCl2.

[0398] D32. The method as described in any one of embodiments A1-A4, B1, C1-C3 and D1-D31, wherein the sequence tag is attached to one or more primers in the amplification primer pair.

[0399] D33. The method as described in any one of embodiments A1-A4, B1, C1-C3, and D1-D32, wherein free Mg 2+ The concentration is 1.0-2.0 mM.

[0400] ***

[0401] All patents, patent applications, publications, and documents cited in this document are incorporated herein by reference in their entirety. Reference to any of these patents, patent applications, publications, and documents does not imply an admission that any of the foregoing content is applicable prior art, nor does it imply an admission of the content or dates of such publications or documents.

[0402] The above content can be modified without departing from the basic aspects of this technology. Although this technology has been described in full detail with reference to one or more specific embodiments, those skilled in the art will recognize that changes can be made to the embodiments specifically disclosed in this application, and such modifications and alterations are within the scope and spirit of this technology.

[0403] The techniques appropriately described herein can be implemented without any element not specifically disclosed herein. Thus, for example, in various examples herein, any one of the terms “comprising,” “basically composed of,” and “composed of” can be replaced by any of the other two. Terms and expressions used herein are illustrative rather than restrictive, and their use does not preclude any equivalents of the features or portions thereof shown and described, or various modifications that may be made within the scope of the claimed technique. The terms “a” or “an” indicate one or more of the elements they modify (e.g., “a reagent” may indicate one or more reagents), unless the context clearly indicates that one or more elements are described. The term “about” as used herein indicates a value within a 10% range of a base parameter (i.e., ±10%), and its use at the beginning of a list of values ​​indicates that each value in that list is modified (i.e., “about 1, 2, and 3” means about 1, about 2, and about 3). For example, a weight of “about 100 grams” can contain a weight of 90 to 110 grams. Therefore, it should be understood that although the present technology has been specifically disclosed through representative embodiments and optional features, those skilled in the art can make modifications and variations to the content disclosed herein, and such modifications and variations should be considered to fall within the scope of the present technology.

[0404] Embodiments of this technology are set forth in the appended claims.

Claims

1. A multiplexing method for identifying the presence or absence of one or more minor nucleic acid species in a nucleic acid sample comprising one or more target nucleic acids, the method comprising: (a) simultaneously amplifying the target nucleic acids of the nucleic acid sample, wherein each target nucleic acid has a major nucleic acid species and one or more minor nucleic acid species, wherein each minor nucleic acid species is a lower frequency or copy number variant of the major nucleic acid species, and the amplification conditions comprise water, a mixture of genomic DNA or minor nucleic acid species and major nucleic acid species, buffer, dNTPs, primer pair, MgCl2, and polymerase, wherein the ratio of the concentration of MgCl2 to the concentration of each of the dNTPs is selected from < 10: 1, < 9: 1, < 8: 1, < 7: 1, < 6: 1, or < 5: 1; (b) contacting the amplified nucleic acids of (a) with one or more extension primers under extension conditions comprising a chain terminator specific for the one or more major nucleic acid species and a chain terminator specific for the one or more minor nucleic acid species, wherein: (i) the one or more major nucleic acid species share a common chain terminator specific for the major nucleic acid species and non-specific for the minor nucleic acid species, (ii) each of the one or more minor nucleic acid species has a chain terminator specific for the minor nucleic acid species and non-specific for the major nucleic acid species, wherein the chain terminator specific for the minor nucleic acid species: (A) is unique to the particular minor nucleic acid species in the amplified nucleic acids and is not shared by other minor nucleic acid species in the amplified nucleic acids, or (B) at least one of the one or more minor nucleic acid species shares a common chain terminator with at least one other minor nucleic acid species in the amplified nucleic acids, and (iii) the concentration of the chain terminator specific for the major nucleic acid species is less than the concentration of each of the chain terminators specific for the one or more minor nucleic acid species, whereby the primers extend to or through different nucleotide positions in the minor nucleic acid species relative to the major nucleic acid species, thereby generating chain-terminated extension products corresponding to the minor nucleic acid species and the major nucleic acid species, respectively, which are present in the sample; and (c) analyzing the extension products of (b), thereby identifying the presence or absence of the one or more minor nucleic acid species, wherein the minor nucleic acid species is present at 0.25% to less than 10%, or 0.5% to less than 10%, or 1% to less than 10% of the copy number or frequency of the major nucleic acid species, or the one or more minor nucleic acid species is less than 30%, 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.8%, 0.75%, or 0.5% of the major nucleic acid species, and wherein the concentration of the chain terminator specific for the major nucleic acid species is 1% to 20%, or 0.1% to 10%, or 0.01% to 5% of the concentration of each of the chain terminators specific for the minor nucleic acid species.

2. The method of claim 1, wherein the nucleic acid sample comprises a plurality of minor nucleic acid species that are variants of a single major nucleic acid species, and the plurality of minor nucleic acid species is identified in a single multiplexed reaction.

3. The method of claim 1, wherein the nucleic acid sample comprises a plurality of primary nucleic acid species, each of which has one or more secondary nucleic acid species, and the secondary nucleic acid species are identified in a single multiplexed reaction.

4. The method of any one of claims 1-3, further comprising identifying the presence or absence of one or more primary nucleic acid species in (c).

5. A nucleic acid analysis method for quantifying one or more secondary nucleic acid species in a nucleic acid sample comprising a target nucleic acid, the method comprising: (a) simultaneously amplifying the target nucleic acid of the sample, wherein the target nucleic acid has a primary nucleic acid species and one or more secondary nucleic acid species, wherein the one or more secondary nucleic acid species are variants of the primary nucleic acid species and each secondary nucleic acid species is a lower frequency or copy number variant of the primary nucleic acid species, and the amplification conditions comprise water, genomic DNA, buffer, dNTPs, a primer pair, MgCl2, and a polymerase, wherein the ratio of the concentration of MgCl2 to the concentration of each of the dNTPs is selected from < 10: 1, < 9: 1, < 8: 1, < 7: 1, < 6: 1, or < 5: 1; (b) contacting the amplified nucleic acid of (a) with an extension primer under extension conditions comprising a chain terminator specific for each of the one or more secondary nucleic acid species and a chain terminator specific for the primary nucleic acid species, whereby the primer extends to or through a different nucleotide position in the secondary nucleic acid species relative to the primary nucleic acid species, thereby generating chain-terminated extension products corresponding to the secondary nucleic acid species and the primary nucleic acid species, respectively, which are present in the sample, wherein: (1) the concentration of each chain terminator is known; and (2) the concentration of the chain terminator specific for the primary nucleic acid species is less than the concentration of the chain terminators specific for the one or more secondary nucleic acid species; (c) determining the proportion of the amount of the extension product corresponding to each of the one or more secondary nucleic acid species relative to the amount of the extension product corresponding to the primary nucleic acid species; and (d) quantifying the amount of the secondary nucleic acid species relative to the primary nucleic acid species based on the proportion of (c), and based on the concentration of the chain terminators specific for the one or more secondary nucleic acid species relative to the concentration of the chain terminator specific for the primary nucleic acid species, wherein each of the one or more secondary nucleic acid species is present at 0.25% to less than 10%, or 0.5% to less than 10%, or 1% to less than 10% of the copy number or frequency of the primary nucleic acid species, or the one or more secondary nucleic acid species is less than 30%, 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.8%, 0.75%, or 0.5% of the primary nucleic acid species, and wherein the concentration of the chain terminator specific for the primary nucleic acid species is 1% to 20%, or 0.1% to 10%, or 0.01% to 5% of the concentration of each of the chain terminators specific for the secondary nucleic acid species.

6. A multiplexed nucleic acid analysis method for quantifying one or more secondary nucleic acid species in a nucleic acid sample comprising one or more target nucleic acids, the method comprising: (a) simultaneously amplifying target nucleic acids of the sample, wherein the target nucleic acids have a major nucleic acid species and one or more minor nucleic acid species, wherein each minor nucleic acid species is a lower frequency or copy number variant of the major nucleic acid species, and the amplification conditions comprise water, genomic DNA, buffer, dNTPs, a primer pair, MgCh, and a polymerase, wherein the ratio of the concentration of MgCh to the concentration of each of the dNTPs is selected from < 10: 1, < 9: 1, < 8: 1, < 7: 1, < 6: 1, or < 5: 1; (b) contacting the amplified nucleic acids of (a) with an extension primer under extension conditions comprising a chain terminator specific to the one or more major nucleic acid species and one or more chain terminators specific to the one or more minor nucleic acid species, wherein: (i) the one or more major nucleic acid species share a common chain terminator specific to the major nucleic acid species and non-specific to the minor nucleic acid species, (ii) each of the one or more minor nucleic acid species has a chain terminator specific to the minor nucleic acid species and non-specific to the major nucleic acid species, wherein the chain terminator specific to the minor nucleic acid species: (A) is unique to the particular minor nucleic acid species in the amplified nucleic acids and is not shared by other minor nucleic acid species in the amplified nucleic acids, or (B) at least one of the one or more minor nucleic acid species shares a common chain terminator with at least one other minor nucleic acid species in the amplified nucleic acids, (iii) the concentration of each chain terminator is known, and the concentration of the chain terminator specific to the major nucleic acid species is less than the concentration of each of the chain terminators specific to the one or more minor nucleic acid species, thereby the primer extends to or through a different nucleotide position in the minor nucleic acid species relative to the major nucleic acid species, thereby generating chain-terminated extension products corresponding to the minor nucleic acid species and the major nucleic acid species, respectively, which are present in the sample; (c) determining the ratio of the amount of the extension product corresponding to each of the one or more minor nucleic acid species relative to the amount of the extension product corresponding to the major nucleic acid species; and (d) quantifying the amount of the minor nucleic acid species relative to the major nucleic acid species based on the ratio of (c), and based on the concentration of the chain terminator specific to the one or more minor nucleic acid species relative to the concentration of the chain terminator specific to the major nucleic acid species, wherein each of the one or more minor nucleic acid species is present at 0.25% to less than 10%, or 0.5% to less than 10%, or 1% to less than 10% of the copy number or frequency of the major nucleic acid species, or the one or more minor nucleic acid species is less than 30%, 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.8%, 0.75%, or 0.5% of the major nucleic acid species, and wherein the concentration of the chain terminator specific to the major nucleic acid species is 1% to 20%, or 0.1% to 10%, or 0.01% to 5% of the concentration of each of the chain terminators specific to the minor nucleic acid species.

7. The method of claim 5 or 6, wherein the nucleic acid sample comprises a plurality of minor nucleic acid species that are variants of the major nucleic acid species, and the plurality of minor nucleic acid species are identified in a single multiplexed reaction.

8. The method of any one of claims 1-3, 5, and 6, wherein the sequences of the minor and major nucleic acid species differ by a single base, and the primers extend to different single bases.

9. The method of any one of claims 1-3, 5, and 6, wherein the ratio of the concentration of MgCl2 to the concentration of each of the dNTPs is selected from < 7:

1.

10. The method of any one of claims 1-3, 5, and 6, wherein the total MgCl2 concentration is 2.6 mM to 4.8 mM MgCl2, 3.0 mM to 4.5 mM MgCl2, or 3.5 mM to 4.0 mM MgCl2.

11. The method of claim 10, wherein the free Mg 2+ concentration is between 1-2 mM, wherein the free Mg 2+ concentration is determined according to the formula: Free Mg 2+ Concentration = total Mg 2+ Concentration - total concentration of all dNTPs used in the amplification conditions.

12. The method of claim 11, wherein the free Mg 2+ concentration is between 1.1-1.9 mM, between 1.2-1.8 mM, between 1.3-1.7 mM, or between 1.4-1.6 mM.

13. The method of claim 11 or 12, wherein the free Mg 2+ concentration is 1.5 mM.

14. The method of any one of claims 1-3, 5, and 6, wherein the sequence of the minor nucleic acid species comprises an insertion or a deletion relative to the sequence of the major nucleic acid species.

15. The method of any one of claims 1-3, 5, and 6, wherein the one or more minor nucleic acid species is a single nucleotide polymorphism (SNP) variant of the major nucleic acid species.

16. The method of any one of claims 1-3, 5, and 6, wherein the minor and major nucleic acid species are, respectively, mutant and wild-type alleles of the same gene, or wherein the one or more minor nucleic acid species is a somatic mutation of the wild-type major nucleic acid species.

17. The method of any one of claims 1-3, 5, and 6, wherein the major nucleic acid species is from a host subject, and the minor nucleic acid species is from a subject other than the host, or the minor nucleic acid species is from the host subject, and the major nucleic acid species is from a subject other than the host.

18. The method of any one of claims 1-3, 5, and 6, wherein the one or more minor nucleic acid species each is present at a copy number or frequency that is less than 10% of the copy number or frequency of the major nucleic acid species.

19. The method of claim 18, wherein the one or more minor nucleic acid species each is present at a copy number or frequency that is 1% to 5%, or 1% to 4%, or 1% to 3.5%, or 1% to 3% of the copy number or frequency of the major nucleic acid species.

20. The method of claim 18, wherein the one or more minor nucleic acid species each is present at a copy number or frequency that is 2% to less than 10% of the copy number or frequency of the major nucleic acid species.

21. The method of claim 18, wherein the one or more minor nucleic acid species each is present at a copy number or frequency that is less than 5% or less than 4% of the copy number or frequency of the major nucleic acid species.

22. The method of claim 21, wherein the one or more minor nucleic acid species each is present at a copy number or frequency that is less than 2% of the copy number or frequency of the major nucleic acid species.

23. The method of any one of claims 1-3, 5, and 6, wherein the concentration of the chain terminating agent specific for the major nucleic acid species is less than 5% of the concentration of each of the chain terminating agents specific for the minor nucleic acid species.

24. The method of any one of claims 1-3, 5, and 6, wherein the concentration of the chain terminating agent specific for the major nucleic acid species is from 0.5% to less than 15% of the concentration of each of the chain terminating agents specific for the minor nucleic acid species.

25. The method of any one of claims 1-3, 5, and 6, wherein the concentration of the chain terminating agent specific for the major nucleic acid species is from 1% to 10%, or from 1% to 7%, of the concentration of each of the chain terminating agents specific for the minor nucleic acid species.

26. The method of claim 25, wherein the concentration of the chain terminating agent specific for the major nucleic acid species is from 1% to 2% of the concentration of each of the chain terminating agents specific for the minor nucleic acid species.

27. The method of any one of claims 1-3, 5, and 6, wherein the ratio of the concentration of the chain terminating agent specific for the major nucleic acid species to the concentration of the chain terminating agent specific for the minor nucleic acid species is selected based on the particular chain terminating agent specific for the major nucleic acid species and the particular chain terminating agent specific for the minor nucleic acid species.

28. The method of claim 27, wherein the ratio of the concentration of the chain terminating agent specific for the major nucleic acid species to the concentration of the chain terminating agent specific for the minor nucleic acid species is 1:15, and each of the minor nucleic acid species is present at a frequency that is less than 10% of the frequency of the major nucleic acid species.

29. The method of claim 27, wherein the ratio of the concentration of the chain terminating agent specific for the major nucleic acid species to the concentration of the chain terminating agent specific for the minor nucleic acid species is 0.2, and each of the minor nucleic acid species is present at a frequency that is 5% of the frequency of the major nucleic acid species.

30. The method of claim 27, wherein the ratio of the concentration of the chain terminating agent specific for the major nucleic acid species to the concentration of the chain terminating agent specific for the minor nucleic acid species is 0.05, and each of the minor nucleic acid species is present at a frequency that is 5%, 2.5%, or 1.25% of the frequency of the major nucleic acid species.

31. The method of any one of claims 1-3, 5, and 6, wherein the chain terminating agent is a chain terminating nucleotide.

32. The method of claim 31, wherein the chain terminating nucleotide is independently selected from the group consisting of ddATP, ddGTP, ddCTP, ddTTP, and ddUTP.

33. The method of claim 31, wherein the chain terminating nucleotide specific for one or more of the minor nucleic acid species consists of 1 chain terminating nucleotide.

34. The method of claim 31, wherein the chain terminating nucleotide specific for one or more of the minor nucleic acid species consists of 2 chain terminating nucleotides.

35. The method of claim 31, wherein the chain terminating nucleotide specific for one or more of the minor nucleic acid species consists of 3 chain terminating nucleotides.

36. The method of any one of claims 1-3, 5, and 6, wherein the chain terminating agent comprises one or more acyclic terminating agents.

37. The method of any one of claims 1-3, 5, and 6, comprising 30 to 45 cycles of PCR amplification in (a).

38. The method of any one of claims 1-3, 5, and 6, wherein the extension conditions in (b) comprise a plurality of extension cycles, wherein the extension reaction cycles 200 to 300 times.

39. The method of any one of claims 1-3, 5, and 6, wherein the extension conditions in (b) comprise a plurality of extension cycles, wherein the extension reaction cycles at least 65, 70, 75, 80, 85, 90, 95, or 100 times.

40. The method of any one of claims 1-3, 5, and 6, wherein the one or more minor nucleic acid species is each present at a copy number or frequency that is 1% of the copy number or frequency of the major nucleic acid species.

41. The method of any one of claims 1-3, 5, and 6, wherein the extension product comprises a distinguishable characteristic indicative of the presence or absence of the extension product.

42. The method of claim 41, wherein the extension product is analyzed by mass spectrometry.

43. The method of claim 5 or 6, wherein the ratio of the amount of extension product corresponding to each of the one or more minor nucleic acid species relative to the amount of extension product corresponding to the major nucleic acid species is determined as the ratio of the signal of the minor nucleic acid species produced by mass spectrometry to the signal of the major nucleic acid species produced by mass spectrometry.

44. The method of any one of claims 1-3, 5, and 6, wherein one or more of the chain terminators comprises a detectable label.

45. The method of claim 44, further comprising detecting the label, thereby identifying or quantifying the one or more minor nucleic acid species.

46. The method of claim 44, wherein the label is a fluorescent label or dye or is a mass label.

47. The method of claim 46, wherein the label is a mass label and detection is by mass spectrometry, or the label is a fluorescent label or dye and detection is by electrophoresis or real-time PCR.

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