WO2023125898A1 - 核酸检测方法与系统 - Google Patents

核酸检测方法与系统 Download PDF

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WO2023125898A1
WO2023125898A1 PCT/CN2022/143769 CN2022143769W WO2023125898A1 WO 2023125898 A1 WO2023125898 A1 WO 2023125898A1 CN 2022143769 W CN2022143769 W CN 2022143769W WO 2023125898 A1 WO2023125898 A1 WO 2023125898A1
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nucleic acid
sequence
amplification
nucleotides
sample
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PCT/CN2022/143769
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English (en)
French (fr)
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王晓岚
杨恩卓
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柏锘（上海）医疗科技有限公司
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Priority to CN202280087030.3A priority Critical patent/CN118489012A/zh
Publication of WO2023125898A1 publication Critical patent/WO2023125898A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present application relates to nucleic acid detection methods and systems.
  • the application relates to the detection of small amounts of nucleic acids.
  • target nucleic acids are an important tool in diagnostic medicine and molecular biology research. Detection of target nucleic acids can be used, for example, to identify pathogens (e.g., bacteria or viruses) in host subjects; determine expression of normal genes and identify mutated genes (e.g., oncogenes); Typing tissues; analyzing homology between genes of different substances; and/or identifying alleles and polymorphisms of gene variants, etc.
  • pathogens e.g., bacteria or viruses
  • mutated genes e.g., oncogenes
  • Typing tissues analyzing homology between genes of different substances
  • identifying alleles and polymorphisms of gene variants etc.
  • the signal from a small number of target molecules may be obscured by the signal from a large number of non-target substances (eg, background signal or noise), and it is often necessary to remove these "background signals" to improve the lower limit of detection.
  • background signal e.g, background signal or noise
  • the signal from the target molecule may be weakened or lost.
  • the present application provides methods and products (eg, detection kits or detection systems) for identifying target nucleic acids.
  • the method and product of the present application can significantly improve the lower limit of detection, and can sensitively and accurately detect the ultra-small amount of nucleic acid present in the sample.
  • the method of the present application can sensitively and accurately detect nucleic acid molecules from circulating tumor cells, such as cfDNA and/or ctDNA.
  • the method of the present application can increase the sensitivity of detection by at least 10 times (for example, at least 20 times, at least 30 times, at least 40 times, At least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times or even more than at least 100 times).
  • the method of the present application can accurately detect nucleic acid molecules (e.g., DNA) whose content in the sample is less than 10ng, for example, the content in the sample is less than 9ng, less than 8ng, less than 7ng, less than 6ng, less than 5ng, less than 4ng, Below 3ng, below 2ng, below 1ng, below 0.9ng, below 0.8ng, below 0.7ng, below 0.6ng, below 0.5ng, below 0.4ng or less.
  • nucleic acid molecules e.g., DNA
  • the content in the sample is less than 9ng, less than 8ng, less than 7ng, less than 6ng, less than 5ng, less than 4ng, Below 3ng, below 2ng, below 1ng, below 0.9ng, below 0.8ng, below 0.7ng, below 0.6ng, below 0.5ng, below 0.4ng or less.
  • the methods described herein can be in vitro or ex vivo methods.
  • the identification results obtained by using the method of the present application, combined with other information are sufficient to obtain an assessment of the subject's health status or disease progression. clear diagnosis.
  • the application provides a method for identifying the presence, absence or content of one or more target nucleic acids in a sample, the method comprising: a) performing linear amplification of nucleic acids derived from the sample processing the sample under conditions to generate a linear amplification product of the nucleic acid; b) exponentially amplifying the linear amplification product to generate an exponential amplification product of the nucleic acid; and c) including a stop core Extended oligonucleotides are generated by contacting the exponentially amplified product of the nucleic acid with an extension primer under conditions for nucleotide extension.
  • the method may further comprise d) analyzing the extended oligonucleotides to identify the presence, absence or amount of the one or more target nucleic acids in the sample.
  • step a) comprises: contacting the sample with an oligonucleotide capable of binding to the nucleic acid to form an oligonucleotide hybrid; and hybridizing the oligonucleotide The body is contacted with the amplification composition under conditions enabling linear amplification to produce a linear amplification product of the nucleic acid.
  • the step a) comprises: ligating the nucleic acid derived from the sample with an adapter sequence to form an adapter-containing nucleic acid; contacting oligonucleotides of sequence to form oligonucleotide hybrids; and contacting said oligonucleotide hybrids with an amplification composition under conditions capable of linear amplification to generate linear amplification of said nucleic acids amplified product.
  • the linker sequence comprises a promoter sequence for RNA polymerase.
  • the linker sequence comprises an SP6 promoter sequence, a T7 promoter sequence and/or a T3 promoter sequence.
  • the oligonucleotide capable of specifically binding to the linker sequence is at least partially complementary to the linker sequence.
  • the oligonucleotide capable of specifically binding to the linker sequence comprises a complementary sequence of an SP6 promoter sequence, a complementary sequence of a T7 promoter sequence and/or a complementary sequence of a T3 promoter sequence.
  • the linker sequence comprises one or more modifications, such that the nucleic acid sequence of the linker sequence does not change after bisulfite treatment.
  • the linker sequence comprises methylation modification of one or more bases.
  • the cytosine contained in the linker sequence is a methylated cytosine.
  • the linker sequence is directly or indirectly linked to the 3' end of the nucleic acid.
  • the 5' end of the linker sequence is directly or indirectly linked to the 3' end of the nucleic acid.
  • ligating the nucleic acid to the linker sequence comprises: repairing the ends of the nucleic acid so that they are blunt-ended.
  • ligating the nucleic acid to the linker sequence comprises repairing the ends of the nucleic acid to be blunt-ended and adding deoxyadenosine dA to the 3' end of the repaired sequence.
  • the linear amplification comprises a nucleic acid transcription reaction, a strand displacement amplification reaction, and/or a rolling circle amplification reaction.
  • the linear amplification is non-target specific amplification not directed against a specific target.
  • the linear amplification product of the nucleic acid comprises RNA encoded by the nucleic acid.
  • RNA polymerase is used in said linear amplification.
  • the RNA polymerase comprises T7 RNA polymerase, SP6 RNA polymerase, and/or T3 RNA polymerase.
  • step a) further comprises: treating said sample with a reagent that modifies a particular unmethylated nucleotide to generate other nucleotides.
  • the reagent that modifies unmethylated specific nucleotides to generate other nucleotides includes a reagent that modifies unmethylated cytosine to generate uracil.
  • the reagents that modify unmethylated specific nucleotides to generate other nucleotides include bisulfite, ⁇ -glucosyltransferase, TET enzyme, pyridine borane and/or A3A deaminase.
  • step a) comprises: ligating nucleic acid derived from said sample to said adapter sequence to form an adapter-containing nucleic acid, wherein said adapter sequence comprises one or more modifications, said modification After the linker sequence is treated with bisulfite, its nucleic acid sequence does not change; the nucleic acid containing the linker is treated with a reagent containing bisulfite to form a converted nucleic acid; the converted nucleic acid is contacting an oligonucleotide capable of specifically binding to the linker sequence to form an oligonucleotide hybrid; and subjecting the oligonucleotide hybrid to the amplification composition under conditions capable of linear amplification Contacted to generate a linear amplification product of the nucleic acid.
  • the methods described herein are used to identify the presence, absence or amount of one or more methylated target nucleic acids in a sample.
  • the methods described herein are used to identify the methylation status of nucleotide loci in one or more target nucleic acids in a sample.
  • said exponential amplification comprises polymerase chain reaction PCR.
  • said b) comprises performing reverse transcription on said linear amplification product, and performing PCR amplification on said reverse transcription product.
  • the exponential amplification comprises reverse transcriptase polymerase chain reaction RT-PCR.
  • the exponential amplification is non-target-specific amplification that is not directed against a specific target.
  • said exponential amplification comprises the use of DNA polymerase and/or reverse transcriptase.
  • the terminating nucleotide is modified at the 3' carbon of its pentose moiety.
  • At least one of said terminating nucleotides is a dideoxynucleotide.
  • the dideoxynucleotide is selected from the group consisting of: ddATP, ddGTP, ddCTP, ddTTP, and ddUTP.
  • the terminating nucleotide is an acyclic nucleotide.
  • the acyclic nucleotide is selected from the group consisting of: acyATP, acyCTP, acyGTP, acyTTP, and acy-bromo-UTP.
  • the method is capable of identifying 2 or more target nucleic acids in the sample.
  • the terminating nucleotide comprises: 1) a terminating nucleotide specific to the other nucleotides generated by modification of the unmethylated specific nucleotide after the treatment and 2) a terminating nucleotide specific for said particular nucleotide being methylated.
  • the extended oligonucleotide comprises a detectable label.
  • the detectable marker is a mass marker.
  • the mass marker is a mass distinguishable tag.
  • said terminating nucleotide comprises said mass distinguishable tag.
  • the mass marker is detected by mass spectrometry.
  • the mass spectrometer is matrix assisted laser desorption ionization (MALDI) mass spectrometry.
  • MALDI matrix assisted laser desorption ionization
  • the nucleic acid derived from said sample comprises cfDNA and/or genomic DNA (gDNA).
  • the nucleic acid derived from the sample comprises ctDNA derived from tumor tissue, and/or DNA derived from exfoliated cells of an organ or fetus.
  • the amount of nucleic acid derived from said sample is less than about 10 ng.
  • the amount of nucleic acid derived from said sample is less than about 1 ng.
  • the sample is a biological sample.
  • biological samples may include: blood, body fluids, and tissues (e.g., organ tissue, formalin-fixed paraffin-embedded FFPE samples, urine, feces, cerebrospinal fluid, ascites, oral rinsing fluid, alveolar lavage fluid, amniotic fluid etc.), and/or in vitro culture samples.
  • the sample is derived from a subject.
  • the extension primer is capable of specifically binding to the target nucleic acid.
  • the nucleic acid sequence of the extension primer is at least partially complementary to the sequence of the target nucleic acid.
  • the methods described herein are used to identify the methylation status of cytosine nucleotides of one or more CpG dinucleotides in the nucleic acid molecule.
  • the one or more CpG dinucleotides are located in a promoter region of a gene.
  • the methods described herein are in vitro or ex vivo methods.
  • the present application provides a method of identifying biomarkers associated with a disease, a disease outcome and/or a treatment regimen outcome, the method comprising the steps of: i) according to the method described herein, identifying a biomarker derived from The presence, absence, or amount of one or more target nucleic acids in one or more samples from one or more subjects with known disease, disease outcome, and/or treatment regimen Test subject; ii) According to the method described in the application, identify the presence, absence or amount of one or more target nucleic acids derived from one or more samples, said one or more samples derived from normal subjects and iii) identifying a difference between the presence, absence or amount of the one or more target nucleic acids in step i) and the presence, absence or amount of the one or more target nucleic acids in step ii) , and accordingly, said difference is identified as a biomarker associated with said disease, disease outcome and/or treatment regimen outcome.
  • the present application provides a method of identifying methylation associated with a disease, a disease outcome and/or a treatment regimen outcome, the method comprising the following steps: i) according to the method described herein, identifying a methylation derived from Methylated or unmethylated nucleotides in one or more target nucleic acids of one or more samples derived from one or more patients with known disease, disease outcome, and/or A subject as a result of a treatment regimen; ii) identifying methylated or unmethylated nucleotides in one or more target nucleic acids derived from one or more samples according to the methods described herein, The one or more samples are derived from normal subjects; and iii) identifying the methylated or unmethylated nucleotides in the one or more target nucleic acids in step i) and step ii) Differences between methylated or unmethylated nucleotides in one or more target nucleic acids, and accordingly, identifying said differentially methylated or
  • the present application provides a kit for identifying the presence, absence or content of one or more target nucleic acids in a sample according to the methods described herein, the kit comprising: a linear amplification component an exponential amplification component; and an extension component comprising a stop nucleotide and an extension primer.
  • the extension component comprises a terminating nucleotide, a thermostable elongase, and an extension primer.
  • the application provides a kit for identifying the presence, absence or amount of one or more target nucleic acids in a sample, the kit comprising: a linear amplification component; an exponential amplification component; and An extension component comprising a terminating nucleotide and an extension primer.
  • the extension component comprises a terminating nucleotide, a thermostable elongase, and an extension primer.
  • said linear amplification component, said exponential amplification component, and said extension component do not mix with each other.
  • the linear amplification component, the exponential amplification component, and the extension component are each independently present in a separate package.
  • kits described herein comprise one or more of the following components: a) reagents that modify specific unmethylated nucleotides to generate other nucleotides; b) one or more nucleic acid polymerases; c) one or more primers; d) a MALDI substrate compound; and e) a MALDI substrate.
  • the present application provides a system comprising the kit described herein, and optionally comprising one or more parts selected from the group consisting of instructions for use, reagents, and instructions for using the described Kit device.
  • the means for using the kit comprises a thermal cycler and/or a mass spectrometer.
  • the step c) may include repeating a plurality of temperature cycles to amplify the amount of the extension product in the reaction.
  • the extension reaction can be cycled 2 or more times.
  • the extension reaction can be cycled about 10, 15, 20, 50, 100, 200, 300, 400, 500, or 600 or more times.
  • the extension reaction can be cycled 20 to 50 times.
  • the extension reaction can be cycled at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times.
  • Figure 1 shows an example of the experimental flow for the method of the present application.
  • 2A-2F show the nucleic acid mass spectrometry detection results of the method of the present application (the sample is 1ng methylated DNA).
  • Figures 3A-3D show exemplary DNA conversion methods.
  • Figure 4 shows a linear amplification reaction based on another strand displacement amplification.
  • Figure 5 shows a linear amplification reaction based on another rolling circle amplification.
  • Figure 6 shows that the detection method of the present application has methylation detection accuracy for clinical response.
  • the term "about” when used to designate certain numerical ranges is not limited to the precise value specified, but may include values that differ from the specified value. For example, values that vary by as much as 10% from the specific values recited may be included. Thus, the term “about” is used to encompass a variation of ⁇ 10% or less, ⁇ 5% or less, ⁇ 1% or less, ⁇ 0.5% or less from the specified value range, or a range of variation of ⁇ 0.1% or less.
  • sample includes, but is not limited to, any quantity of material from a currently living individual, or a formerly living individual.
  • the living individuals include, but are not limited to, humans, mice, rats, monkeys, dogs, rabbits and other animals, or plants.
  • Such substances include, but are not limited to, blood (eg, whole blood), plasma, serum, cerebrospinal fluid, interstitial fluid (eg, ascites), lavage fluid (eg, alveolar), urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes , monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen, etc.
  • the sample may also include living tissue and/or processed tissue, such as biopsied tissue (eg, liquid biopsy tissue), formalin-fixed and paraffin-embedded (FFPE) tissue, and the like.
  • biopsied tissue eg, liquid biopsy tissue
  • FFPE formalin-fixed and paraffin-embedded
  • the sample e.g., a biological sample
  • Samples can be from any source, such as biological specimens or environmental sources.
  • a biological specimen includes any tissue or material obtained from a living or dead organism that may contain an analyte or nucleic acid in or from an analyte.
  • biological samples include respiratory tissue, exudate (e.g., bronchoalveolar lavage fluid), biopsy, sputum, peripheral blood, plasma, serum, lymph nodes, gastrointestinal tissue, stool, urine, cerebrospinal fluid, interstitial fluid (e.g., , pleural effusion), lavage fluid (eg, alveoli), or other fluid, tissue, or material.
  • exudate e.g., bronchoalveolar lavage fluid
  • biopsy sputum
  • peripheral blood plasma
  • serum serum
  • lymph nodes e.g., gastrointestinal tissue, stool, urine, cerebrospinal fluid, interstitial fluid (e.g., pleural effusion), lavage fluid (eg, alveoli), or other fluid, tissue, or material.
  • interstitial fluid e.g., pleural effusion
  • lavage fluid eg, alveoli
  • Examples of environmental samples include water, ice, soil, suspensions, debris, biofilms, atmospheric dust particles, and aerosols
  • Additional processing of the sample may include treatment that physically or mechanically disrupts tissue, cell aggregates, or cells, thereby releasing intracellular components, including nucleic acids, into a solution that may contain other components, such as enzymes , buffers, salts, detergents, etc.
  • the sample can also include tissue, eg, biopsied tissue (eg, liquid biopsy tissue), formalin-fixed and paraffin-embedded (FFPE) tissue, and the like.
  • contacting generally means at least partially intermixing two or more components. Contacting can be achieved by mixing the components in a fluid or semi-fluid mixture. Contact can also be achieved when one or more components are brought into physical contact with one or more other components on a solid surface, such as a solid tissue section or matrix.
  • nucleotide generally includes natural and unnatural nucleotides.
  • Nucleotides include, but are not limited to, naturally occurring nucleoside monophosphates, nucleoside diphosphates, and nucleoside triphosphates; deoxyadenosine monophosphates, deoxyadenosine diphosphates, and deoxyadenosine triphosphates; deoxyguanosine monophosphates, Deoxyguanosine diphosphate and deoxyguanosine triphosphate; deoxythymidine monophosphate, deoxythymidine diphosphate and deoxythymidine triphosphate; deoxycytidine monophosphate, deoxycytidine diphosphate and deoxycytidine triphosphate; deoxyurine glycoside monophosphate, deoxyuridine diphosphate and deoxyuridine triphosphate; and deoxyinosine monophosphate, deoxyinosine diphosphate and deoxyinosine triphosphate (referred to as
  • Nucleotides also include, but are not limited to, modified nucleotides and nucleotide analogs.
  • Modified nucleotides and nucleotide analogs include, but are not limited to, dideoxynucleotides, acyclic nucleotides, deazapurine nucleotides, e.g., 7-deaza-deoxyguanosine (7-deaza -dG) monophosphate, diphosphate and triphosphate and 7-deaza-deoxyadenosine (7-deaza-dA) monophosphate, diphosphate and triphosphate, deuterium-deoxythymidine (deuterium-dT) monophosphate, Diphosphates and triphosphates, methylated nucleotides, eg, 5-methyldeoxycytidine triphosphate, 13C/15N labeled nucleotides and deoxyinosine monophosphates, diphosphates and triphosphates.
  • nucleic acid generally refers to polynucleotide compounds including oligonucleotides comprising nucleosides or nucleoside analogs having Nitrogen-containing heterocyclic bases or base analogs covalently linked by phosphodiester bonds or other bonds.
  • Nucleic acids include RNA, DNA, chimeric DNA-RNA polymers, or the like.
  • the backbone can be composed of a variety of linkages, including one or more of sugar phosphodiester linkages, peptide nucleic acid (PNA) linkages, phosphorothioate linkages, methylphosphonate linkages, or combinations thereof.
  • PNA peptide nucleic acid
  • the sugar moiety in the nucleic acid can be ribose, deoxyribose, or similar compounds with substituents such as 2'methoxy and 2'halide (eg, 2'-F) substituents.
  • Nitrogenous bases can be conventional bases (A, G, C, T, U), their analogs (e.g., inosine), derivatives of purine or pyrimidine bases (e.g., N4-methyldeoxyguanosine , deaza or azapurines, deaza or azapyrimidines, pyrimidines or purines with altered or substituted substituents at any of a variety of chemical positions (e.g., 2-amino-6-methylaminopurine , 06-methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and 04-alkyl-pyrimidine), or pyrazole compounds, such as unsubstituted or 3 -substit
  • Nucleic acids can include "abasic" positions, wherein the backbone does not have a nitrogenous base at one or more positions, e.g., one or more abasic positions can form a linker region that separates individual oligonucleotides nucleotide sequences joined together. Nucleic acids may contain only conventional sugar groups, bases and linkages as found in conventional RNA and DNA, or may include conventional components and substituents (such as conventional bases linked by a 2' methoxy backbone, or conventional base and a mixture of one or more base analogues).
  • LNA locked nucleic acid
  • LNA locked nucleic acid
  • nucleic acid generally refers to two or more nucleotides or nucleotide analogs linked by a covalent bond.
  • the nucleic acid can be any type of nucleic acid suitable for use with the methods described herein.
  • the nucleic acid can be DNA (such as complementary DNA (cDNA), genomic DNA (gDNA), plasmid and vector DNA, etc.), RNA (such as viral RNA, messenger RNA (mRNA), short inhibitory RNA (siRNA), ), ribosomal RNA (rRNA), tRNA, etc.), and/or DNA or RNA analogs (for example, comprising base analogs, sugar analogs and/or unnatural backbones, etc.).
  • DNA such as complementary DNA (cDNA), genomic DNA (gDNA), plasmid and vector DNA, etc.
  • RNA such as viral RNA, messenger RNA (mRNA), short inhibitory RNA (siRNA), ), ribosomal RNA (rRNA), tRNA, etc.
  • a nucleic acid can be in any form useful for performing the methods described herein (eg, linear, circular, supercoiled, single-stranded, double-stranded, etc.).
  • the nucleic acid may be or may be derived from a plasmid, bacteriophage, autonomously replicating sequence (ARS), centromere, artificial chromosome, chromosome, cell, nucleus, or cytoplasm of a cell.
  • the nucleic acid is from a single chromosome (eg, a nucleic acid sample can be from one chromosome of a sample obtained from a diploid organism).
  • the nucleic acid may be from the paternal allele, the maternal allele, or both maternal and paternal alleles.
  • oligonucleotide and “oligomer” are used interchangeably and generally refer to nucleic acid polymers generally consisting of less than 1,000 nucleotides (nt), Polymers ranging in length from about 2 nt to about 900 nt are included.
  • the oligonucleotide may comprise 5 nt to 500 nt, eg may comprise 10 nt to 150 nt.
  • the oligonucleotides can be prepared synthetically using any known in vitro chemical or enzymatic methods, and can be purified after synthesis using standard methods including, for example, high performance liquid chromatography (HPLC).
  • representative oligonucleotides include, for example, primers, promoters, detection probe oligonucleotides, target capture oligonucleotides, and the like.
  • primer generally refers to an oligonucleotide whose at least 3' end is complementary to a nucleic acid template, and which complexes with the template (for example, by hydrogen bonding or hybridization) to obtain a primer- A template complex suitable for priming synthesis by an RNA- or DNA-dependent nucleic acid polymerase (eg, DNA polymerase or RNA polymerase).
  • a primer can be extended by adding a covalently bonded nucleotide base to the 3' end of the primer, which base is complementary to the template. What is obtained is a primer extension product.
  • primers may be at least 10 nucleotides in length, and may extend to a length of about 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides.
  • tagged oligonucleotide generally refers to an oligonucleotide comprising at least a first region and a second region, wherein the first region comprises a "target hybridization sequence” capable of interacting with The target nucleic acid sequence of interest hybridizes, and wherein the second region comprises a "tag sequence” located 5' to the target hybridizing sequence and unable to stably hybridize or bind to a target nucleic acid comprising the target nucleic acid sequence.
  • Hybridization of the target hybridizing sequence to the target nucleic acid sequence results in a "tagged target nucleic acid sequence.”
  • the "tag sequence” or “heterologous tag sequence” can be essentially any sequence provided that the sequence does not hybridize stably to the target nucleic acid sequence of interest.
  • the tag sequence cannot stably hybridize to any sequence obtained from the genome of the organism to be tested, or cannot hybridize to any target nucleic acid under the reaction conditions.
  • the tag sequence present in the tagged oligonucleotide is designed not to substantially impair or prevent the ability of the target hybridizing sequence to hybridize to its target sequence.
  • the tag sequence can be of sufficient length such that once the complement of the tag sequence is incorporated into the initial DNA primer extension product, tag-specific primers can then be used to engage in subsequent amplification.
  • the tag sequences of the present application are typically at least 10 nucleotides in length, and can be extended to, for example, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length.
  • the tagged oligonucleotide is a "tagged primer" that includes a tag sequence and a target hybridization sequence.
  • the tagged oligonucleotide is a "tagged promoter oligonucleotide" comprising a tag sequence, a target hybridization sequence, and a promoter sequence, which may be located 5' to the tag sequence end and acts to initiate transcription from this 5' end.
  • the term "amplification" of a target nucleic acid generally refers to the process of constructing in vitro a nucleic acid strand identical or complementary to at least a part of a target nucleic acid sequence or a general sequence or a tag sequence as an alternative sequence of the target nucleic acid sequence, This process can only occur if the target nucleic acid is present in the sample.
  • nucleic acid amplification uses one or more nucleic acid polymerases and/or transcriptases to produce multiple copies of a target polynucleotide or fragment thereof, or multiple copies of a sequence complementary to the target polynucleotide or fragment thereof , or multiple copies of a universal sequence or tag sequence that have been introduced into the amplification system as a surrogate sequence for the target polynucleotide, for example in the detection step to indicate a certain The presence of the target polynucleotide at the moment, either serves as a site for further priming in an amplification reaction, or for a sequencing-related process or sequencing reaction.
  • TMA transcription-mediated amplification
  • NASBA nucleic acid sequence-based amplification
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription - PCR
  • LCR ligase chain reaction
  • linear amplification refers to an amplification process designed to increase the production of a target nucleic acid in a reaction linearly proportional to the amount of the target nucleic acid.
  • transcription-related reactions can be used to make multiple copies of RNA from target DNA, where the increase in copy number can be described by a linear factor (e.g., starting copy of template x 100).
  • the linear amplification of the first stage can result in at least a 10-fold increase in the initial number of the target nucleic acid strand or the complementary strand of the target nucleic acid strand before the second stage amplification reaction begins, e.g. At least 100 times, such as 10 to 1000 times.
  • linear amplification refers to an amplification reaction that does not result in exponential amplification of a target nucleic acid sequence.
  • linear amplification does not refer to a method of making only a single copy of a nucleic acid strand.
  • linear amplification can be based on known reactions involving nucleic acid transcription reactions, strand displacement amplification reactions, and/or rolling circle amplification reactions, among others.
  • the term "exponential amplification” generally refers to an amplification process designed to increase the production of target nucleic acid in a reaction that is geometrically proportional to the amount of target nucleic acid.
  • PCR produces one strand of DNA for each original target strand and for each synthetic strand present.
  • transcription-associated amplification produces multiple RNA transcripts for each original target strand as well as for each subsequently synthesized strand.
  • the amplification is exponential as the synthesized strand is used as template in subsequent rounds of amplification.
  • An amplification reaction need not actually produce an exponentially increasing amount of nucleic acid to be considered exponentially amplified, so long as the amplification reaction is designed to produce such an increase.
  • transcription-associated amplification methods amplify a target sequence by generating multiple transcripts from a nucleic acid template.
  • Such methods typically employ one or more oligonucleotides (one of which provides the promoter sequence, deoxynucleoside triphosphates (dNTPs), ribonucleoside triphosphates (rNTPs)) and RNA polymerase and DNA polymerase activity to generate a functional promoter sequence near the target sequence and then transcribe the target sequence from the promoter.
  • oligonucleotides one of which provides the promoter sequence, deoxynucleoside triphosphates (dNTPs), ribonucleoside triphosphates (rNTPs)
  • RNA polymerase and DNA polymerase activity to generate a functional promoter sequence near the target sequence and then transcribe the target sequence from the promoter.
  • transcription-associated amplification examples include transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), single-primer transcription-associated amplification, and self-sustaining sequence amplification (3SR), among others.
  • TMA transcription-mediated amplification
  • NASBA nucleic acid sequence-based amplification
  • 3SR self-sustaining sequence amplification
  • single primers can be used to amplify nucleic acids in vitro by making transcripts indicative of the presence of a target nucleic acid.
  • the single primer approach can use an oligonucleotide primer and optionally a blocker molecule (eg, a termination oligonucleotide) to terminate DNA extending from the target strand.
  • a blocker molecule eg, a termination oligonucleotide
  • the method can synthesize multiple copies of a target sequence by treating a target nucleic acid comprising the target sequence with: 1) an oligonucleotide primer that hybridizes to the 3' end of the target sequence so that primers can be primed from the 3' end An extension reaction, and 2) a blocker molecule that binds to the target nucleic acid adjacent to or near the 5' end of the target sequence.
  • the oligonucleotide primer is extended by using a DNA polymerase in a primer extension reaction to obtain a DNA primer extension product complementary to the target sequence, wherein the DNA primer extension product has a characteristic determined by the blocker molecule and is compatible with the target sequence
  • the 5' end is complementary to the 3' end.
  • a primer is hybridized to a target DNA at a position in the 3' portion of the target sequence and a primer is hybridized to a stop nucleotide at a position in the 5' portion of the target sequence. to synthesize multiple copies of the target sequence.
  • examples of single-primer transcription-associated amplification methods do not require the use of stop (oligo)nucleotides.
  • target nucleic acid generally refers to any nucleic acid comprising a sequence to be detected.
  • the target nucleic acid can be DNA or RNA.
  • Target nucleic acid can be of any origin, such as genomic DNA, mRNA, cDNA, cfDNA, ctDNA, and the like.
  • Target nucleic acids can be naturally occurring or synthetic (eg, amplification products, vectors, etc.).
  • a target nucleic acid can be, but need not be, purified or isolated.
  • the target nucleic acid can be derived from plant or animal tissue, or taken from a reaction mixture.
  • the target nucleic acid is not limited in length, although the target nucleic acid may be exposed to a restriction endonuclease prior to detection or identification by the methods of the present application.
  • label generally refers to a molecular moiety or compound that can be detected or produces a detectable response, which can be directly or indirectly attached to a labeled nucleic acid molecule.
  • Direct labeling can use bonds or interactions to link the label and the nucleic acid molecule to be labeled, including covalent bonds, non-covalent interactions (hydrogen bonds, hydrophobic and ionic interactions), or Chelate or coordination complex.
  • Indirect labeling can use directly or indirectly labeled bridging moieties or linkers (eg, antibodies, oligonucleotides, or other compounds) that amplify the signal. Labels include any detectable moiety.
  • detectable moieties include radionuclides, ligands (e.g., biotin or avidin), enzymes, enzyme substrates, reactive groups, chromophores (detectable dyes, particles or beads), Fluorophores, or luminescent compounds (eg, bioluminescent labels, phosphorescent labels, or chemiluminescent labels).
  • ligands e.g., biotin or avidin
  • enzymes e.g., enzyme substrates, reactive groups, chromophores (detectable dyes, particles or beads), Fluorophores, or luminescent compounds (eg, bioluminescent labels, phosphorescent labels, or chemiluminescent labels).
  • sample generally refers to the removal of one or more components of a mixture (eg, a sample) to separate from one or more other components of the mixture.
  • Sample components include nucleic acids, which may include cell fragments, proteins, carbohydrates, lipids, and other compounds.
  • the term "multiple target nucleic acids” generally refers to more than one target nucleic acid.
  • the plurality of target nucleic acids can be about 2 to about 10,000 nucleic acids, about 2 to about 1000 nucleic acids, about 2 to about 500 nucleic acids, or, for example, 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 or 10000 target nucleic acids.
  • multiple target nucleic acids are in one or more reaction vessels, and each reaction vessel contains more than one target nucleic acid. In certain embodiments, multiple target nucleic acids are in one reaction vessel. In certain embodiments, the plurality of target nucleic acids is from about 2 to about 100 target nucleic acids. In certain embodiments, from about 2 to about 100 target nucleic acids are in a single reaction vessel.
  • hybridizing sequence generally refers to an oligonucleotide that is capable of being specific to an amplification product or part thereof, a target nucleic acid or part thereof, a target nucleic acid or part thereof, a variant of a target nucleic acid or part thereof, or its complement.
  • sexually hybridized nucleotide sequences The hybridizing sequence is easy to design and select, and the sequence can be of a length suitable for hybridizing to the amplification product described herein, the target sequence or its complement.
  • the hybridizing sequence in each oligonucleotide is about 5-about 200 nucleotides in length (e.g., about 5-10, about 10-15, about 15-20, about 20- 25, about 25-30, about 30-35, about 35-40, about 40-45 or about 45-50, about 50-70, about 80-90, about 90-110, about 100-120, about 110- 130, about 120-140, about 130-150, about 140-160, about 150-170, about 160-180, about 170-190, about 180-200 nucleotides).
  • hybridization conditions generally refers to conditions under which two nucleic acids having complementary nucleotide sequences can interact with each other.
  • Hybridization conditions can be of high stringency, medium stringency or low stringency, and conditions of varying degrees of stringency are known. Depending on the application of interest, hybridization conditions are generally chosen to allow amplification and/or extension.
  • specifically hybridize refers to hybridizing to a certain amplification product or target nucleic acid in a sample without substantially hybridizing to other amplification product substances or target nucleic acids in the sample.
  • terminating nucleotide is used interchangeably with “terminating nucleotide”, “chain terminator (chain terminator) reagent” or “chain terminator” and generally refers to the A molecule that stops the extension reaction when it is in the extension reaction.
  • Chain terminators may include nucleotide analogs which, when present in a polynucleotide chain or oligonucleotide, prevent further extension of the chain or oligonucleotide.
  • the terminating nucleotide is a terminating nucleotide.
  • the terminating nucleotide is a modified nucleotide that, when incorporated into the 3' end of a nucleic acid molecule (e.g., an oligonucleotide) during an extension reaction, will not allow the nucleotide to be The oligonucleotide is further incorporated.
  • the terminating nucleotide is not removed from the oligonucleotide or polynucleotide chain in the presence of an enzyme having 3'-5' exonuclease activity.
  • the '3OH of the pentose sugar of a nucleotide can be replaced by a moiety that produces a chain-terminating nucleotide and is also resistant to the formation of nucleotides by an enzyme with 3'-5' exonuclease activity. Removal is resistant.
  • the 3' position of the pentose sugar of the terminating nucleotide is modified to replace the OH with another moiety, including but not limited to, a phosphoryl group, an acetyl group, 3'-O-methyl, 3' -O-(2-nitrobenzyl), 3'-O-allyl, 3'-azido and 3'-amino.
  • the 3'OH group is replaced with hydrogen.
  • the modified nucleotides are dideoxynucleotides.
  • the modified nucleotides are acyclic nucleotides. Examples of terminating nucleotides include: dideoxynucleotides such as ddA (dideoxyadenine), ddT (dideoxythymine), ddC (dideoxycytosine), ddG (dideoxyguanine), and ddU (dideoxyguanine). deoxyuracil) and acyclic nucleotides such as acyATP, acyCTP, acyGTP, acyTTP and acy-bromo-UTP.
  • the term “signal-to-noise ratio” generally refers to the quantitative measurement of signal quality by quantifying the ratio of the intensity of signal relative to noise when using a detection process (eg, mass spectrometry).
  • a detection process eg, mass spectrometry
  • an intensity peak on one spectrum has a higher signal-to-noise ratio than a lower intensity peak produced by the same analyte (eg, an extended oligonucleotide species) on another spectrum.
  • analyte eg, an extended oligonucleotide species
  • the methods of the present application comprise contacting the oligonucleotide hybrid with an extension composition comprising one or more terminating nucleotides under extension conditions.
  • sensitivity generally refers to the amount of analyte that can be detected for a given signal-to-noise ratio using a detection method (eg, mass spectrometry). In some embodiments, sensitivity can be improved by reducing background noise levels.
  • cfDNA cell-free DNA
  • cfDNA can include both normal cells and DNA derived from cancer cells.
  • cfDNA is usually obtained from blood or plasma ("circulating").
  • cfDNA can be released into the circulation through secretion or through cell death processes such as necrosis or apoptosis.
  • some cfDNA can be ctDNA.
  • circulating tumor DNA or "circulating cancer DNA” refers to the cell-free DNA (cfDNA) fraction derived from tumors.
  • the present application provides a method for identifying the presence, absence or amount of one or more target nucleic acids in a sample.
  • the method of the present application may comprise: a) processing the sample under conditions that enable linear amplification of nucleic acid derived from the sample to generate a linear amplification product of the nucleic acid.
  • the method may further comprise: b) performing exponential amplification on the linear amplification product to generate an exponential amplification product of the nucleic acid.
  • the method may further comprise: c) contacting the exponentially amplified product of the nucleic acid with an extension primer under extension conditions that include a stop nucleotide, thereby generating an extended oligonucleotide.
  • the method may further comprise: d) analyzing the extended oligonucleotides, thereby identifying the presence, absence or amount of the one or more target nucleic acids in the sample.
  • the present application provides a method of identifying a biomarker associated with a disease, a disease outcome, and/or a treatment regimen outcome.
  • the method may comprise the steps of: i) identifying the presence, absence or amount of one or more target nucleic acids derived from one or more samples according to the methods described herein, the one or more samples originating from From one or more subjects with known disease, disease outcome, and/or outcome of a treatment regimen; ii) identifying one or more target nucleic acids derived from one or more samples according to the methods described herein The presence, absence or amount of said one or more samples is derived from a normal subject; and iii) the presence, absence or amount of said one or more target nucleic acids in identifying step i) is consistent with step ii) The difference between the presence, absence or amount of the one or more target nucleic acids in, and accordingly, the difference is identified as a biomarker associated with the disease, disease outcome and/or treatment regimen outcome .
  • the application provides a method of identifying methylation associated with a disease, disease outcome, and/or treatment regimen outcome.
  • the method may comprise the steps of: i) identifying methylated or unmethylated nucleotides in one or more target nucleic acids derived from one or more samples according to the methods described herein, said The one or more samples are derived from one or more subjects with known disease, disease outcome, and/or treatment regimen outcome; ii) according to the methods described herein, identifying a sample derived from the one or more samples methylated or unmethylated nucleotides in one or more target nucleic acids, said one or more samples being derived from a normal subject; and iii) identifying said one or more targets in step i) The difference between the methylated or unmethylated nucleotides in the nucleic acid and the methylated or unmethylated nucleotides in the one or more target nucleic acids in step ii), and accordingly, The differentially methylated or unmethylated nucle
  • the present application provides a kit for identifying the presence, absence or amount of one or more target nucleic acids in a sample according to the methods described herein.
  • the kit may comprise: a linear amplification component; an exponential amplification component; and an extension component comprising a terminating nucleotide and an extension primer.
  • the present application provides a kit for identifying the presence, absence or amount of one or more target nucleic acids in a sample.
  • the kit may comprise: a linear amplification component; an exponential amplification component; and an extension component comprising a terminating nucleotide and an extension primer.
  • terminator nucleotides, a thermostable elongase, and an extension primer can be included in the extension component.
  • the present application provides a system.
  • the system may comprise a kit as described herein, and optionally one or more parts selected from the group consisting of instructions for use, reagents and means for using the kit.
  • the methods and products (eg, kits, systems, etc.) of the present application can be used to identify the presence, absence or amount of one or more target nucleic acids in a sample.
  • the nucleic acids identified may be oligonucleotides or polynucleotides, including but not limited to natural nucleic acids (such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA)), synthetic nucleic acids, non-natural nucleic acids (such as peptide nucleic acid (PNA)), unmodified nucleic acid, modified nucleic acid (eg, methylated DNA, labeled nucleic acid, nucleic acid molecule with one or more modified nucleotides), etc.
  • PNA peptide nucleic acid
  • polynucleotide refers to two or more nucleotides or nucleotide analogs linked by a covalent bond.
  • the nucleic acid can be any type of nucleic acid suitable for use in the methods described herein.
  • the nucleic acid can be DNA (such as complementary DNA (cDNA), genomic DNA (gDNA), cfDNA, ctDNA, plasmid and vector DNA, etc.), or a nucleic acid analog (for example, containing base analogs, sugar analogs substances and/or non-natural frameworks, etc.).
  • a nucleic acid can be in any form useful for performing the methods described herein (eg, linear, circular, supercoiled, single-stranded, double-stranded, etc.).
  • the nucleic acid may be, or may be derived from, a plasmid, bacteriophage, autonomously replicating sequence (ARS), centromere, artificial chromosome, chromosome, cell, nucleus, or cytoplasm of a cell.
  • the nucleic acid can be from a single chromosome (eg, a nucleic acid sample can be from one chromosome of a sample obtained from a diploid organism).
  • the nucleic acid may be from the paternal allele, the maternal allele, or both maternal and paternal alleles.
  • the nucleic acid described herein can be cell-free DNA, such as cfDNA in a tissue or sample, such as ctDNA from a tumor or cancerous tissue/site.
  • the target nucleic acid molecule to be detected or identified can be of any length.
  • it may comprise at least 1 (for example, it may comprise at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 , 80, 85, 90, 95, 100 or more than 100) nucleotides.
  • a target nucleic acid molecule to be detected or identified may comprise one or more modifications, for example it may comprise one or more methyl groups or methylation states, one or more phosphate groups, one or more One and or more deletions, additions or substitutions of multiple acetyl groups and 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 specific mutations, the presence of nucleotide substitutions (e.g., single nucleotide polymorphisms (SNPs)) presence or absence of repetitive sequences (e.g., dinucleotide, trinucleotide, tetranucleotide, pentanucleotide repeats), presence or absence of markers (e.g., microsatellites) and distinguishing sequences (e.g. , the presence or absence of sequences that distinguish one organism from another (eg, sequences that distinguish one strain of virus from another strain of virus).
  • SNPs single nucleotide polymorphisms
  • markers e.g., microsatellites
  • distinguishing sequences e.g. , the presence or absence of sequences that distinguish one organism from another (eg, sequences that distinguish one strain of virus from another strain of
  • variants of a target nucleic acid may be present in a sample at approximately equal (eg, SNP) frequencies or copy numbers. In certain embodiments, variants of a target nucleic acid may be present in a sample at different frequencies or copy numbers. In certain embodiments, one variant may be present in greater abundance than other variants. In certain embodiments, a more abundant variant is referred to as a wild type, and a less abundant variant is referred to as a mutant. In some cases, the target nucleic acid comprises a first and a second variant, wherein the first or second variant exhibits a greater abundance than the other variant, i.e.
  • a variant exhibiting greater abundance is typically present in a higher concentration or represented by a higher number of molecules (eg, copy) number when compared to another variant.
  • Higher concentrations can be 2-fold or more.
  • the higher concentration is 10-fold or more.
  • the higher concentration is 100-fold, 1000-fold, or 10000-fold or more.
  • one variant represents the wild-type sequence and is present at a concentration 100-fold or more greater than the other variant.
  • the concentration of a variant (low abundance variant) is significantly lower than another variant (eg, wild type, high abundance variant).
  • the method of the application can be used to detect less than 30%, 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2% of the total nucleic acid in the sample , 1%, 0.8%, 0.75%, 0.5%, 0.1%, 0.05%, 0.01% or less of the presence or absence of low abundance target nucleic acid (eg, cfDNA or ctDNA).
  • the methods of the present application can be used to detect the presence or absence of low-abundance target nucleic acids (eg, cfDNA or ctDNA) that account for about 1% to about 10% of the total nucleic acid in a sample.
  • the methods of the present application can be used to detect the presence or absence of a low-abundance target nucleic acid (eg, cfDNA or ctDNA) that accounts for about 5% or less of the total nucleic acid in a sample. In certain embodiments, the methods of the present application can be used to detect the presence or absence of low-abundance target nucleic acids (eg, cfDNA or ctDNA) accounting for about 5%-0.75% of the total nucleic acid quantity in a sample.
  • a low-abundance target nucleic acid eg, cfDNA or ctDNA
  • the methods of the present application can be used to detect the presence or absence of low-abundance target nucleic acids (eg, cfDNA or ctDNA) accounting for about 5% to about 0.1% of the total nucleic acid quantity in a sample. In certain embodiments, the methods of the present application can be used to detect the presence or absence of low-abundance target nucleic acid (eg, cfDNA or ctDNA) accounting for about 1% or less of the total nucleic acid amount in a sample.
  • low-abundance target nucleic acids eg, cfDNA or ctDNA
  • the methods of the present application can be used to detect the presence or absence of low-abundance target nucleic acids (eg, cfDNA or ctDNA) accounting for about 0.1% to about 0.001% of the total nucleic acid quantity in a sample.
  • target nucleic acids eg, cfDNA or ctDNA
  • the content of target nucleic acid (for example, DNA, such as cfDNA, ctDNA, etc.) in the sample can be less than about 100ng (for example, less than about 90ng, less than about 80ng, less than about 70ng, less than about 60ng, less than About 50 ng, less than about 40 ng, less than about 30 ng, less than about 20 ng, less than about 15 ng, less than about 10 ng, less than about 9 ng, less than about 8 ng, less than about 7 ng, less than about 6 ng, less than About 5 ng, less than about 4 ng, less than about 3 ng, less than about 2 ng, less than about 1 ng, less than about 0.9 ng, less than about 0.8 ng, less than about 0.7 ng, less than about 0.6 ng, less than about 0.5 ng, less than about 0.45 ng, less than about 0.4 ng, less than about 0.35 ng, less than about 0.3 ng, less than about 0.2 ng or
  • the content of target nucleic acid (e.g., DNA, such as cfDNA, ctDNA, etc.) in the sample can be about 20% or less of the total nucleic acid content, such as about 15% or less, about 10% or less, about 9% or less, about 8% or less , about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.9% or less, about 0.8% or less, about 0.7% or less , about 0.6% or less, about 0.5% or less, about 0.4% or less, about 0.3% or less, about 0.2% or less, about 0.1% or less, about 0.05% or less, about 0.04% or less, about 0.03% or less, about 0.02% or less , about 0.01% or less.
  • the percentages may be mass percent, volume percent and/or mole percent.
  • the sample can comprise a mixture of one or more target nucleic acids (each target nucleic acid can have low-abundance and high-abundance variants), or can be obtained by combining more than one sample comprising one or more target nucleic acids Combined to form a mixture (each target nucleic acid may have low-abundance and high-abundance variants).
  • Low-abundance variants may be variants of high-abundance variants and may include, but are not limited to, mutants (low-abundance variants) of the wild-type (high-abundance variant) allele, present in more than one variants of genes of the host species (e.g., viral oncogenes (low-abundance genes), which are variants of normal healthy genes (high-abundance variants), polymorphisms, including single nucleotide polymorphisms ( SNPs), insertions, deletions, or other mutant forms of high-abundance variants.
  • mutants low-abundance variants of the wild-type (high-abundance variant) allele
  • genes of the host species e.g., viral oncogenes (low-abundance genes)
  • high-abundance variants low-abundance genes
  • polymorphisms including single nucleotide polymorphisms ( SNPs),
  • the methods of the present application can be used to simultaneously identify multiple target nucleic acids that may exist in a sample.
  • the plurality of target nucleic acids may refer to more than one target nucleic acid.
  • the plurality of target nucleic acids can be about 2 to about 10,000 target nucleic acids, about 2 to about 1000 nucleic acids, about 2 to about 500 nucleic acids, or 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 or 10000 target nucleic acids.
  • the plurality of target nucleic acids are located in one or more reaction vessels, and each reaction vessel may contain more than one target nucleic acid. In certain embodiments, the multiple target nucleic acids are all located in the same reaction vessel. In certain embodiments, the plurality of target nucleic acids is about 2 to about 200 target nucleic acids. In certain embodiments, the about 2 to about 200 target nucleic acids are in a single reaction vessel.
  • nucleic acid detection or identification can detect one or more loci (e.g., about 2 to about 10,000 loci, about 2 to about 1,000 loci) in DNA (especially cfDNA, such as ctDNA) sites, about 2 to about 500 sites, or 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 or 10000 loci, can It includes different sites of the same nucleic acid molecule, and may also include whether methylation exists or the methylation status at the same site in different nucleic acid molecules).
  • loci e.g., about 2 to about 10,000 loci, about
  • a sample comprising nucleic acids may be derived from one or more sources and may comprise a mixture of target nucleic acids, each of which may have different copy numbers of one or more high abundance variants and low-abundance variants. Samples can also be pooled to generate a mixture that includes different target nucleic acids, which can have different abundances, and/or different copy numbers.
  • samples of the present application may be collected from biological, mineral or geological sites (eg, soil, rocks, deposits, fossils) or forensic sites (eg, crime scenes, contraband or suspected contraband).
  • the source can be environmental, such as geological, agricultural, battlefield or soil sources.
  • the source can also be any type of organism such as any plant, fungus, protozoa, prokaryote, virus or animal, including but not limited to: human, non-human, mammalian, reptile, bovine, cat, dog, goat, Pigs, monkeys, apes, orangutans, bulls, cows, bears, horses, sheep, poultry, mice, rats, fish, dolphins, whales, and sharks, etc., or any animal or organism with detectable nucleic acid.
  • Source can also refer to different parts of an organism, such as internal, external, living or dead cells, tissues, fluids, etc.
  • a sample may be a "biological sample,” which may be any material obtained from a source that is or was once living, such as an animal such as a human or other mammal, plant, bacterium, fungus, protist, or virus.
  • the source may be in any form, including, but not limited to, solid material such as tissue, cells, cell aggregates, cell extracts, or biopsy samples, or biological fluids such as urine, blood, saliva, amniotic fluid, exudates from areas of infection or inflammation fluid, or mouthwash containing oral cells, hair, cerebrospinal and joint fluid, and organs.
  • samples may include respiratory tissue, exudates (eg, bronchoalveolar lavage fluid), biopsies, sputum, peripheral blood, plasma, serum, lymph nodes, gastrointestinal tissue, feces, Urine, cerebrospinal fluid, interstitial fluid (eg, ascites), lavage fluid (eg, alveoli), or other fluid, tissue, or material.
  • a sample can be a processed specimen or material, such as obtained by treating the sample using filtration, centrifugation, sedimentation, or adsorption to a medium (eg, a matrix or carrier).
  • Additional processing of the sample may include treatment that physically or mechanically disrupts tissue, cell aggregates, or cells, thereby releasing intracellular components, including nucleic acids, into a solution that may contain other components, such as enzymes , buffers, salts, detergents, etc.
  • the sample can also include tissue, eg, biopsied tissue (eg, liquid biopsy tissue), formalin-fixed and paraffin-embedded (FFPE) tissue, and the like.
  • a sample can also be isolated at a different time point from another sample, where each sample can be from the same or different source.
  • a nucleic acid can be from a nucleic acid library, such as a DNA library.
  • a nucleic acid may be the product of nucleic acid purification or isolation and/or amplification of nucleic acid molecules in a sample.
  • the nucleic acid provided by the application for sequencing analysis may contain samples from one or from two or more samples (such as 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 1000 or more samples) of nucleic acid.
  • Nucleic acids can be treated in a variety of ways during, before or after the methods provided herein.
  • the length or mass of a nucleic acid can be reduced (e.g., by shearing, nuclease or restriction enzyme digestion, dephosphorylation, demethylation), the size or mass of a nucleic acid can be increased (e.g., by phosphorylation, reaction with a methylation specific reagent, attachment of a detectable label, etc.), can be treated with a nucleic acid cleavage inhibitor, etc.
  • unprocessed nucleic acid is provided for analysis according to the methods described herein.
  • the processed nucleic acid is provided for use in practicing the methods described herein.
  • nucleic acid can be extracted, isolated, purified or amplified from a sample.
  • a nucleic acid can be removed from its original environment (eg, the natural environment in which it is naturally produced or a host cell in which it is exogenously expressed) so that the nucleic acid is altered by "man-made" manipulation from its original environment.
  • Isolated nucleic acid generally has fewer non-nucleic acid components (eg, proteins, lipids, etc.) than the amount of components in the sample from which it was derived.
  • a composition comprising an isolated nucleic acid can be substantially isolated (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% free from Contains non-nucleic acid components).
  • purified nucleic acid refers to a nucleic acid provided that contains fewer non-nucleic acid components than the sample source from which it was derived.
  • a composition comprising a nucleic acid can be substantially purified (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% free of other nucleic acid).
  • the methods of the present application may include treating the sample under conditions that enable linear amplification of nucleic acid derived from the sample to generate a linear amplification product of the nucleic acid.
  • the nucleic acid in the sample can be separated, for example, a capture agent and/or a carrier (eg, a solid carrier, such as magnetic beads) containing the capture agent can be used.
  • a capture agent and/or a carrier eg, a solid carrier, such as magnetic beads
  • nucleic acids in a sample can be captured onto a solid support by interacting with immobilized capture probes.
  • the nucleic acid in the sample can be captured onto a solid support as a member of a trimolecular complex wherein the nucleic acid in the sample and the immobilized capture probe are bridged by a target capture oligonucleotide.
  • the solid support typically includes a plurality of magnetic or magnetizable particles or beads that can be manipulated using a magnetic field.
  • the step of isolating nucleic acid in the sample may also include washing the target capture oligonucleotide:target nucleic acid hybrid to remove undesired components that may interfere with subsequent amplification.
  • the nucleic acids in the sample are not further isolated prior to the linear amplification process.
  • the treatment for obtaining a linear amplification product may include: bringing the sample into contact with an oligonucleotide capable of binding to the nucleic acid to form an oligonucleotide hybrid; and making the oligonucleotide The nucleotide hybrid is contacted with the amplification composition under conditions enabling linear amplification to produce a linear amplification product of the nucleic acid.
  • the linear amplification is not sequence-specific amplification capable of amplifying only specific target sequences in a sample (e.g., the linear amplification is indifferent sequence amplification, also referred to as non-biased sexual or unbiased sequence amplification).
  • the linear amplification can amplify both nucleic acids present in the sample (eg, amplify nucleic acids present in the sample by substantially the same factor and/or ratio).
  • the conditions under which the linear amplification is performed generally do not support the exponential amplification of the nucleic acid in the sample.
  • the processing for obtaining a linear amplification product may include: ligating nucleic acid derived from the sample with an adapter sequence to form an adapter-containing nucleic acid; contacting oligonucleotides of sequence to form oligonucleotide hybrids; and contacting said oligonucleotide hybrids with an amplification composition under conditions capable of linear amplification to generate linear amplification of said nucleic acids amplified product.
  • the linker sequence can be ligated to substantially all nucleic acid molecules in the sample substantially indiscriminately (e.g., it can be ligated to nucleic acid molecules (e.g. DNA molecules such as cfDNA or ctDNA) in the sample one or both ends of the DNA molecule), for example, it can be ligated to the upstream or downstream, 5' or 3' end of the DNA molecule.
  • the ligation can be performed using a ligase (eg, T4 DNA ligase).
  • the nucleic acid in the sample e.g., isolated nucleic acid or unisolated nucleic acid, e.g., isolated or unisolated DNA in a sample, such as cfDNA
  • the nucleic acid in the sample can be end-repaired, e.g., rendered Flat ends.
  • deoxyadenosine dA can also be added to the 3' end of nucleic acid (e.g., DNA, such as cfDNA or ctDNA) in the sample (e.g., after performing said end repair).
  • a phosphate group P can also be added to the 5' end of nucleic acid (e.g., DNA, such as cfDNA or ctDNA) in the sample.
  • nucleic acids e.g., DNA, such as cfDNA or ctDNA
  • the ends of nucleic acids in a sample can be blunt-filled or trimmed using nucleic acid polymerase, and the 5' hydroxyl group can be converted to a 5' hydroxyl group using polynucleotide kinase in the presence of dNTPs. Phosphate group and converts 3' phosphate group to 3' hydroxyl group.
  • dATP is added to the 3' end of the nucleic acid molecule (eg, DNA) using a polymerase that does not have 3'-5' exo-activity in the presence of excess dATP.
  • the nucleic acid e.g., the 3' end of the nucleic acid
  • the linker sequence can be aligned after the end repair, the addition of the 3' dA and/or the 5' P Ligated to form the adapter-containing nucleic acid.
  • the linker sequence can be linked directly or indirectly (e.g., through other polynucleotide fragments or sequences) to the 3' end of the nucleic acid.
  • the 5' end of the linker sequence is directly or indirectly linked to the 3' end of the nucleic acid.
  • the linker sequence may be a double-stranded nucleic acid (eg, DNA) sequence.
  • the linker sequence may be a single-stranded nucleic acid sequence, such as a single-stranded DNA sequence.
  • the linker sequence may comprise about 1-100 nucleotides or base pairs, for example about 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-35, 1 -30, 1-25, 1-20, 1-15 or 1-10 nucleotides or base pairs.
  • the promoter sequence of RNA polymerase can be included in the described linker sequence, such as SP6 promoter sequence, T7 promoter sequence and/or T3 promoter sequence.
  • the linker sequence (e.g., the promoter sequence of the RNA polymerase therein) may comprise one or more modifications that allow the linker sequence to undergo bisulfite treatment , its nucleic acid sequence does not change.
  • the promoter sequence of the RNA polymerase e.g., the SP6 promoter sequence, T7 promoter sequence and/or T3 promoter sequence
  • the cytosine contained in the linker sequence may be a methylated cytosine.
  • the adapter sequence used may contain a methylated RNA polymerase promoter sequence (for example, a methylated SP6 promoter sequence, a methylated methylated T7 promoter sequence and/or methylated T3 promoter sequence).
  • a methylated RNA polymerase promoter sequence for example, a methylated SP6 promoter sequence, a methylated methylated T7 promoter sequence and/or methylated T3 promoter sequence.
  • one or more bases in the promoter sequence of the RNA polymerase eg, the SP6 promoter sequence, T7 promoter sequence and/or T3 promoter sequence
  • the cytosine contained in the linker sequence eg, the promoter sequence of the RNA polymerase therein
  • the linker sequence may comprise the nucleic acid sequence shown in any one of SEQ ID NO: 1-2 and 19-20.
  • the linker-containing nucleic acid can be specifically bound to the linker sequence under conditions that allow nucleic acid hybridization (for example, when the linker sequence is a double-stranded nucleic acid, it can specifically bind to the linker sequence).
  • oligonucleotides which are also referred to as "linear amplification primers" in this application
  • the oligonucleotides (“linear amplification primers") capable of specifically binding to the adapter sequence may or may not contain modified nucleotides. In certain instances, the oligonucleotides are not methylated.
  • the oligonucleotide capable of specifically binding to the linker sequence may be at least partially complementary to the linker sequence.
  • the oligonucleotide may be at least partially complementary to the sense strand of the adapter sequence.
  • the oligonucleotide can be at least partially complementary to the antisense strand of the linker sequence.
  • the oligonucleotide capable of specifically binding to the linker sequence may be substantially complementary to the linker sequence (e.g., fully complementary, or at least 80% complementary, such as at least 85% complementary, at least 90% complementary, At least 91% complementary, at least 92% complementary, at least 93% complementary, at least 94% complementary, at least 95% complementary, at least 96% complementary, at least 97% complementary, at least 98% complementary or at least 99% complementary, e.g., 100 % complementary).
  • the oligonucleotide may be a single-stranded nucleic acid sequence, such as a single-stranded DNA sequence (for example, it may comprise about 1-100 nucleotides, such as about 1-90, 1-80, 1-70, 1 -60, 1-50, 1-40, 1-35, 1-30, 1-25, 1-20 or 1-15 nucleotides, for example at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides nucleotides nucle
  • the oligonucleotide capable of specifically binding to the linker sequence may comprise the nucleic acid sequence shown in SEQ ID NO:3.
  • the oligonucleotide hybrid can include one or more nucleotides that do not hybridize to the template (eg, the linker sequence).
  • the oligonucleotide hybrids may include one or more mismatched nucleotides (e.g., non-complementary nucleotides) and, sometimes, non-hybridizing 5' and/or 3' regions of the nucleic acid.
  • the oligonucleotide hybrid can comprise a tag (eg, a mass distinguishable tag, sequence tag, luminescent tag, or radioactive tag).
  • the oligonucleotide hybrid can comprise a capture agent (eg, biotin other capture agent).
  • the oligonucleotide hybrid may comprise a terminating nucleotide.
  • said linear amplification can be performed on a molecule of interest (eg, a nucleic acid molecule) comprising said oligonucleotide hybrid.
  • a molecule of interest eg, a nucleic acid molecule
  • the linear amplification reaction is performed under conditions that do not support exponential amplification of the nucleic acid sequence of interest (eg, the nucleic acid molecule in the sample).
  • the linear amplification reaction will typically result in about 2-fold to about 10,000-fold amplification of the nucleic acid sequence, such as 10-fold to about 10,000-fold amplification of the nucleic acid sequence (e.g., about 50-fold, about 100-fold, about 200 times, about 300 times, about 400 times, about 500 times, about 600 times, about 700 times, about 800 times, about 900 times, about 1000 times, about 1500 times, about 2000 times, about 3000 times, about 5000 times , about 8000 times, about 10000 times or more nucleic acid sequence amplification).
  • 10-fold to about 10,000-fold amplification of the nucleic acid sequence e.g., about 50-fold, about 100-fold, about 200 times, about 300 times, about 400 times, about 500 times, about 600 times, about 700 times, about 800 times, about 900 times, about 1000 times, about 1500 times, about 2000 times, about 3000 times, about 5000 times , about 8000 times, about 10000 times or more nucleic acid sequence amplification.
  • the linear amplification can be substantially isothermal.
  • the linear amplification can include a nucleic acid transcription reaction.
  • the linear amplification can comprise a nucleic acid transcription reaction, a strand displacement amplification reaction (SDA), and/or a rolling circle amplification reaction (RCA).
  • the linear amplification can comprise non-enzymatic isothermal strand displacement and amplification (NISDA).
  • NISDA non-enzymatic isothermal strand displacement and amplification
  • the linear amplification is non-target-specific amplification that is not directed to a specific target.
  • the linear amplification can result in nucleic acid molecules in the sample being amplified substantially indiscriminately, in substantially the same or similar proportions.
  • the obtained linear amplification product may comprise RNA encoded by the nucleic acid (for example, the nucleic acid in the sample).
  • the linear amplification reaction may not involve the thermal cycling process characteristic of PCR and other common amplification techniques.
  • a linear amplification reaction can involve contacting a nucleic acid sequence to be amplified (e.g., a molecule of interest comprising the oligonucleotide hybrid) with an amplification composition (e.g., a linear amplification composition) that An augmenting composition (eg, a linear amplification composition) supports linear amplification of a target nucleic acid sequence but lacks at least one component required to exponentially amplify the target nucleic acid sequence.
  • the amplification composition (eg, a linear amplification composition) can include an amplification enzyme selected from the group consisting of reverse transcriptase, polymerase, and combinations thereof.
  • the polymerase may be selected from RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, DNA-dependent RNA polymerase, and combinations thereof.
  • RNA polymerase is included in the amplification composition (eg, a linear amplification composition).
  • the RNA polymer can be selected from T7 RNA polymerase, SP6 RNA polymerase and T3 RNA polymerase.
  • SP6 RNA polymerase can be included in the amplification composition (eg, a linear amplification composition).
  • a T7 promoter sequence is included in the linker sequence and/or the oligonucleotide hybrid
  • T7 RNA polymerase may be included in the amplification composition (eg, a linear amplification composition).
  • T3 RNA polymerase may be included in the amplification composition (eg, a linear amplification composition).
  • the linear amplification reaction preferably cannot support exponential amplification reaction.
  • the linear amplification reaction preferably cannot support exponential amplification reaction.
  • the conditions under which the reaction was run e.g., temperature, etc.
  • the components required for the exponential amplification and/or the inhibitors and/or the conditions under which the reaction is run can be selected from the group consisting of: amplification oligonucleotides (e.g., 5 'promoter sequence amplification oligonucleotides, non-promoter amplification oligonucleotides, or combinations thereof), enzymes (e.g., polymerases, such as DNA polymerases), nucleases (e.g., exonucleases enzymes, endonucleases, lyases, ribonucleases, phosphorylases, glycosylases, etc.), enzyme cofactors, chelating agents (e.g., EDTA or EGTA), ribonucleoside triphosphates (rNTP), deoxynucleoside Glycoside triphosphates (dNTPs), Mg 2+ , salts, buffers, enzyme inhibitors, blocking oligonucleotides (
  • the product obtained after the linear amplification reaction is a linear amplification product.
  • the linear amplification product may include RNA molecules transcribed from the nucleic acid molecules in the sample.
  • exponential amplification may be performed on the linear amplification product to generate an exponential amplification product of the nucleic acid.
  • an amplification composition (e.g., an exponential amplification composition) may be used, the components of which may include, but are not limited to: nucleotides (e.g., nucleotide triphosphates), modified core Nucleotides, oligonucleotides (e.g., primer oligonucleotides for polymerase-based amplification and oligonucleotide building blocks for ligase-based amplification), one or more salt (for example, magnesium-containing salt), one or more buffers, one or more polymerizing agents (for example, ligase, polymerase), one or more nicking enzymes (for example, for cutting double-stranded nucleic acid one chain enzyme) and one or more nucleases (eg, exonuclease, endonuclease, RNase).
  • nucleotides e.g., nucleotide triphosphates
  • oligonucleotides e.g., primer
  • Amplification conditions can also include certain reaction conditions, such as isothermal or temperature cycling conditions. Methods of cycling temperature in amplification methods are known, such as by thermal cycling devices.
  • the term "cycling" generally refers to amplification (eg, an amplification reaction or an extension reaction) using a single primer or multiple primers, in which temperature cycling is used.
  • amplification conditions can also include an emulsifier (eg, oil) that is used to form multiple reaction compartments in which a single nucleic acid molecular species can be amplified.
  • the exponential amplification reaction is a reaction performed under conditions that allow for exponential amplification of nucleic acid molecules in the sample or system.
  • the linear amplification product can be contacted with an amplification composition (eg, an exponential amplification composition) such that under suitable conditions exponential amplification of the nucleic acid can occur.
  • the exponential amplification composition typically includes minimal amounts of one or more components required for exponential amplification that are absent from the linear amplification composition.
  • one or more of the following components or conditions may be included in the exponential amplification composition: amplification oligonucleotide, reverse transcriptase, polymerase, nuclease, phosphorylase, enzyme cofactor, Chelating agents, ribonucleoside triphosphates (rNTPs), deoxynucleoside triphosphates (dNTPs), Mg2 + , pH optima, temperature optima, salts, and combinations thereof.
  • the polymerase is typically selected from RNA-dependent DNA polymerases, DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, and combinations thereof.
  • reverse transcriptase and DNA polymerase are included in the exponential amplification composition.
  • the exponential amplification comprises polymerase chain reaction PCR.
  • the exponential amplification reaction is a RT-PCR reaction, such as a one-step RT-PCR reaction, or a two-step RT-PCR reaction.
  • reverse transcription can provide cDNA templates for PCR amplification and downstream experiments, and reverse transcriptases can be selected with the highest efficiency for all samples, including difficult-to-transcribe RNA samples such as those that are degraded, inhibitor-residual, or have a high degree of secondary structure RNA samples.
  • the one-step RT-PCR can include combining first-strand cDNA synthesis (RT) and subsequent PCR reactions in a single reaction (eg, in a single reaction tube).
  • Two-step RT-PCR can involve two separate reactions, for example, first-strand cDNA synthesis (RT) can be performed first, and then the cDNA obtained in the first step can be amplified by PCR.
  • RT first-strand cDNA synthesis
  • the exponential amplification is non-target-specific amplification that is not directed against a particular target.
  • the obtained amplification products or the amplification products may have different nucleic acid sequences from each other (eg, because they are each amplified based on different template sequences, respectively).
  • the amplified products are often identical to the template sequence.
  • the exponential amplification can amplify substantially all nucleic acid molecules (eg, nucleic acid molecules that differ in sequence) present in a sample or system in a similar ratio or factor.
  • the exponential amplification is substantially free of specific amplification primers for a specific target or for a specific target sequence, e.g., it does not specifically amplify only the Some specific nucleic acid molecules or gene fragments, while substantially not amplifying or amplifying at a significantly lower ratio other nucleic acid molecules or gene fragments present in the sample or reaction system.
  • the exponential amplification may be sequence specific, ie it may be an exponential amplification of a sequence shared by multiple molecules or molecules in the sample.
  • polymerase-based amplification is achieved through the use of universal primers.
  • a hybridizing region to which one or more universal primers hybridize is introduced into a template nucleic acid.
  • such hybridization reagents can incorporate (i) a primer that hybridizes to and is extended by a target nucleic acid, (ii) an oligonucleotide that is ligated (e.g., ligated using a ligase) to the target nucleic acid or the product of (i), and /or (iii) a primer with a universal sequence made at the 5' end of the gene-specific sequence.
  • Amplification methods using universal primers can offer the advantage, for example, of using only one or two amplification primers to amplify multiple target nucleic acids (also referred to in this application as "non-target-specific amplification not targeting a specific target").
  • the exponential amplification comprises repeating multiple temperature cycles to amplify the amount of target nucleic acid.
  • the amplification reaction is cycled 2 or more times.
  • the amplification reaction is cycled 10 or more times.
  • the amplification reaction is cycled about 10, 15, 20, 50, 100, 200, 300 or more times.
  • the amplification reaction is cycled 20 to 50 times.
  • the amplification reaction is cycled 30 to 45 times.
  • strands of a single-stranded nucleic acid target can be amplified (eg, the exponential amplification can be performed using an RNA molecule as a template), and one or both strands of a double-stranded nucleic acid target can be amplified.
  • the amplification product is about 10 to about 10000 nucleotides in length, for example about 10 to about 1000 nucleotides in length, about 10 to about 500 nucleotides in length, 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 in length ,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 nucleotides.
  • the methods of the present application can be used to identify the presence, absence or amount of one or more methylated target nucleic acids in a sample. In certain instances, the methods of the present application can be used to identify the methylation status of nucleotide loci in one or more target nucleic acids in a sample.
  • the process of linearly amplifying nucleic acid in a sample further includes the step of: treating the sample with a reagent that can modify specific unmethylated nucleotides to generate other nucleotides.
  • the reagents that modify unmethylated specific nucleotides to generate other nucleotides may include reagents that modify unmethylated cytosines to generate uracils.
  • the reagents that modify unmethylated specific nucleotides to generate other nucleotides can include bisulfite, ⁇ -glucosyltransferase, TET enzyme, pyridine borane and/or A3A deaminase .
  • a nucleic acid molecule such as DNA
  • a nucleic acid molecule can be denatured and treated with bisulfite, wherein unmethylated C (cytosine) is converted to U (uracil), and methylated Modified 5mC remains 5mC.
  • the specific base position of the target region will be U or C
  • U represents that the target region has no methylation modification (and thus is converted to U)
  • C represents that the target region has a methyl group U modification (and therefore not converted to U).
  • the reagents that modify specific unmethylated nucleotides to generate other nucleotides may include reagents other than bisulfite (see Figures 3A-3D).
  • Figure 3A shows the process of using bisulfite (Bisulfite) for treatment. Bisulfite treatment can deaminate C to U, which is read as T when read (for example, sequencing), while 5mC and 5hmC are not affected. Affected, it is still read as C when read (for example, sequencing).
  • Figure 3B shows the process of TAPS sequencing.
  • 5hmC was protected with a sugar using ⁇ -glucosyltransferase ( ⁇ -GT), 5mC was oxidized to 5caC using TETase, and 5caC was reduced to dihydrouracil DHU using pyridine borane, read (e.g., Sequencing) is read as T.
  • Figure 3C shows the process of the TAB-Seq method.
  • 5hmC is protected with sugars using ⁇ -glucosyltransferase ( ⁇ -GT)
  • ⁇ -GT ⁇ -glucosyltransferase
  • 5mC is oxidized to 5caC using TETase
  • bisulfite treatment can deaminate C and 5caC to U, read (e.g., sequenced) It is read as T when it is read, but 5hmC is not affected, and it is still read as C when read (for example, sequencing).
  • Figure 3D shows the process of the ACE-seq method.
  • ⁇ -glucosyltransferase ⁇ -GT
  • A3A deaminase to deaminate C and 5mC to generate U
  • read for example , sequenced
  • step a) (e.g., a step for generating a linear amplification product of the nucleic acid) may comprise:
  • a nucleic acid derived from the sample is ligated to the adapter sequence of the present application to form the adapter-containing nucleic acid, wherein the adapter sequence comprises one or more modifications (e.g., methylation modifications, such as 5mC, 5hmC, 5ghmC, etc.), the modification makes the nucleic acid sequence of the linker sequence unchanged after bisulfite treatment; the nucleic acid containing the linker is treated with a reagent containing bisulfite to form a converted nucleic acid; contacting the converted nucleic acid with the oligonucleotide capable of specifically binding to the linker sequence to form the oligonucleotide hybrid; and contacting the oligonucleotide hybrid with The amplification composition is contacted under conditions enabling linear amplification to produce a linear amplification product of the nucleic acid.
  • modifications e.g., methylation modifications, such as 5mC, 5hmC, 5ghmC, etc.
  • methylation patterns Changes in methylation patterns are often early events in the development and progression of cancer and other diseases. In many cancers, certain genes are inappropriately repressed or activated due to abnormal methylation. The ability of a methylation pattern to repress or activate transcription can be inherited.
  • the methods of the present application can be used to detect sequence variation that is an epigenetic change in a target sequence, such as a change in methylation pattern in a target sequence.
  • Covalent addition of methyl groups to cytosine occurs primarily on CpG dinucleotides (microsatellites).
  • CpG islands in promoter regions have received special attention because their methylation status regulates the transcription and expression of related genes. Methylation of promoter regions leads to silencing of gene expression. This silencing is permanent and persists through the process of mitosis.
  • DNA methylation Due to its essential role in gene expression, DNA methylation has implications for developmental processes, imprinting and X chromosome inactivation, as well as tumorigenesis, aging, and repression of parasitic DNA. Methylation is thought to be involved in carcinogenesis of a wide range of tumors such as lung, breast and colon cancers as well as leukemias. There is also an association between methylation and protein dysfunction (long Q-T syndrome) or metabolic disease (transient neonatal diabetes, type 2 diabetes).
  • Bisulfite treatment of DNA can be used to analyze the location of methylated cytosine residues in the DNA.
  • Treatment of nucleic acids with bisulfite deaminates cytosine residues to uracil residues, while methylated cytosines remain unchanged.
  • the sequence of a target nucleic acid that has not been treated with bisulfite with the sequence of a nucleic acid treated with bisulfite according to the methods of the present application, it is possible to infer the degree of methylation in the methylated nucleic acid and the degree of cellularity. The position where the pyrimidine is methylated.
  • TAPS sequencing, TAB-Seq, ACE-seq, etc. can also be used to analyze the position of methylated cytosine residues (eg, 5mC, 5hmC, 5ghmC, etc.) in DNA.
  • methylated cytosine residues eg, 5mC, 5hmC, 5ghmC, etc.
  • the exponential amplification product of the nucleic acid may be contacted with an extension primer under extension conditions that include a stop nucleotide, thereby generating an extended oligonucleotide (ie, performing an extension reaction).
  • the extension reaction of the present application may be a single base extension reaction.
  • the extension reaction is terminated when the extension primer is extended by only one base (eg, single-base extension using a stop nucleotide).
  • a nucleic acid polymerase can add one or more nucleotides to the 3' end of a primer (eg, oligonucleotide) in a template-specific manner.
  • a primer eg, oligonucleotide
  • a primer is annealed (ie, hybridized) to a target nucleic acid to form a primer-template complex.
  • the primer-template complex is contacted with a DNA polymerase and one or more free nucleotides under suitable conditions to allow the addition of one or more nucleotides to the 3' end of the primer.
  • the primers do not directly hybridize to the nucleic acid site desired to be detected (e.g., a target site, such as a methylated modification site desired to be identified), but instead hybridize to a position adjacent to the site (e.g., the 5' end of this position).
  • a primer hybridizes directly to a region adjacent to a target site (eg, a methylation modification site one wishes to identify).
  • a stop nucleotide may be used in a primer extension reaction to terminate primer extension.
  • hybridization site refers to a specific site on an amplification product or a target nucleic acid (eg, a nucleic acid molecule to be detected in a sample).
  • the termini of the oligonucleotide are adjacent to or substantially adjacent to a site on an amplification product or target nucleic acid that has a sequence that differs from another amplification product or target nucleic acid.
  • An oligonucleotide end is "adjacent" to a site when there are no nucleotides between the site and the end of the oligonucleotide.
  • the end of the oligonucleotide is The loci are "substantially contiguous.”
  • Extension reactions are typically performed under extension conditions.
  • Extension conditions may/reagents include, but are not limited to: one or more oligonucleotides, extending nucleotides (e.g., nucleotide triphosphates (dNTPs)), terminating nucleotides (e.g., one or more double deoxynucleotide triphosphate (ddNTP) or acyclic nucleotide), one or more salts (e.g., magnesium-containing salts), one or more buffers (e.g., containing ⁇ -NAD, TritonX-100 ) and one or more polymerizing agents (eg, DNA polymerase, RNA polymerase).
  • dNTPs nucleotide triphosphates
  • ddNTP double deoxynucleotide triphosphate
  • acyclic nucleotide e.g., acyclic nucleotide
  • salts e.g., magnesium-containing salt
  • extension reactions can be used to distinguish SNP alleles or specific methylation sites by adding deoxynucleotides and/or terminator nucleotides (e.g., dideoxynucleotides, acyclic nucleotides) Incorporated into extension oligonucleotides that hybridize to regions adjacent to specific sites in the target nucleic acid.
  • the primer is typically extended by a polymerase.
  • the oligonucleotide is extended by only one deoxynucleotide or terminator nucleotide (eg, a dideoxynucleotide or an acyclic nucleotide) that is complementary to the site to be detected.
  • oligonucleotides are extended by incorporation of dNTPs and terminated by ddNTPs or acyclic nucleotides, or in certain embodiments, oligonucleotides are terminated by incorporation of ddNTPs or acyclic nucleotides No dNTP extensions.
  • an oligonucleotide can be extended by any of five stop nucleotides (eg, ddATP, ddUTP, ddTTP, ddGTP, ddCTP).
  • the target nucleic acid, or its corresponding amplification product can be used as a template and can determine in part which stop nucleotide is added to the oligonucleotide in the extension reaction.
  • other terminating nucleotides eg, acyclic nucleotides or terminators
  • extension can be performed under isothermal conditions or in a non-isothermal environment (eg, thermal cycling conditions).
  • One or more target nucleic acids can be extended in an extension reaction, and one or more variants of each target nucleic acid can be extended.
  • the nucleic acid can be extended by one or more nucleotides, and in some embodiments, the extension product is about 10 nucleotides to about 10,000 nucleotides in length, about 10 to about 1000 nucleotides in length, About 10 to about 500 nucleotides, about 10 to about 100 nucleotides in length, and sometimes about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 in length ,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 nucleotides.
  • Incorporation of terminating nucleotides can determine the length of the oligonucleotide extension.
  • amplification and extension reactions are performed during the same detection process.
  • 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 2 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, 100, 200, 300, 400, 500, or 600 or more times. In some embodiments, the extension reaction is cycled 20 to 50 times. In some embodiments, the extension reaction is cycled 20 to 100 times. In some embodiments, the extension reaction is cycled 20 to 300 times. In some embodiments, the extension reaction is cycled 200 to 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.
  • an oligonucleotide that hybridizes to a target nucleic acid (eg, an exponential amplification product of the present application) is extended by one nucleotide in the presence of an extension composition.
  • the extension composition may include one or more buffers, salts, enzymes (e.g., polymerase, Klenow, etc.), water, templates (e.g., DNA, RNA, amplification products, etc.), primers (e.g., oligonucleotides ), nucleotide triphosphates, glycerol, macromolecular exclusion molecules (macromolecular exclusion molecule) and any other additives used in the art.
  • enzymes e.g., polymerase, Klenow, etc.
  • templates e.g., DNA, RNA, amplification products, etc.
  • primers e.g., oligonucleotides
  • nucleotide triphosphates glycerol
  • macromolecular exclusion molecules
  • the extension composition may include terminating nucleotides (e.g., dideoxynucleotides (e.g., ddNTP) or pentacyclic nucleotides), non-terminating or extending nucleotides (e.g., dNTPs) or terminating nucleotides and non-terminating nucleotides.
  • terminating nucleotides e.g., dideoxynucleotides (e.g., ddNTP) or pentacyclic nucleotides
  • non-terminating or extending nucleotides e.g., dNTPs
  • terminating nucleotides and non-terminating nucleotides e.g., dNTPs
  • the extension composition consists essentially of one or more specific terminating nucleotides and may contain any other components of the extension composition (e.g., buffers, salts, templates, etc.),
  • an extension composition consisting essentially of ddTTP and ddCTP does not contain ddATP, ddGTP or any other dNTPs.
  • the only nucleotides in the extension composition are stop nucleotides and oligonucleotides that hybridize to a target nucleic acid, or an amplification product thereof, that is extended by one nucleotide.
  • the extension composition consists essentially of terminating nucleotides (eg, ddNTPs, acyclic nucleotides).
  • the presence (or in some embodiments absence) of the terminating nucleotide in the extension composition determines which terminating nucleotide is added to the oligonucleotide.
  • the extension composition comprises one or more terminating nucleotides (eg, ddNTPs or acyclic nucleotides).
  • an extension composition comprises one or more terminating nucleotides and one or more non-terminating nucleotides (eg, dNTPs).
  • the extension composition includes a terminating nucleotide that corresponds to a particular variant (eg, a first variant, a low-abundance variant, or a low-abundance variant), and is thus only capable of extending the particular variant.
  • a terminating nucleotide capable of allowing extension of the second variant eg, wild-type, high-abundance variant, or high-abundance variant is included in the extension composition, thereby allowing extension of the second variant.
  • the methods of the present application comprise contacting the hybridized oligonucleotide species with an extension composition comprising one or more terminating nucleotides under extension conditions, thereby generating an extended oligonucleotide species, wherein oligonucleotides that hybridize to a first variant (e.g., low-abundance variant, less-abundant variant, low-abundance variant) are extended by a stop nucleotide, and hybridized to a second variant (e.g., wild-type , high-abundance variants, high-quantity variants) hybridized oligonucleotides are extended from the stop nucleotide.
  • a first variant e.g., low-abundance variant, less-abundant variant, low-abundance variant
  • a second variant e.g., wild-type , high-abundance variants, high-quantity variants
  • the extension primer is capable of specifically binding to the target nucleic acid (eg, a specific site or target nucleic acid that one wishes to identify).
  • the extension primer may comprise at least about 1 nucleotide, for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, Nucleic acids of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more nucleotides Molecules (eg, single-stranded nucleic acid molecules, such as single-stranded DNA molecules).
  • the extension primer may be substantially fully complementary (e.g., at least 80% complementary, such as at least 85% complementary, at least 90% complementary, at least 91% complementary, at least 92% complementary, at least 93% complementary, at least 94% complementary, at least 95% complementary, at least 96% complementary, at least 97% complementary, at least 98% complementary or at least 99% or more complementary, e.g., 100% complementary).
  • a DNA polymerase can be used in the extension reaction (for example, a single base extension reaction), such as Therminator TM DNA polymerase from NEB Company, Therminator DNA polymerase is a product of 9°N TM DNA polymerase Mutants, engineered enzymes that recognize and incorporate modified bases such as ddNTP. Due to the thermostability of the enzyme, the use efficiency of the primer can be fully improved and the yield can be increased by repeated denaturation, annealing and extension.
  • Therminator TM DNA polymerase from NEB Company
  • Therminator DNA polymerase is a product of 9°N TM DNA polymerase Mutants, engineered enzymes that recognize and incorporate modified bases such as ddNTP. Due to the thermostability of the enzyme, the use efficiency of the primer can be fully improved and the yield can be increased by repeated denaturation, annealing and extension.
  • T7 DNA polymerases can also be used, for example, genetically engineered T7 DNA polymerases can also be used for single base extension reactions.
  • the DNA polymerase can be a genetically engineered form of T7 DNA polymerase (eg, Sequenase). Unlike the wild-type enzyme, it can be substantially devoid of 3' ⁇ 5' exonuclease activity.
  • the DNA polymerase may comprise two subunits, one of which may be Escherichia coli thioredoxin, and the other may be a genetically engineered version of bacteriophage T7 gene 5 protein. Genetic alterations to this subunit (eg, deletion of 28 amino acids by in vitro mutation) abolished all measurable exonuclease activity but did not alter DNA polymerase activity.
  • the terminating nucleotide may be modified at the 3' carbon of its pentose moiety.
  • at least one of said terminating nucleotides may be a dideoxynucleotide.
  • the dideoxynucleotides may be selected from: ddATP, ddGTP, ddCTP, ddTTP and ddUTP.
  • the terminating nucleotide is an acyclic nucleotide.
  • the acyclic nucleotide may be selected from: acyATP, acyCTP, acyGTP, acyTTP and acy-bromo-UTP.
  • the terminating nucleotides include: 1) terminating nucleotides specific to the other nucleotides modified from unmethylated specific nucleotides after the treatment; and 2) terminating nucleotides specific to The specific nucleotides that are methylated have specific terminating nucleotides.
  • the extended oligonucleotide comprises a detectable label.
  • the detectable marker may be a mass marker.
  • the mass marker may be a mass distinguishable tag.
  • the terminating nucleotide may comprise the mass distinguishable tag.
  • the mass marker can be detected by mass spectrometry.
  • the mass spectrometer can be, for example, a matrix assisted laser desorption ionization (MALDI) mass spectrometer.
  • MALDI matrix assisted laser desorption ionization
  • the detectable label generally refers to a label that can be distinguished from one another and used to identify the nucleic acid to which the label is attached.
  • Various types of markers can be selected and used in the methods of the present application.
  • oligonucleotides, amino acids, small organic molecules, light-emitting molecules, light-absorbing molecules, light-scattering molecules, luminescent molecules, isotopes, enzymes, etc. can be used as detectable labels.
  • oligonucleotides, amino acids, and/or small molecule organic molecules of various lengths, various charge-to-mass ratios, various electrophoretic mobilities (capillary electrophoretic mobility), and/or various masses can also be used. Molecules serve as detectable markers.
  • fluorophores radioactive isotopes
  • chromogenic agents luminescent agents
  • chemiluminescent agents light scattering agents, and the like
  • the choice of label depends on the desired sensitivity, ease of conjugation to the nucleic acid, stability requirements, and available equipment.
  • the detectable label is linked to a terminating nucleotide.
  • suitable detectable labels can be selected and/or designed to achieve optimal on-flight performance in mass spectrometry and allow label differentiation at higher multiplex levels.
  • the marker is a fluorescent marker or dye detected by electrophoresis or by performing PCR.
  • Detection devices suitable for detecting mass distinguishable tags include, but are not limited to, certain mass spectrometry and gel electrophoresis devices.
  • 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 mass spectrometry (OTOF MS; two-dimensional), laser desorption mass spectrometry (LDMS), electrospray (ES) mass spectrometry, ion cyclotron resonance (ICR) mass spectrometry and Fourier transform mass spectrometry.
  • MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight
  • OTOF MS MALDI orthogonal TOF mass spectrometry
  • LDMS laser desorption mass spectrometry
  • ES electrospray
  • ICR ion cyclotron resonance
  • ionization MS e.g., MALDI-TOF MS, LDMS, ESMS, linear TOF, OTOF
  • Orthogonal ion extraction MALDI-TOF and axial MALDI-TOF are capable of producing relatively high resolution and, therefore, relatively high levels of multiplexing.
  • Detection devices suitable for detecting light-emitting, light-absorbing and/or light-scattering labels include, but are not limited to, certain photodetectors and photodetectors (eg, to detect fluorescent, chemiluminescent, absorbing and/or light-scattering labels).
  • extension products obtained by the methods of the present application can be detected by various methods.
  • the extension primer (UEP) and/or the terminating nucleotide can be labeled with any type of chemical group or moiety that allows detection and/or quantification of the signal, including, but 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, e.g. complexes of iron, ruthenium or osmium moieties, moieties with detectable electron spin resonance, capacitance, permittivity, or conductivity, or any combination of markers thereof.
  • mass distinguishable label refers to a marker whose mass is used as a distinguishing feature.
  • the detectable label consists of nucleotides, and sometimes the label is from about 5 nucleotides to about 50 nucleotides in length.
  • the detectable label is a nucleotide complex, sometimes from about 5 nucleotides to about 35 nucleotides in length.
  • the detectable label is a peptide, sometimes from about 5 amino acids to about 100 amino acids in length.
  • the detectable label is a concatemer of organic molecular units.
  • the marker is a concatemer of trityl molecules.
  • the mass distinguishable tags are chain terminating nucleotides.
  • mass distinguishable tags such as complexes, amino acids and/or concatemers
  • Nucleotide strings eg, nucleic acids; complexes
  • amino acid strings eg, peptides; polypeptides; complexes
  • concatemers of different lengths and/or compositions can be distinguished by mass and used as markers. Any number of units may be employed in a mass distinguishable tag, and the upper and lower limits of such units depend in part on the mass window and resolution of the system used to detect and distinguish such markers.
  • the length and composition of the mass distinguishable tags can be selected based in part on the mass window used to detect and distinguish the markers and the resolution of the detector.
  • a detectable label can be released from a nucleic acid product (eg, an extended oligonucleotide).
  • the link between the detectable label and the nucleic acid can be of any type capable of being transcribed and cleaved, cleaved and allowing detection of the released label or labels.
  • the label can be separated from other parts of the molecule to which the label is attached.
  • linkages that can be cleaved by nucleases e.g., ribonucleases, endonucleases
  • linkages that can be cleaved by chemical means linkages that can be cleaved by physical treatment
  • photocleavable linkers that can be cleaved by light e.g. , o-nitrobenzyl, 6-nitroveratryloxycarbonyl (6-nitroveratryloxycarbonyl), 2-nitrobenzyl group).
  • Photocleavable linkers are advantageous when using light-emitting detection systems (eg, matrix-assisted laser desorption ionization (MALDI) mass spectrometry involving the excitation emission of light) because cleavage and detection are combined and performed in one step.
  • detection systems eg, matrix-assisted laser desorption ionization (MALDI) mass spectrometry involving the excitation emission of light
  • dG/(dA+dG) may represent the ratio or content of the nucleic acid molecule comprising methylation modification.
  • Multiplexing refers to the simultaneous detection of more than one target nucleic acid (or more than one specific site, such as a methylated site).
  • Conventional methods for multiplexing reactions coupled with mass spectrometry are known (see eg WO1997037041A2).
  • Multiplexing provides the advantage that multiple target nucleic acids and their variants (eg, variants with different sequence variations) can be identified in a single mass spectrum, compared to having to perform separate mass spectrometric analyzes for each target nucleic acid.
  • the methods of the present application can be used in high-throughput, highly automated processes for rapid and accurate analysis of target sequences (eg, DNA methylation, particularly methylation of small amounts of DNA).
  • target sequences eg, DNA methylation, particularly methylation of small amounts of DNA.
  • the methods of the present application can be highly multiplexed in a single reaction. Multiplexing is useful when the genotype at the locus is unknown, and in some embodiments, the genotype at the locus is known.
  • the number of multiplexed target nucleic acids includes, but is not limited to, about 2 to about 1,000, about 2 to about 500, about 2 to about 100, for example, about 1-5, 5 -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
  • Design methods for obtaining resolved mass spectra using multiplex analysis may include primer and oligonucleotide design methods and reaction design methods.
  • primer and oligonucleotide design in multiplex assays the same general guidelines for primer design are used as for singleplex reactions, such as avoiding false priming and primer-dimers, except that more primers are involved in multiplex reactions.
  • analyte peaks in the mass spectrum of one experiment can be fully resolved from the products of any experiments with which the experiment is multiplexed, including pausing peaks and any other by-product peaks.
  • the analyte peak preferably falls within a user-specified mass window, eg, in the range of 5000-8500 Da.
  • extension oligonucleotides can be designed relative to a specific target sequence.
  • the length is typically between a range that can be user-specified (e.g., 17-24 bases or 17-26 bases), and typically does not include unspecified bases in the target sequence .
  • the strength of hybridization is sometimes measured by calculating the sequence-dependent melting (or hybridization/dissociation) temperature, Tm.
  • Methods and software for designing extension primers are known and include, for example, SpectroDESIGNER (Sequenom).
  • the multiplex assays provided herein are designed for single base extensions.
  • an oligonucleotide e.g., an extended oligonucleotide in the present application
  • an amplification product derived from a target nucleic acid e.g., an amplification product in the present application, such as an exponential amplification product
  • Hybridization at a position 5' to a single base position that differs between variants of the target nucleic acid eg, target nucleic acids with and without the methylation modification.
  • the methods and products of the present application can be used in the diagnosis of a disease or disorder (including the prognosis of a disease or disorder), for example, by performing the diagnosis of the disease or disorder non-invasively.
  • the disease or disorder may be a disease or disorder associated (e.g., judged based on the methylation status) with changes (e.g., methylation modifications or SNPs) in genetic material (e.g., nucleic acid, such as DNA), Such as tumor or cancer.
  • genetic material e.g., nucleic acid, such as DNA
  • the genetic material may be cfDNA, such as ctDNA, derived from a sample (eg, tissue or bodily fluid) of the subject.
  • the methods and products of the present application can be used to improve, adjust or determine a treatment regimen suitable for a particular subject.
  • the present application provides a kit and/or system, which can be used to implement the methods described herein.
  • the kit may include: the linear amplification component described in this application, the exponential amplification component described in this application, and the extension component described in this application.
  • the linear amplification component, the exponential amplification component and the extension component are immiscible with each other in the kit.
  • the linear amplification component, the exponential amplification component, and the extension component can each be present independently in separate packages.
  • one or more of the following components may be included in the kit: a) reagents that modify specific unmethylated nucleotides to generate other nucleotides; b) one or more A nucleic acid polymerase; c) one or more primers; d) a MALDI matrix compound; and e) a MALDI substrate.
  • the means for using the kit can include a thermal cycler and/or a mass spectrometer.
  • Kits described herein generally comprise one or more containers containing one or more components described herein.
  • a kit includes one or more components in any number of individual containers, packs, tubes, vials, multiwell plates, etc., or the components may be combined in various combinations within these containers.
  • the kit may include, for example, one or more of the following components: (i) one or more nucleotides (e.g., terminating and/or non-terminating nucleotides); one or more of which may comprise detection label; (ii) one or more oligonucleotides, one or more of which may include a detection label (e.g., an amplification primer, one or more extension primers, a label-containing oligonucleotide nucleotides); (iii) one or more enzymes (e.g., polymerase, endonuclease, restriction enzyme, exonuclease, etc.); (v) control components (e.g., control DNA, primers, synthetic template , target
  • Kits are sometimes used in conjunction with certain methods, and may include instructions for performing one or more methods and/or a description of one or more compositions. Kits can be used to perform the methods described herein. Instructions and/or descriptions may be in tangible form (eg, paper, etc.) or electronic form (eg, a computer-readable file on a tangible medium, etc. (eg, compact disc)), and may be included in a kit insert. Kits can also include written instructions providing internet locations for such instructions or descriptions.
  • kits of the present application include one or more stop nucleotides that, when at the 3' end of the oligonucleotide, inhibit the enzyme on the activity.
  • the terminating nucleotide is a dideoxynucleotide.
  • the dideoxynucleotide is selected from the group consisting of: ddATP, ddGTP, ddCTP, ddTTP, and ddUTP.
  • the terminating nucleotide is an acyclic nucleotide.
  • the acyclic nucleotide is selected from the group consisting of: acyATP, acyCTP, acyGTP, acyTTP, and acy-bromo-UTP.
  • the terminating nucleotide includes a mass distinguishable tag.
  • the kit includes one or more of the following: an oligonucleotide, a polymerase, a reverse transcriptase, one or more buffers, and one or more reaction controls.
  • Embodiment 1 sample synthesis
  • the reference sequences for the synthetic double strands are:
  • Reference sequence 1 (internal reference 1): its forward nucleic acid sequence is shown in SEQ ID NO: 4, and the reverse nucleic acid sequence complementary to the forward nucleic acid sequence is shown in SEQ ID NO: 5. In all reference sequence 1 samples, the content of nucleic acid molecules containing 5-methylcytosine (ie 5mC) was 0%.
  • Reference sequence 2 (internal reference 2): its forward nucleic acid sequence is shown in SEQ ID NO: 7, and the reverse nucleic acid sequence complementary to the forward nucleic acid sequence is shown in SEQ ID NO: 8. In all reference sequence 2 samples, the content of nucleic acid molecules containing 5-methylcytosine (ie 5mC) was 25%.
  • Reference sequence 3 (internal reference 3): its forward nucleic acid sequence is shown in SEQ ID NO: 10, and the reverse nucleic acid sequence complementary to the forward nucleic acid sequence is shown in SEQ ID NO: 11. In all reference sequence 3 samples, the content of nucleic acid molecules containing 5-methylcytosine (ie 5mC) was 50%.
  • Reference sequence 4 (internal reference 4): its forward nucleic acid sequence is shown in SEQ ID NO: 13, and the reverse nucleic acid sequence complementary to the forward nucleic acid sequence is shown in SEQ ID NO: 14. In all reference sequence 4 samples, the content of nucleic acid molecules containing 5-methylcytosine (ie 5mC) was 75%.
  • Reference sequence 5 (internal reference 5): its forward nucleic acid sequence is shown in SEQ ID NO: 16, and the reverse nucleic acid sequence complementary to the forward nucleic acid sequence is shown in SEQ ID NO: 17. In all reference sequence 5 samples, the content of nucleic acid molecules containing 5-methylcytosine (ie 5mC) was 100%.
  • the reference sequence 1-reference sequence 5 in Example 1 were respectively end-repaired. Briefly, prepare the end repair and A tailing reaction mix in PCR tubes as follows:
  • the above reaction mixture was mixed by pipetting and centrifuged quickly.
  • Example 2.2 Using the DNA Clean&Concentrator-5 kit of Zymo Research, according to the description of the instructions, the ligation product in Example 2.2 was purified to obtain the adapter-containing nucleic acid.
  • the purified adapter-containing nucleic acid obtained in Example 2.2 was subjected to bisulfite treatment using the EZ DNA Methylation-Gold kit from Zymo Research. Among them, cytosine (C) in the DNA sample molecule is converted to uracil (U), and 5-methylcytosine (5mC) remains unchanged.
  • the converted nucleic acid molecules are linearly amplified using T7 RNA polymerase.
  • the reaction mixture was divided into two tubes, each containing 12 ⁇ l.
  • Reverse transcription was performed on the linear amplification product obtained in Example 4.1.
  • the primer for reverse transcription is the nucleic acid sequence shown in SEQ ID NO:3.
  • the cDNA product obtained after the reverse transcription reaction was purified using the DNA Clean&Concentrator-5 kit from Zymo Research.
  • the product of the PCR reaction was purified using the AxyPrep PCR Cleanup Kit (AP-PCR-250G).
  • Embodiment 6 single base extension
  • the PCR product obtained in Example 5 was added into nuclease-free pure water, treated at 98° C. for 10 min, and then quickly placed on ice.
  • Add 1 ⁇ l of extension primers to each tube of reaction mixture (the extension primer of reference sequence 1 is shown in SEQ ID NO: 6, the extension primer of reference sequence 2 is shown in SEQ ID NO: 9, the extension primer of reference sequence 3 The primer is shown in SEQ ID NO:12, the extension primer of reference sequence 4 is shown in SEQ ID NO:15, and the extension primer of reference sequence 5 is shown in SEQ ID NO:18).
  • Embodiment 7 MALDI-TOF analysis
  • Desalination is performed first. Desalination can be done in 2 ways.
  • Oligo Clean & Concentrator (D4061) from Zymo Research is used, purified according to the instructions, and finally eluted with pure water.
  • Mass spectrometry was performed using a MassARRAY small analyzer (MALD1-TOF mass spectrometer) to obtain the detection results.
  • MALD1-TOF mass spectrometer MALD1-TOF mass spectrometer
  • 1 ng of cfDNA can be extracted from about 2 ml of whole blood.
  • the detection results of internal reference 1 to internal reference 5 are shown in Figures 2A-2E, respectively, and the results are summarized in Figure 2F.
  • Figure 2F shows the molecular weight of each internal reference-specific primer before and after single-base extension, and the accuracy of the methylation content at a specific site calculated using micronucleic acid mass spectrometry. It can be seen that the detection accuracy for internal reference 1 to internal reference 5 is at least about 65% or higher.
  • Example 8 Amplification reaction based on another strand displacement amplification
  • the adapter can be double-stranded DNA, and the adapter can contain at least one nickase (Nickase) that can recognize the conserved sequence, and the corresponding number of protected bases; Dicer recognition sequence. At the same time, in the nickase recognition sequence of the linker, the C base on at least one chain is protected by methylation at position 5.
  • the 3' end of one of the strands of the adapter can have an extra T tail that does not match the complementary strand, so as to facilitate connection with the DNA fragment after end repair and A base tail added to the 3' end.
  • the linker contains an endonuclease recognition sequence
  • the nucleotides on one of the chains on both sides or near the two sides of the endonuclease corresponding to the cleavage position are phosphorothioated (Phosphorothioate), or Other modifications that can resist endonuclease activity, so as to achieve the effect that one of the DNA double strands is cut by endonuclease, while the other remains intact.
  • a DNA ligase system can be used.
  • the ligated DNA fragments are treated with sulfite to convert unmethylated C bases into T bases, and the processed product should be single-stranded DNA.
  • a complementary single-stranded primer that is added to a DNA sequence that has been treated with sulfite on one strand of the double-stranded linker, and forms a partial double-stranded structure with the single-stranded DNA that has been treated with sulfite after annealing.
  • This part of the double-stranded DNA should contain at least Nickase or endonuclease recognition sequence and its corresponding number of protected bases.
  • the obtained linear amplification product is used in the nucleic acid detection method of the present application, such as downstream PCR reaction, sequencing, or detection of nucleic acid mass spectrometry, which can achieve the detection accuracy of the methylation content of a specific site.
  • the linker can be double-stranded DNA, the length of the linker can be no less than 5 bp, and the linker does not contain C bases or contains C bases but is protected by 5-methylation.
  • the 3' end of one of the strands of the adapter can have an extra T tail that does not match the complementary strand, so as to facilitate connection with the DNA fragment after end repair and A base tail added to the 3' end.
  • a DNA ligase system can be used.
  • the ligated DNA fragments are treated with sulfite to convert unmethylated C bases into T bases, and the processed product should be single-stranded DNA.
  • An enzyme with DNA ligation function is added to connect the two ends of the single-stranded DNA with adapters to form circular DNA.
  • DNA polymerase with strand displacement function to start rolling circle amplification; in this system, DNA single-strand binding protein can be added to stabilize the reaction system.
  • the rolling circle amplification product is the linear amplification product.
  • the obtained linear amplification product is used in the nucleic acid detection method of the present application, such as downstream PCR reaction, sequencing, or nucleic acid mass spectrometry detection, which can achieve the detection accuracy of the methylation content of a specific site.
  • Embodiment 10 clinical sample detection result
  • the methylation status of septin9 was detected according to the following steps.
  • the cfDNA in the plasma was extracted with the Yibien Biotechnology Extraction Kit, and then according to the detection method described in this application, the cfDNA obtained with the KAPA Hyper Prep Library Construction Kit was used for end repair and T7 methylation linker ligation.
  • Adapter-ligated cfDNA was then purified using DNA Clean & Concentrator-5 (Zymo Research).
  • the purified product was then subjected to sulfite treatment using the EZ DNA Methylation-Gold (Zymo Research) kit.
  • the sulfitation product is then purified.
  • the purified product after sulfite treatment was linearly amplified using T7 RNA polymerase and reverse transcriptase, and the specific operation was as shown in Example 4.
  • forward and reverse are PCR amplification primers corresponding to the site
  • extension is an extension primer corresponding to the site.
  • the number of primer pairs used for PCR amplification of Septin9, a total of seven sites, seven pairs of primers, the primer concentration in each reaction is 2.5 ⁇ M.
  • the number of extension primers used for Septin9 includes seven sites and seven primers, and the primer concentration in each reaction is 0.5 ⁇ M.
  • the desalted product was analyzed using a nucleic acid time-of-flight mass spectrometer. As shown in Figure 6, the methylation modifications of the 7 septin9 methylation sites in the patient were all detected.

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Abstract

提供了核酸检测的方法和系统。具体而言，提供了鉴别样品中的一种或多种靶核酸存在、不存在或其含量的方法。

Description

核酸检测方法与系统 技术领域
本申请涉及核酸检测方法与系统。特别地，本申请涉及少量核酸的检测。
背景技术
对靶核酸进行特异性检测是诊断医学和分子生物学研究中的重要工具。对靶核酸的检测可以用于，例如鉴定宿主对象中的病原体(例如，细菌或病毒)；确定正常基因的表达和鉴定突变基因(例如，癌基因)；在进行组织移植前，针对相容性对组织分型；分析不同物质的基因之间的同源性；和/或鉴定基因变体的等位基因和多态性等。
在这些应用场景中，通常需要从含有大量非靶标核酸的核酸样品或混合物中检测和/或鉴定出少量存在的目标核酸的能力。
目前常用的检测方法检测灵敏性或检测下限不足。例如，来自少量目标分子的信号可能被源自大量非靶标物质的信号遮蔽(例如，背景信号或噪音)，往往需要去除这些“背景信号”，来提高检测下限。然而，在去除背景信号的过程中，可能导致来自目标分子的信号减弱或丢失。
此外，为了扩大来自少量目标分子的信号，常常需要对样品中的分子进行扩增。PCR等指数扩增方法往往会造成扩增偏差(bias)，从而影响检测结果的准确性。另外，在涉及多种目标分子的情况下，进行多重此类指数扩增会带来更多问题。
因此，亟需开发更加准确和灵敏的核酸检测方法，特别是针对样品中少量或超少量的目标分子进行检测。
发明内容
本申请提供了鉴别靶核酸的方法和产品(例如，检测试剂盒或检测系统)。本申请的方法和产品能够显著改善检测下限，且能够灵敏、准确地检测样品中存在的超小量核酸。例如，本申请的方法能够灵敏、准确地检测到来自循环肿瘤细胞的核酸分子，如cfDNA和/或ctDNA等。例如，与现有方法(e.g.，使用PCR法，如qPCR进行的检测)相比，本申请的方法能够将检测的灵敏度提升至少10倍(例如，至少20倍，至少30倍，至少40倍，至少50倍，至少60倍，至少70倍，至少80倍，至少90倍甚至超过至少100倍)。例如，本申请的方法能够准确地检测到样品中含量为10ng以下的核酸分子(e.g.，DNA)，例如，样品中含量为9ng以下、8ng以下、7ng以下、6ng以下、5ng以下、4ng以下、3ng以下、2ng以下、1ng以 下、0.9ng以下、0.8ng以下、0.7ng以下、0.6ng以下、0.5ng以下、0.4ng以下或更少量的核酸分子。
本申请所述的方法可以是体外或离体方法。在某些情形中，利用本申请的方法所获得的鉴别结果，结合其他信息(例如，受试者的其他信息，或者其他临床参数/指标)才足以得出对受试者健康状况或疾病进展的明确诊断结果。
一方面，本申请提供了一种鉴别样品中一种或多种靶核酸存在、不存在或其含量的方法，所述方法包括：a)在使得源自所述样品的核酸能够线性扩增的条件下处理所述样品，以生成所述核酸的线性扩增产物；b)对所述线性扩增产物进行指数扩增，以生成所述核酸的指数扩增产物；和c)在包括终止核苷酸的延伸条件下使所述核酸的指数扩增产物与延伸引物接触，从而生成延伸的寡核苷酸。所述方法还可包括d)分析所述延伸的寡核苷酸，从而鉴别所述样品中所述一种或多种靶核酸存在、不存在或其含量。
在某些实施方式中，所述步骤a)包括：使所述样品与能够结合于所述核酸的寡核苷酸接触，以形成寡核苷酸杂交体；和使所述寡核苷酸杂交体与扩增组合物在能够发生线性扩增的条件下接触，以生成所述核酸的线性扩增产物。
在某些实施方式中，所述步骤a)包括：使源自所述样品的核酸与接头序列连接，以形成含接头的核酸；使所述含接头的核酸与能够特异性结合于所述接头序列的寡核苷酸接触，以形成寡核苷酸杂交体；和使所述寡核苷酸杂交体与扩增组合物在能够发生线性扩增的条件下接触，以生成所述核酸的线性扩增产物。
在某些实施方式中，所述接头序列包含RNA聚合酶的启动子序列。
在某些实施方式中，所述接头序列包含SP6启动子序列、T7启动子序列和/或T3启动子序列。
在某些实施方式中，所述能够特异性结合于所述接头序列的寡核苷酸与所述接头序列至少部分互补。
在某些实施方式中，所述能够特异性结合于所述接头序列的寡核苷酸包含SP6启动子序列的互补序列、T7启动子序列的互补序列和/或T3启动子序列的互补序列。
在某些实施方式中，所述接头序列包含一种或多种修饰，所述修饰使得所述接头序列经亚硫酸氢盐处理后，其核酸序列不发生改变。
在某些实施方式中，所述接头序列包含一种或多种碱基的甲基化修饰。
在某些实施方式中，所述接头序列中包含的胞嘧啶为经甲基化修饰的胞嘧啶。
在某些实施方式中，所述接头序列直接或间接连接于所述核酸的3’端。
在某些实施方式中，所述接头序列的5’端直接或间接连接于所述核酸的3’端。
在某些实施方式中，使所述核酸与所述接头序列连接包括：修复所述核酸的末端，使其成为平末端。
在某些实施方式中，使所述核酸与所述接头序列连接包括：修复所述核酸的末端，使其成为平末端，并且在所述经修复的序列的3’末端添加脱氧腺苷dA。
在某些实施方式中，所述线性扩增包含核酸转录反应、链置换扩增反应和/或滚环扩增反应。
在某些实施方式中，所述线性扩增为不针对特定靶标的非靶标特异性扩增。
在某些实施方式中，所述核酸的线性扩增产物包含由所述核酸编码的RNA。
在某些实施方式中，所述线性扩增中使用RNA聚合酶。
在某些实施方式中，所述RNA聚合酶包括T7 RNA聚合酶，SP6 RNA聚合酶，和/或T3 RNA聚合酶。
在某些实施方式中，步骤a)还包括：用可修饰未甲基化的特定核苷酸以生成其他核苷酸的试剂处理所述样品。
在某些实施方式中，所述可修饰未甲基化的特定核苷酸以生成其他核苷酸的试剂包括可将未甲基化的胞嘧啶修饰生成尿嘧啶的试剂。
在某些实施方式中，所述可修饰未甲基化的特定核苷酸以生成其他核苷酸的试剂包括亚硫酸氢盐，β-葡萄糖基转移酶，TET酶，吡啶硼烷和/或A3A脱氨酶。
在某些实施方式中，步骤a)包括：使源自所述样品的核酸与所述接头序列连接，以形成含接头的核酸，其中所述接头序列包含一种或多种修饰，所述修饰使得所述接头序列经亚硫酸氢盐处理后，其核酸序列不发生改变；用包含亚硫酸氢盐的试剂处理所述含接头的核酸，以形成经转变的核酸；使所述经转变的核酸与能够特异性结合于所述接头序列的寡核苷酸接触，以形成寡核苷酸杂交体；和使所述寡核苷酸杂交体与扩增组合物在能够发生线性扩增的条件下接触，以生成所述核酸的线性扩增产物。
在某些实施方式中，本申请所述的方法用于鉴别样品中一种或多种甲基化靶核酸的存在、不存在或其含量。
在某些实施方式中，本申请所述的方法用于鉴别样品中一种或多种靶核酸中核苷酸基因座的甲基化状态。
在某些实施方式中，所述指数扩增包括聚合酶链式反应PCR。
在某些实施方式中，所述b)包括对所述线性扩增产物进行逆转录，并对所述逆转录产物 进行PCR扩增。
在某些实施方式中，所述指数扩增包括逆转录酶聚合酶链式反应RT-PCR。
在某些实施方式中，所述指数扩增为不针对特定靶标的非靶标特异性扩增。
在某些实施方式中，所述指数扩增包括使用DNA聚合酶和/或逆转录酶。
在某些实施方式中，所述终止核苷酸在其戊糖部分的3’碳处被修饰。
在某些实施方式中，至少一种所述终止核苷酸是双脱氧核苷酸。
在某些实施方式中，所述双脱氧核苷酸选自：ddATP、ddGTP、ddCTP、ddTTP和ddUTP。
在某些实施方式中，所述终止核苷酸是无环核苷酸。
在某些实施方式中，所述无环核苷酸选自：acyATP、acyCTP、acyGTP、acyTTP和acy-溴-UTP。
在某些实施方式中，所述方法能够鉴别所述样品中的2种或更多种靶核酸。
在某些实施方式中，所述终止核苷酸包含：1)对经所述处理后由未甲基化的特定核苷酸修饰生成的所述其他核苷酸具有特异性的终止核苷酸；以及2)对甲基化的所述特定核苷酸具有特异性的终止核苷酸。
在某些实施方式中，所述延伸的寡核苷酸包含可检测标记物。
在某些实施方式中，所述可检测标记物是质量标记物。
在某些实施方式中，所述质量标记物是质量可区分标签。
在某些实施方式中，所述终止核苷酸包含所述质量可区分标签。
在某些实施方式中，在本申请的所述方法中，通过质谱检测所述质量标记物。
在某些实施方式中，所述质谱是基质辅助激光解吸电离(MALDI)质谱。
在某些实施方式中，源自所述样品的核酸包含cfDNA和/或基因组DNA(gDNA)。
在某些实施方式中，源自所述样品的核酸包含源自肿瘤组织的ctDNA，和/或源自器官或胎儿的脱落细胞的DNA。
在某些实施方式中，源自所述样品的核酸的含量为低于约10ng。
在某些实施方式中，源自所述样品的核酸的含量为低于约1ng。
在某些实施方式中，所述样品为生物样品。所述生物样品可包括：血液，体液和组织(例如，器官组织，福尔马林固定石蜡包埋的FFPE样品，尿，粪便，脑脊液，胸腹水，口腔涮洗液，肺泡灌洗液，羊水等)，和/或体外培养样品。
在某些实施方式中，所述样品源自受试者。
在某些实施方式中，所述延伸引物能够与所述靶核酸特异性结合。
在某些实施方式中，所述延伸引物的核酸序列与所述靶核酸的序列至少部分互补。
在某些实施方式中，本申请所述的方法用于鉴别所述核酸分子中一个或多个CpG二核苷酸的胞嘧啶核苷酸的甲基化状态。
在某些实施方式中，所述一个或多个CpG二核苷酸位于基因的启动子区。
在某些实施方式中，本申请所述的方法为体外或离体方法。
另一方面，本申请提供了一种鉴别与疾病、疾病结果和/或治疗方案结果相关的生物标记物的方法，所述方法包括以下步骤：i)根据本申请所述的方法，鉴别源自一个或多个样品的一种或多种靶核酸的存在、不存在或含量，所述一个或多个样品源自一个或多个具有已知疾病、疾病结果和/或治疗方案的结果的受试者；ii)根据本申请所述的方法，鉴别源自一个或多个样品的一种或多种靶核酸的存在、不存在或含量，所述一个或多个样品源自正常受试者；以及iii)鉴定步骤i)中所述一种或多种靶核酸的存在、不存在或含量与步骤ii)中所述一种或多种靶核酸的存在、不存在或含量之间的差异，并据此，将所述差异鉴定为与所述疾病、疾病结果和/或治疗方案结果相关的生物标记物。
另一方面，本申请提供了一种鉴别与疾病、疾病结果和/或治疗方案结果相关的甲基化的方法，所述方法包括以下步骤：i)根据本申请所述的方法，鉴别源自一个或多个样品的一种或多种靶核酸中甲基化或未甲基化的核苷酸，所述一个或多个样品源自一个或多个具有已知疾病、疾病结果和/或治疗方案的结果的受试者；ii)根据本申请所述的方法，鉴别源自一个或多个样品的一种或多种靶核酸中甲基化或未甲基化的核苷酸，所述一个或多个样品源自正常受试者；以及iii)鉴定步骤i)中所述一种或多种靶核酸中甲基化或未甲基化的核苷酸与步骤ii)中所述一种或多种靶核酸中甲基化或未甲基化的核苷酸之间的差异，并据此，将所述差异的甲基化或未甲基化的核苷酸鉴别为与所述疾病、疾病结果和/或治疗方案结果相关的甲基化。
另一方面，本申请提供了用于根据本申请所述的方法来鉴别样品中一种或多种靶核酸存在、不存在或其含量的试剂盒，所述试剂盒包含：线性扩增组分；指数扩增组分；以及延伸组分，所述延伸组分包含终止核苷酸以及延伸引物。在某些情形中，所述延伸组分包含终止核苷酸，热稳定性延伸酶，以及延伸引物。
另一方面，本申请提供了用于鉴别样品中一种或多种靶核酸存在、不存在或其含量的试剂盒，所述试剂盒包含：线性扩增组分；指数扩增组分；以及延伸组分，所述延伸组分包含终止核苷酸以及延伸引物。在某些情形中，所述延伸组分包含终止核苷酸，热稳定性延伸酶，以及延伸引物。
在某些实施方式中，在本申请所述的试剂盒中，所述线性扩增组分，所述指数扩增组分， 以及所述延伸组分互相不混合。
在某些实施方式中，在本申请所述的试剂盒中，所述线性扩增组分，所述指数扩增组分，以及所述延伸组分各自独立地存在于单独的包装中。
在某些实施方式中，本申请所述的试剂盒包含下述组分中的一种或多种：a)试剂，其可修饰未甲基化的特定核苷酸以生成其他核苷酸；b)一种或多种核酸聚合酶；c)一种或多种引物；d)MALDI基质化合物；和e)MALDI底物。
另一方面，本申请提供了一种系统，所述系统包含本申请所述的试剂盒，以及任选地包含选自下组的一个或多个部分：用法说明书、试剂和用于使用所述试剂盒的装置。
在某些实施方式中，在本申请所述的系统中，所述用于使用所述试剂盒的装置包括热循环仪和/或质谱仪。
在本申请的方法中，所述步骤c)可包括重复多个温度循环以扩增反应中延伸产物的量。例如，该延伸反应可循环2次或更多次。例如，该延伸反应可循环约10，15，20，50，100，200，300，400，500或600次或更多次。例如，该延伸反应可循环20至50次。例如，该延伸反应可循环至少50、55、60、65、70、75、80、85、90、95或100次。
本领域技术人员能够从下文的详细描述中容易地洞察到本公开的其它方面和优势。下文的详细描述中仅显示和描述了本公开的示例性实施方式。如本领域技术人员将认识到的，本公开的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示了本申请的方法的实验流程示例。
图2A-2F显示了本申请的方法的核酸质谱检测结果(样本为1ng经甲基化修饰的DNA)。
图3A-3D显示了示例性的DNA转变方法。
图4显示了基于另一种链置换扩增的线性扩增反应。
图5显示了基于另一种滚环扩增的线性扩增反应。
图6显示了本申请检测方法用于临床反应具有甲基化检测准确性。
具体实施方式
以下由特定的具体实施方式说明本申请涉及的发明，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
除非另有说明，否则本申请中所使用的科学和技术术语具有与分子生物学领域内的技术人员基于技术文献或者与分子生物学相关的其他熟知的技术出版物通常所理解的相同含义，所述技术文献包括，例如Dictionary of Microbiology and Molecular Biology，2nd ed.(Singleton et al.，1994，John Wiley&Sons，New York，NY)(Singleton等人，《微生物学和分子生物学词典》，第2版，1994年)。
除非另有说明，否则本申请中使用或考虑的技术是分子生物学领域中熟知的标准方法。
在本申请中，术语“约”当用于指定某些数值范围时，不限于所指定的精确值，而是可包括与该指定值不同的值。例如，可包括与所列举的特定数值有多达10％的差异的值。因此，术语“约”用于涵盖自特定值±10％或更少的变化范围、±5％或更少的变化范围、±1％或更少的变化范围、±0.5％或更少的变化范围、或±0.1％或更少的变化范围。
在本申请中，术语“样品”以其最广泛的含义使用。术语“生物学样品”包括但不限于，来自当前有生命的个体、或者曾经有生命的个体的任何数量的物质。所述有生命的个体包括但不限于，人、小鼠、大鼠、猴、狗、兔和其他动物，或者植物。所述物质包括但不限于，血液(例如全血)、血浆、血清、脑脊液、组织液(例如，胸腹水)、灌洗液(例如，肺泡)、尿、羊膜液、滑液、内皮细胞、白细胞、单核细胞、其他细胞、器官、组织、骨髓、淋巴结和脾等。所述样品也可以包括活组织和/或经过处理的组织，例如活检的组织(如液体活检的组织)、福尔马林固定和石蜡包埋(FFPE)的组织等。
例如，所述样品(例如，生物学样品)可以是包含所关注的待分析物质的标本，例如，微生物、病毒、核酸(如基因)，或者它们的组分，所述组分可包含分析物中的或者从分析物取得的核酸。样品可来自任何来源，例如生物标本或者环境来源。生物标本包括从活生物体或死亡生物体取得的任何组织或材料，该组织或材料可包含分析物或者分析物中的或从分析物取得的核酸。生物样品的例子包括呼吸组织、渗出物(例如，支气管肺泡灌洗液)、活组织标本、痰、外周血、血浆、血清、淋巴结、胃肠组织、粪便、尿液、脑脊液、组织液(例如，胸腹水)、灌洗液(例如，肺泡)或者其他流体、组织或材料。环境样品的例子包括水、冰、土壤、混悬液、残渣、生物膜、大气尘粒子以及气溶胶。样品可为经处理的标本或材料，例如通过使用过滤、离心、沉淀或者吸附至介质(例如，基体或载体)处理样品而获得。样品的其他处理可包括物理或机械地破碎组织、细胞凝聚体或细胞的处理，从而将包括核酸在 内的细胞内组分释放到可包含其他组分的溶液中，所述其他组分诸如酶、缓冲液、盐、洗涤剂等等。所述样品也可以包括组织，例如活检的组织(如液体活检的组织)、福尔马林固定和石蜡包埋(FFPE)的组织等。
在本申请中，术语“接触”通常是指使两种或更多种组分至少部分地相互混合。可以通过在流体或半流体混合物中混合各组分来实现接触。当一种或多种组分在固体表面上与一种或多种其他组分发生物理接触时也可实现接触，该固体表面例如固体组织切片或者基质。
在本申请中，术语“核苷酸”通常包括天然和非天然核苷酸。核苷酸包括但不限于，天然存在的核苷单磷酸、核苷二磷酸和核苷三磷酸；脱氧腺苷单磷酸、脱氧腺苷二磷酸和脱氧腺苷三磷酸；脱氧鸟苷单磷酸、脱氧鸟苷二磷酸和脱氧鸟苷三磷酸；脱氧胸苷单磷酸、脱氧胸苷二磷酸和脱氧胸苷三磷酸；脱氧胞苷单磷酸、脱氧胞苷二磷酸和脱氧胞苷三磷酸；脱氧尿苷单磷酸、脱氧尿苷二磷酸和脱氧尿苷三磷酸；以及脱氧肌苷单磷酸、脱氧肌苷二磷酸和脱氧肌苷三磷酸(本申请分别称为dA、dG、dT、dC、dU和dI，或A、G、T、C、U和I)。核苷酸还包括但不限于，修饰的核苷酸和核苷酸类似物。修饰的核苷酸和核苷酸类似物包括但不限于，双脱氧核苷酸，无环核苷酸，脱氮嘌呤核苷酸，例如，7-脱氮-脱氧鸟苷(7-脱氮-dG)单磷酸、二磷酸和三磷酸以及7-脱氮-脱氧腺苷(7-脱氮-dA)单磷酸、二磷酸和三磷酸，氘-脱氧胸苷(氘-dT)单磷酸、二磷酸和三磷酸，甲基化核苷酸，例如，5-甲基脱氧胞苷三磷酸、13C/15N标记的核苷酸和脱氧肌苷单磷酸、二磷酸和三磷酸。能够使用多种官能团和连接位置的组合得到修饰的核苷酸、富含同位素的核苷酸、损耗的核苷酸、标签和标记的核苷酸以及核苷酸类似物。
在本申请中，术语“核酸”通常是指包括寡核苷酸的多核苷酸化合物，所述寡核苷酸包含核苷或者核苷类似物，所述核苷或者核苷类似物具有通过标准的磷酸二酯键或者其他键而共价连接的含氮杂环碱基或碱基类似物。核酸包括RNA、DNA、嵌合的DNA-RNA聚合物或其类似物。在核酸中，主链可由多种键组成，包括糖磷酸二酯键、肽核酸(PNA)键、硫代磷酸酯键、甲基磷酸酯键或其组合中的一者或多者。核酸中的糖基部分可为核糖、脱氧核糖或者具有取代基的类似化合物，所述取代基例如，2'甲氧基和2'卤离子(例如，2'-F)取代基。含氮碱基可为常规的碱基(A、G、C、T、U)、其类似物(例如，肌苷)、嘌呤或嘧啶碱基的衍生物(例如，N4-甲基脱氧鸟苷、脱氮或者氮杂嘌呤、脱氮或者氮杂嘧啶、在多个化学位置的任一者处具有改变的或者置换的取代基的嘧啶或嘌呤(例如，2-氨基-6-甲胺基嘌呤、06-甲基鸟嘌呤、4-硫代-嘧啶、4-氨基-嘧啶、4-二甲基肼-嘧啶，以及04-烷基-嘧啶)，或者吡唑化合物，例如未取代的或者3-取代的吡唑[3，4-d]嘧啶)。核酸可包括“脱碱基”位置，其中主链在 一个或多个位置处不具有含氮碱基，例如，一个或多个脱碱基位置可形成接头区，该接头区将单独的寡核苷酸序列接合到一起。核酸可仅包含如存在于常规RNA和DNA中的常规糖基、碱基和键，或可包括常规组分和取代物(例如由2'甲氧基主链连接的常规碱基，或包含常规碱基与一种或多种碱基类似物的混合物的聚合物)。所述术语包括“锁核酸”(LNA)，该“锁核酸”可包含一种或多种LNA核苷酸单体，该单体具有锁定为RNA模拟糖构象的双环呋喃糖单元，该构象增强了ssRNA、ssDNA或者dsDNA中互补序列的杂交亲和力。
此外，以“多核苷酸”提及核酸通常是指通过共价键连接的两个或多个核苷酸或核苷酸类似物。核酸可以是适于本申请所述的方法使用的任意类型的核酸。在某些实施方式中，核酸可以是DNA(例如互补DNA(cDNA)、基因组DNA(gDNA)、质粒和载体DNA等)、RNA(例如病毒RNA、信使RNA(mRNA)、短抑制性RNA(siRNA)、核糖体RNA(rRNA)、tRNA等)、和/或DNA或RNA类似物(例如，包含碱基类似物、糖类似物和/或非天然骨架等)。核酸可以是可用于进行本申请所述方法的任何形式(例如，线性、环状、超螺旋、单链、双链等)。在某些实施方式中，核酸可以是或可以来自质粒、噬菌体、自主复制序列(ARS)、着丝粒、人工染色体、染色体、细胞、细胞核或细胞的胞质。在某些实施方式中，核酸来自单一染色体(例如，核酸样品可以来自获自二倍体生物的样品的一个染色体)。在胎儿核酸的情况中，所述核酸可以来自父本等位基因、母本等位基因或母本和父本等位基因。
在本申请中，术语“寡核苷酸”和“低聚物(oligomer)”可互换地使用，通常是指一般由少于1，000个核苷酸(nt)构成的核酸聚合物，包括长度为约2nt至约900nt的聚合物。例如，所述寡核苷酸可包含5nt至500nt，例如可包含10nt至150nt。例如，所述寡核苷酸可通过使用任何公知的体外化学或酶促方法合成制备的，并且可在合成之后通过使用标准方法而纯化，所述标准方法包括例如高效液相色谱(HPLC)。在本申请中，代表性的寡核苷酸包括，例如引物，启动子，检测探针寡核苷酸，靶捕获寡核苷酸等。
在本申请中，术语“引物”通常是指是寡核苷酸，其至少3'端与核酸模板互补，并且该寡核苷酸与模板复合(例如，通过氢键或杂交)以得到引物-模板复合物，该复合物适合于通过RNA或DNA依赖性的核酸聚合酶(例如，DNA聚合酶或RNA聚合酶)引发合成。引物可通过将共价键合的核苷酸碱基添加到该引物的3'端而延伸，该碱基与所述模板互补。所获得的是引物延伸产物。在本申请中，引物的长度可以为至少10个核苷酸，并且可延长至约15、20、25、30、35、40、45、50或者更多个核苷酸的长度。
在本申请中，术语“带标签的寡核苷酸”通常是指包含至少第一区和第二区的寡核苷酸，其中第一区包含“靶杂交序列”，该靶杂交序列能够与所关注的靶核酸序列杂交，并且其中第 二区包含位于该靶杂交序列的5’端并且无法稳定地杂交或结合至包含该靶核酸序列的靶核酸的“标签序列”。该靶杂交序列与该靶核酸序列的杂交产生“带标签的靶核酸序列”。该“标签序列”或者“异源标签序列”可为基本上任何序列，前提条件是该序列无法稳定地杂交至所关注的靶核酸序列。例如，该标签序列无法稳定地杂交至从待测生物体的基因组中获得的任何序列，或者无法在反应条件下杂交至任何靶核酸。存在于带标签的寡核苷酸中的标签序列被设计为不会实质上损害或者妨碍该靶杂交序列杂交至其靶序列的能力。此外，该标签序列可具有足够的长度，使得一旦将该标签序列的互补序列掺入到初始的DNA引物延伸产物中，随后就可使用标签特异性的引物来参与后续的扩增。本申请的标签序列的长度通常为至少10个核苷酸，并且可延长至例如15、20、25、30、35、40、45、50或者更多个核苷酸的长度。在某些情形中，带标签的寡核苷酸是包含标签序列和靶杂交序列的“带标签的引物”。在其他情形中，带标签的寡核苷酸是包含标签序列、靶杂交序列和启动子序列的“带标签的启动子寡核苷酸”，所述启动子序列可位于该标签序列的5'端并且作用为从该5'端引发转录。
在本申请中，术语靶核酸的“扩增”通常是指在体外构建与靶核酸序列或者作为该靶核酸序列的替代序列的通用序列或标签序列的至少一部分相同或互补的核酸链的过程，仅当该靶核酸存在于样品中时才能发生此过程。通常，核酸扩增使用一种或多种核酸聚合酶和/或转录酶来产生靶多核苷酸或者其片段的多个拷贝，或者与该靶多核苷酸或者其片段互补的序列的多个拷贝，或者已经引入到该扩增体系中充当该靶多核苷酸的替代序列的通用序列或者标签序列的多个拷贝，所述通用序列或者标签序列例如在检测步骤中用于指示在测定的某一时刻靶多核苷酸的存在，或者作为位点用于在扩增反应中进一步引发，或者用于测序相关的过程或者测序反应。体外核酸扩增技术包括转录相关的扩增方法，例如转录介导的扩增(TMA)或者基于核酸序列的扩增(NASBA)，以及其他方法，例如聚合酶链式反应(PCR)、逆转录-PCR(RT-PCR)、复制酶介导的扩增，以及连接酶链式反应(LCR)。
在本申请中，术语“线性扩增”是指经设计以在反应中与靶核酸的含量成线性比例地使靶核酸的产生增长的扩增过程。例如，可使用转录相关的反应从靶DNA制备多个RNA拷贝，其中拷贝数量的增加可用线性因子(例如，模板的起始拷贝X 100)来描述。例如，在多阶段扩增程序中，第一阶段的线性扩增可使得在第二阶段扩增反应开始前，靶核酸链或该靶核酸链的互补链的起始数量增加至少10倍，例如至少100倍，例如10至1000倍。线性扩增体系的一个例子是基于T7的DNA线性扩增。本申请中记载了其他方法。因此，术语“线性扩增”是指不会导致靶核酸序列的指数级扩增的扩增反应。术语“线性扩增”并非是指仅制备核酸链的单拷贝的方法。例如，线性扩增可以基于包含核酸转录反应、链置换扩增反应和/或滚环扩 增反应等已知的反应。
在本申请中，术语“指数级扩增“通常是指经设计以在反应中与靶核酸的量成几何比例地使靶核酸的产生增长的扩增过程。例如，PCR针对每一原始靶链以及针对存在的每一合成链产生一条DNA链。类似地，转录相关的扩增针对每一原始靶链以及针对每一随后合成的链产生多个RNA转录物。该扩增是指数级的，因为所合成的链被用作后续轮扩增中的模板。扩增反应不需要实际上产生指数级增长量的核酸才能被视为指数级扩增，只要该扩增反应被设计为应产生此类增长即可。
在本申请中，“转录相关的扩增”方法通过从核酸模板产生多个转录物来扩增靶序列。此类方法通常使用一种或多种寡核苷酸(其中一种寡核苷酸提供启动子序列、脱氧核苷三磷酸(dNTP)、核糖核苷三磷酸(rNTP))和具有RNA聚合酶和DNA聚合酶活性的酶，以在靶序列附近生成功能性启动子序列，然后从启动子转录靶序列。转录相关扩增的例子包括转录介导的扩增(TMA)、基于核酸序列的扩增(NASBA)、单引物转录相关的扩增以及自动维持序列扩增(3SR)等。本领域内的普通技术人员将会意识到，基于寡核苷酸序列的聚合酶介导的延伸的替代性扩增方法也可与本申请所述的组合物和/或方法步骤一起使用。
在本申请中，可使用单引物(而非成对引物)通过制备指示靶核酸存在的转录物来在体外扩增核酸。例如，该单引物方法可使用寡核苷酸引物以及任选地阻断剂分子(例如，终止寡核苷酸)来终止从该靶链延伸DNA。该方法可通过用以下物质处理包括靶序列的靶核酸来合成靶序列的多个拷贝：1)寡核苷酸引物，其杂交至该靶序列的3'端使得可以从该3'端引发引物延伸反应，以及2)阻断剂分子，其结合至与该靶序列的5'端邻近或者在该靶序列的5'端附近的靶核酸。通过使用DNA聚合酶在引物延伸反应中延伸该寡核苷酸引物，以获得与该靶序列互补的DNA引物延伸产物，其中该DNA引物延伸产物具有由该阻断剂分子确定并且与该靶序列的5'端互补的3'端。
在另一个实例中，通过使引物在该靶序列的3'部分中的某一位置处杂交至靶DNA以及使引物在该靶序列的5'部分中的某一位置处杂交至终止核苷酸来合成靶序列的多个拷贝。
在某些情形中，单引物转录相关的扩增方法的实例不需要使用终止(寡)核苷酸。
在本申请中，术语“靶核酸”通常指任何包括待检测序列的核酸。靶核酸可以是DNA或RNA。靶核酸可以是任意来源，例如基因组DNA、mRNA、cDNA、cfDNA，ctDNA等。靶核酸可以是天然存在的或合成的(如扩增产物、载体等)。靶核酸可以是但不需要是纯化或分离的。取决于希望测定的性质，靶核酸可以是来源于植物或动物组织，或取自反应混合物。靶核酸在长度上没有限制，尽管靶核酸可以在由本申请的方法检测或鉴定之前暴露于限制性内 切核酸酶。
在本申请中，术语“标记物”通常是指可以被检测或产生可检测响应的分子部分或化合物，所述分子部分或化合物可直接或间接地接合至被标记的核酸分子。直接标记可使用键或相互作用来连接标记物和待标记的核酸分子，所述键或相互作用包括共价键、非共价的相互作用(氢键、疏水的和离子的相互作用)，或者螯合物或配位络合物。间接标记可使用直接或间接标记的桥联部分或接头(例如抗体、寡核苷酸或者其他化合物)，该桥联部分或接头可放大信号。标记物包括任何可检测的部分。可用的可检测部分的例子包括放射性核素、配体(例如，生物素或者亲和素)、酶、酶底物、反应基团、发色团(可检测的染料、粒子或者珠粒)、荧光团，或者发光化合物(例如，生物发光标记物、磷光标记物，或者化学发光标记物)。
在本申请中，术语“分离”或“纯化”通常是指移除混合物(例如，样品)中的一种或多种组分，以与该混合物中的一种或多种其他组分分离。样品组分包括核酸，所述样品组分可包括细胞片段、蛋白质、碳水化合物、脂质，以及其他化合物。
在本申请中，术语“多种靶核酸”通常是指超过一种靶核酸。在某些实施方式中，多种靶核酸可以是约2种至约10000种核酸，约2中至约1000种核酸，约2种至约500种核酸，或者，例如约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或10000种靶核酸。在某些实施方式中，多种靶核酸处于一个或多个反应容器中，并且各反应容器包含超过一种靶核酸。在某些实施方式中，多种靶核酸处于一个反应容器中。在某些实施方式中，多种靶核酸是约2种至约100种靶核酸。在某些实施方式中，约2种至约100种靶核酸处于单一反应容器中。
在本申请中，术语“杂交序列”通常是指寡核苷酸中能够与扩增产物或其部分、靶核酸或其部分、靶核酸或其部分、靶核酸变体或其部分或其补体特异性杂交的核苷酸序列。杂交序列易于设计和选择，并且该序列能够是适于与本申请所述的扩增产物、靶序列或其互补分子杂交的长度。在某些实施方式中，在每个寡核苷酸中的杂交序列长约5-约200个核苷酸(例如，长约5-10、约10-15、约15-20、约20-25、约25-30、约30-35、约35-40、约40-45或约45-50、约50-70、约80-90、约90-110、约100-120、约110-130、约120-140、约130-150、约140-160、约150-170、约160-180、约170-190、约180-200个核苷酸)。
在本申请中，术语“杂交条件”通常是指在该条件下两条具有互补核苷酸序列的核酸能够彼此相互作用的条件。杂交条件可以是高严谨性、中等严谨性或低严谨性的，并且这些变化 的严谨性程度的条件是已知的。根据感兴趣的应用，通常选择允许扩增和/或延伸的杂交条件。
在本申请中，“特异性杂交”是指与样品中的某种扩增产物或靶核酸杂交而基本不与该样品中的其他扩增产物物质或靶核酸杂交。
在本申请中，术语“终止核苷酸”可与“终止核苷酸”、“链终止子(chain terminator)试剂”或“链终止子”互换地使用，并且通常是指当将其添加到延伸反应中时，使延伸反应停止的分子。链终止子可以包括核苷酸类似物，当其存在于多核苷酸链或寡核苷酸时阻止链或寡核苷酸的进一步延伸。在某些实施方式中，终止核苷酸是终止核苷酸。在某些实施方式中，终止核苷酸是经修饰的核苷酸，当其在延伸反应中被掺入核酸分子(例如，寡核苷酸)的3'端时将不允许核苷酸被进一步掺入该寡核苷酸。在某些实施方式中，在存在具有3'-5'外切核酸酶活性的酶时，不将终止核苷酸从寡核苷酸或多核苷酸链移除。在某些实施方式中，核苷酸戊糖的'3OH可以被这样的部分取代，所述部分产生链终止的核苷酸并且还对通过具有3'-5'外切核酸酶活性的酶的移除具有抗性。在某些实施方式中，修饰终止核苷酸的戊糖3'位置以用另一部分替换OH，包括但不限于，磷酰基基团，乙酰基基团，3'-O-甲基，3'-O-(2-硝基苄基)，3'-O-烯丙基，3'-叠氮基和3'-氨基。在某些实施方式中，3'OH基团被氢取代。在某些实施方式中，修饰的核苷酸是双脱氧核苷酸。在某些实施方式中，修饰的核苷酸是无环核苷酸。终止核苷酸的示例包括：双脱氧核苷酸，例如ddA(双脱氧腺嘌呤)，ddT(双脱氧胸腺嘧啶)，ddC(双脱氧胞嘧啶)，ddG(双脱氧鸟嘌呤)和ddU(双脱氧尿嘧啶)和无环核苷酸，例如，acyATP、acyCTP、acyGTP、acyTTP和acy-溴-UTP。
在本申请中，术语“信噪比”通常是指通过在使用检测过程(例如，质谱)时量化信号相对于噪声的强度的比例来定量测量信号质量。在某些实施方式中，一个谱图上的强度峰具有比另一谱图上相同分析物(例如，延伸的寡核苷酸物质)产生的低强度峰更高的信噪比。例如，使用质谱时。本申请所述“信噪比”中使用的术语“信号”指延伸的寡核苷酸物质的信号峰的强度。在某些实施方式中，本申请的方法包括将寡核苷酸杂交体与延伸组合物在延伸条件下接触，所述延伸组合物包含一种或多种终止核苷酸。
在本申请中，术语“灵敏度”通常是指使用检测方法(例如，质谱)时能够检测到给定信噪比的分析物的量。在某些实施方式中，能够通过减少背景噪声水平改善灵敏度。
在本申请中，术语“无细胞DNA(cfDNA)”通常是指样品中的DNA，当采集时该DNA没有包含在细胞内。该术语不是指通过细胞或组织的体外破裂而使其无细胞的DNA。cfDNA可以包括正常细胞和源自癌细胞的DNA两者。cfDNA通常获自血液或血浆(“循环”)。cfDNA可以通过分泌或细胞死亡过程，如细胞坏死或凋亡释放到循环中。例如，某些cfDNA可以是 ctDNA。
在本申请中，术语“循环肿瘤DNA(ctDNA)”或“循环癌DNA”是指源自肿瘤的无细胞DNA(cfDNA)组分。
一方面，本申请提供了一种鉴别样品中一种或多种靶核酸存在、不存在或其含量的方法。本申请的方法可包括：a)在使得源自所述样品的核酸能够线性扩增的条件下处理所述样品，以生成所述核酸的线性扩增产物。该方法还可包括：b)对所述线性扩增产物进行指数扩增，以生成所述核酸的指数扩增产物。所述方法还可包括：c)在包括终止核苷酸的延伸条件下使所述核酸的指数扩增产物与延伸引物接触，从而生成延伸的寡核苷酸。所述方法还可包括：d)分析所述延伸的寡核苷酸，从而鉴别所述样品中所述一种或多种靶核酸存在、不存在或其含量。
另一方面，本申请提供了一种鉴别与疾病、疾病结果和/或治疗方案结果相关的生物标记物的方法。所述方法可包括以下步骤：i)根据本申请所述的方法，鉴别源自一个或多个样品的一种或多种靶核酸的存在、不存在或含量，所述一个或多个样品源自一个或多个具有已知疾病、疾病结果和/或治疗方案的结果的受试者；ii)根据本申请所述的方法，鉴别源自一个或多个样品的一种或多种靶核酸的存在、不存在或含量，所述一个或多个样品源自正常受试者；以及iii)鉴定步骤i)中所述一种或多种靶核酸的存在、不存在或含量与步骤ii)中所述一种或多种靶核酸的存在、不存在或含量之间的差异，并据此，将所述差异鉴定为与所述疾病、疾病结果和/或治疗方案结果相关的生物标记物。
另一方面，本申请提供了一种鉴别与疾病、疾病结果和/或治疗方案结果相关的甲基化的方法。所述方法可包括以下步骤：i)根据本申请所述的方法，鉴别源自一个或多个样品的一种或多种靶核酸中甲基化或未甲基化的核苷酸，所述一个或多个样品源自一个或多个具有已知疾病、疾病结果和/或治疗方案的结果的受试者；ii)根据本申请所述的方法，鉴别源自一个或多个样品的一种或多种靶核酸中甲基化或未甲基化的核苷酸，所述一个或多个样品源自正常受试者；以及iii)鉴定步骤i)中所述一种或多种靶核酸中甲基化或未甲基化的核苷酸与步骤ii)中所述一种或多种靶核酸中甲基化或未甲基化的核苷酸之间的差异，并据此，将所述差异的甲基化或未甲基化的核苷酸鉴别为与所述疾病、疾病结果和/或治疗方案结果相关的甲基化。
另一方面，本申请提供了用于根据本申请所述的方法来鉴别样品中一种或多种靶核酸存在、不存在或其含量的试剂盒。所述试剂盒可包含：线性扩增组分；指数扩增组分；以及延伸组分，所述延伸组分包含终止核苷酸以及延伸引物。
另一方面，本申请提供了用于鉴别样品中一种或多种靶核酸存在、不存在或其含量的试剂盒。所述试剂盒可包含：线性扩增组分；指数扩增组分；以及延伸组分，所述延伸组分包含终止核苷酸以及延伸引物。例如，所述延伸组分中可包含终止核苷酸，热稳定性延伸酶，以及延伸引物。
另一方面，本申请提供了一种系统。所述系统可包含本申请所述的试剂盒，以及任选地包含选自下组的一个或多个部分：用法说明书、试剂和用于使用所述试剂盒的装置。
样品及靶核酸
本申请的方法和产品(例如，试剂盒、系统等)可用于鉴别样品中一种或多种靶核酸存在、不存在或其含量。
在本申请中，所鉴别的核酸可以是寡核苷酸或多核苷酸，包括但不限于天然核酸(例如脱氧核糖核酸(DNA)、核糖核酸(RNA))、合成核酸、非天然核酸(例如肽核酸(PNA))、未修饰的核酸、修饰的核酸(例如，甲基化DNA，经标记的核酸，具有一个或多个经修饰的核苷酸的核酸分子)等。在本申请中，“多核苷酸”是指通过共价键连接的两个或多个核苷酸或核苷酸类似物。核酸可以是适用于本申请所述方法的任意类型的核酸。例如，所述核酸可以是DNA(例如互补DNA(cDNA)、基因组DNA(gDNA)、cfDNA，ctDNA，质粒和载体DNA等)，也可以是核酸类似物(例如，包含碱基类似物、糖类似物和/或非天然骨架等)。核酸可以是可用于进行本申请所述方法的任何形式(例如，线性、环状、超螺旋、单链、双链等)。例如，所述核酸可以是或可以来自，质粒、噬菌体、自主复制序列(ARS)、着丝粒、人工染色体、染色体、细胞、细胞核或细胞的胞质。在某些情形中，核酸可来自单一染色体(例如，核酸样品可以来自获自二倍体生物的样品的一个染色体)。在胎儿核酸的情况中，所述核酸可以来自父本等位基因、母本等位基因或母本和父本等位基因。例如，本申请所述的核酸可以是无细胞DNA，例如组织或样本中的cfDNA，如来自肿瘤或癌变组织/部位的ctDNA。
在本申请中，待检测或鉴别的靶核酸分子可以为任意长度。例如，其可包含至少1个(例如，其可包含至少2、3、4、5、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100或超过100个)核苷酸。
在本申请中，待检测或鉴别的靶核酸分子可包含一种或多种修饰，例如其可包含一个或多个甲基基团或甲基化状态、一个或多个磷酸基团、一个或多个乙酰基基团和一个或多个核苷酸的一个和或多个缺失、添加或取代。一个或多个核苷酸的一个或多个缺失、添加或取代的示例包括但不限于特定突变的存在与否、核苷酸取代(例如，单核苷酸多态性(SNP))的存在与否、重复序列(例如，双核苷酸、三核苷酸、四核苷酸、五核苷酸重复)的存在与否、标志物 (例如，微卫星)的存在与否和区分序列(例如，区分一种生物与另一种生物的序列(例如，区分一种病毒毒株与另一种病毒毒株的序列))的存在与否。可通过任意已知的方式区分靶核酸的不同核酸和不同的靶核酸，例如，如本申请所述，通过质量、结合、可区分标签等。
在某些实施方式中，靶核酸的变体可以大致相等(例如，SNP)的频率或拷贝数存在于样品中。在某些实施方式中，靶核酸的变体可以不同的频率或拷贝数存在于样品中。在某些实施方式中，一个变体可以比其他变体更大的丰度存在。在某些实施方式中，丰度较大的变体被称为野生型，而丰度较小的变体被称为突变体。在某些情形中，靶核酸包含第一和第二变体，其中第一或第二变体表现出比其他变体更大的丰度，即高丰度变体和低丰度变体或大量(major)变体和少量(minor)变体。当与另一变体进行比较时，表现出较大丰度的变体通常以较高的浓度存在或者通过更高数量的分子(例如，拷贝)数来表示。较高的浓度可以是2倍或更多。在某些实施方式中，较高的浓度是10倍或更多。在某些实施方式中，较高的浓度是100倍、1000倍或10000倍或更多。在某些实施方式中，一个变体表示野生型序列，并且浓度比另一变体高100倍或更多。在某些实施方式中，变体(低丰度变体)的浓度显著低于另一变体(例如，野生型，高丰度变体)。
在某些实施方式中，本申请的方法可以用于检测占样品中总核酸数量的不到30％、20％、15％、10％、8％、5％、4％、3％、2％、1％、0.8％、0.75％、0.5％、0.1％、0.05％、0.01％或更少的低丰度靶核酸(例如，cfDNA或ctDNA)的存在与否。在某些实施方式中，本申请的方法可以用于检测占样品中总核酸数量约1％-约10％的低丰度靶核酸(例如，cfDNA或ctDNA)的存在与否。在某些实施方式中，本申请的方法可以用于检测占样品中总核酸数量约5％或更少的低丰度靶核酸(例如，cfDNA或ctDNA)的存在与否。在某些实施方式中，本申请的方法可以用于检测占样品中总核酸数量的约5％-0.75％的低丰度靶核酸(例如，cfDNA或ctDNA)的存在与否。在某些实施方式中，本申请的方法可以用于检测占样品中总核酸数量的约5％-约0.1％的低丰度靶核酸(例如，cfDNA或ctDNA)的存在与否。在某些实施方式中，本申请的方法可以用于检测占样品中总核酸数量的约1％或更少的低丰度靶核酸(例如，cfDNA或ctDNA)的存在与否。在某些实施方式中，本申请的方法可以用于检测占样品中总核酸数量的约0.1％-约0.001％的低丰度靶核酸(例如，cfDNA或ctDNA)的存在与否。
例如，样品中靶核酸(例如，DNA，如cfDNA，ctDNA等)的含量可以为低于约100ng(例如，低于约90ng，低于约80ng，低于约70ng，低于约60ng，低于约50ng，低于约40ng，低于约30ng，低于约20ng，低于约15ng，低于约10ng，低于约9ng，低于约8ng，低于约7ng， 低于约6ng，低于约5ng，低于约4ng，低于约3ng，低于约2ng，低于约1ng，低于约0.9ng，低于约0.8ng，低于约0.7ng，低于约0.6ng，低于约0.5ng，低于约0.45ng，低于约0.4ng，低于约0.35ng，低于约0.3ng，低于约0.2ng或更低)。
例如，样品中靶核酸(例如，DNA，如cfDNA，ctDNA等)的含量可以为总核酸含量的约20％以下，例如约15％以下，约10％以下，约9％以下，约8％以下，约7％以下，约6％以下，约5％以下，约4％以下，约3％以下，约2％以下，约1％以下，约0.9％以下，约0.8％以下，约0.7％以下，约0.6％以下，约0.5％以下，约0.4％以下，约0.3％以下，约0.2％以下，约0.1％以下，约0.05％以下，约0.04％以下，约0.03％以下，约0.02％以下，约0.01％以下或更低。所述百分比可以为质量百分比，体积百分比和/或摩尔百分比。
例如，所述样品可以包含一种或多种靶核酸的混合物(各靶核酸可以具有低丰度和高丰度变体)，或者可以通过将一种以上包含一种或多种靶核酸的样品组合来形成混合物(各靶核酸可以具有低丰度和高丰度变体)。低丰度变体可以是高丰度变体的变体并且可以包括但不限于，野生型(高丰度变体)等位基因的突变体(低丰度变体)，存在于多于一种宿主的基因的变体(例如，病毒致癌基因(低丰度基因)，其是正常健康基因(高丰度变体)的变体)，多态性，包括单核苷酸多态性(SNP)、插入、缺失或高丰度变体的其他突变形式。
本申请的方法可用于同时鉴别样品中可能存在的多种靶核酸。所述多种靶核酸可以指超过一种靶核酸。例如，所述多种靶核酸可以是约2-约10000种靶核酸，约2-约1000种核酸，约2-约500种核酸，或约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或10000种靶核酸。在某些实施方式中，所述多种靶核酸位于一个或多个反应容器中，并且各反应容器可包含超过一种靶核酸。在某些实施方式中，所述多种靶核酸均位于同一个反应容器中。在某些实施方式中，所述多种靶核酸是约2-约200种靶核酸。在某些实施方式中，所述约2-约200种靶核酸位于单一反应容器中。
例如，本申请所述方法所提供的核酸检测或鉴定可以检测DNA(特别是cfDNA，如ctDNA)中一个或多个位点(例如，约2-约10000个位点，约2-约1000个位点，约2-约500个位点，或约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或10000个位点，可以包括同一核酸分子的不同位点，也可以包括不同核酸分子中的相同位点)处的甲基化是否存在或者甲基化状态。
在本申请中，包含核酸(例如，靶核酸)的样品可以源自一个或多个来源并且可以包含靶核酸的混合物，所述靶核酸各自可以具有不同拷贝数的一种或多种高丰度变体和低丰度变体。还可以将样品合并以生成包括不同靶核酸的混合物，所述不同靶核酸可具有不同的丰度，和/或不同的拷贝数。
例如，本申请的样品可以收集自生物体、矿物或地质场所(例如，土壤、岩石、矿床、化石)或法医场所(例如，犯罪现场、违禁品或可疑违禁品)。因此，来源可以是环境的，例如地质、农业、战场或土壤来源。来源也可以为任何类型的生物体，如任意植物、真菌、原生动物、原核生物、病毒或动物，包括但不限于：人、非人、哺乳动物、爬行动物、牛、猫、狗、山羊、猪、猴、猿、猩猩、公牛、母牛、熊、马、绵羊、家禽、小鼠、大鼠、鱼、海豚、鲸鱼和鲨鱼等，或具有可检测核酸的任意动物或生物体。来源也可指生物体的不同部分，如内部、外部、活细胞或死细胞、组织、液体等。因此，样品可以是“生物样品”，其可以是获自活体或曾经为活体的来源，例如动物如人或其它哺乳动物、植物、细菌、真菌、原生生物或病毒的任何材料。来源可为任何形式，包括但不限于，固体材料如组织、细胞、细胞团块、细胞提取物、或活检样品，或者生物体液如尿液、血液、唾液、羊水、感染或炎症区域的渗出液、或含有口腔细胞的漱口水、毛发、脑脊液和关节液，以及器官。例如，样品(如生物学样品)的例子可包括呼吸组织、渗出物(例如，支气管肺泡灌洗液)、活组织标本、痰、外周血、血浆、血清、淋巴结、胃肠组织、粪便、尿液、脑脊液、组织液(例如，胸腹水)、灌洗液(例如，肺泡)或者其他流体、组织或材料。例如，样品可为经处理的标本或材料，例如通过使用过滤、离心、沉淀或者吸附至介质(例如，基体或载体)处理样品而获得。样品的其他处理可包括物理或机械地破碎组织、细胞凝聚体或细胞的处理，从而将包括核酸在内的细胞内组分释放到可包含其他组分的溶液中，所述其他组分诸如酶、缓冲液、盐、洗涤剂等等。所述样品也可以包括组织，例如活检的组织(如液体活检的组织)、福尔马林固定和石蜡包埋(FFPE)的组织等。
样品还可以在与另一样品不同的时间点分离得到，其中各样品可来自相同或不同来源。核酸可来自核酸库，例如DNA库。核酸可以是样品中核酸分子的核酸纯化或分离和/或扩增的产物。本申请所提供的用于测序分析的核酸可含有来自一个或来自两个或更多个样品(例如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或1000个或更多样品)的核酸。
在本申请所提供的方法之中、之前或之后，可采用多种方式处理核酸。例如，可以减小核酸的长度或质量(例如，通过剪切、核酸酶或限制性酶消化、去磷酸化、去甲基化)、可以增加核酸的大小或质量(例如，通过磷酸化、与甲基化特异性试剂反应、与可检测标记物连接等)、可以用核酸切割抑制剂处理等。
在某些实施方式中，提供未处理的核酸用于根据本申请所述的方法进行分析。在某些实施方式中，提供处理后的核酸用于实施本申请所述的方法。例如，可从样品提取、分离、纯化或扩增核酸。例如，可以将核酸从其原始环境中取出(例如，天然产生核酸的天然环境或外源表达核酸的宿主细胞)，因此核酸从其原始环境通过“人工”操作而被改变。与其所源自的样品中的组分含量相比，分离的核酸一般带有较少的非核酸组分(例如，蛋白质、脂质等)。包含分离的核酸的组合物可以是基本分离的(例如，约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或大于99％不含非核酸组分)。在本申请中，经纯化的核酸是指提供的核酸与其所源自的样品来源相比包含更少的非核酸组分。包含核酸的组合物可以是基本纯化的(例如，约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或大于99％不含其它核酸)。
线性扩增
本申请的方法可包括，在使得源自所述样品的核酸能够线性扩增的条件下处理所述样品，以生成所述核酸的线性扩增产物。
在进行所述线性扩增之前，可以分离样品中的核酸，例如可以使用捕获剂和/或包含捕获剂的载体(例如，固体载体，如磁珠)。例如，可使样品中的核酸与经固定的捕获探针相互作用而将所述核酸捕获到固体载体上。或者，可使样品中的核酸被捕获到固体载体上作为三分子复合物的成员，其中通过靶捕获寡核苷酸桥接所述样品中的核酸和经固定的捕获探针。所述固体载体通常包括多个磁性或者可磁化的粒子或珠粒，所述粒子或珠粒可使用磁场来操纵。分离样品中的核酸的步骤还可包括洗涤靶捕获寡核苷酸：靶核酸杂交体以除去可能干扰后续扩增的不期望的组分。
在某些情况下，在进行所述线性扩增处理之前，不另行对样品中的核酸进行分离。
在本申请中，用于获得线性扩增产物的处理可包括：使所述样品与能够结合于所述核酸的寡核苷酸接触，以形成寡核苷酸杂交体；以及使所述寡核苷酸杂交体与扩增组合物在能够发生线性扩增的条件下接触，以生成所述核酸的线性扩增产物。
在某些实施方式中，所述线性扩增不是仅仅能够扩增样品中的特定靶序列的序列特异性 扩增(例如，所述线性扩增为无差别的序列扩增，也称为非偏好性的或unbiased序列扩增)。例如，所述线性扩增可以对样品中存在的核酸均进行扩增(例如，以基本上相同的倍数和/或比例对样品中存在的核酸进行扩增)。
在本申请中，所述线性扩增进行的条件通常不支持样品中核酸的指数级扩增。
例如，用于获得线性扩增产物的处理可包括：使源自所述样品的核酸与接头序列连接，以形成含接头的核酸；使所述含接头的核酸与能够特异性结合于所述接头序列的寡核苷酸接触，以形成寡核苷酸杂交体；和使所述寡核苷酸杂交体与扩增组合物在能够发生线性扩增的条件下接触，以生成所述核酸的线性扩增产物。
例如，所述接头序列可被基本上无差别地连接到所述样品中的基本上所有的核酸分子上(例如，其可以被连接到样品中的核酸分子(例如DNA分子，如cfDNA或ctDNA)的一端或两端)，例如其可被连接到DNA分子的上游或下游，5’端或3’端。可使用连接酶(例如，T4DNA连接酶)进行所述连接。
在进行所述连接之前，可先对样品中的核酸(例如，经分离的核酸或未分离的核酸，例如，样品中的分离或未分离的DNA，如cfDNA)进行末端修复，例如使其成为平末端。此外，还可向样品中的核酸(例如，DNA，如cfDNA或ctDNA)的3’端添加脱氧腺苷dA(例如，在进行所述末端修复之后)。在某些情况下，还可向样品中的核酸(例如，DNA，如cfDNA或ctDNA)的5’端添加磷酸基团P。
例如，可以在dNTP的存在下，利用核酸聚合酶将样品中核酸(例如，DNA，如cfDNA或ctDNA)的末端补平或切平，利用多聚核苷酸激酶将5’羟基转变成5’磷酸基团且将3’磷酸基团转变成3’羟基。然后，在过量dATP存在下，利用不具有3’-5’外切活性的聚合酶在该核酸分子(例如，DNA)的3’末端加上dATP。
在某些情况下，当进行了所述末端修复、添加了所述3’的dA和/或5’的P之后，可使该核酸(例如，该核酸的3’端)与所述接头序列连接，以形成所述含接头的核酸。
例如，所述接头序列可直接或间接(例如，通过其他多核苷酸片段或序列)连接于所述核酸的3’端。例如，所述接头序列的5’端直接或间接连接于所述核酸的3’端。
在本申请中，所述接头序列可以是双链的核酸(例如，DNA)序列。在某些情形中，所述接头序列可以是单链的核酸序列，例如单链DNA序列。所述接头序列可包含约1-100个核苷酸或碱基对，例如约1-90，1-80，1-70，1-60，1-50，1-40，1-35，1-30，1-25，1-20，1-15或1-10个核苷酸或碱基对。
所述接头序列中可包含RNA聚合酶的启动子序列，例如SP6启动子序列、T7启动子序 列和/或T3启动子序列。
在某些情形中，所述接头序列(例如，其中的所述RNA聚合酶的启动子序列)可包含一种或多种修饰，所述修饰可使得所述接头序列经亚硫酸氢盐处理后，其核酸序列不发生改变。例如，所述RNA聚合酶的启动子序列(例如，所述SP6启动子序列、T7启动子序列和/或T3启动子序列)可以是经甲基化的，比如其中的一种或多种碱基包含甲基化修饰。在某些情形中，所述接头序列(例如，其中的所述RNA聚合酶的启动子序列)中包含的胞嘧啶可以是经甲基化修饰的胞嘧啶。
例如，当希望检测或鉴别样品中的核酸的甲基化情况时，使用的接头序列中可包含经甲基化的RNA聚合酶启动子序列(例如，经甲基化的SP6启动子序列、经甲基化的T7启动子序列和/或经甲基化的T3启动子序列)。例如，所述RNA聚合酶的启动子序列(例如，所述SP6启动子序列、T7启动子序列和/或T3启动子序列)中的一种或多种碱基可包含甲基化修饰。在某些情形中，所述接头序列(例如，其中的所述RNA聚合酶的启动子序列)中包含的胞嘧啶可以是经甲基化修饰的胞嘧啶。
在某些实施方式中，所述接头序列可包含SEQ ID NO:1-2和19-20中任一项所示的核酸序列。
在本申请的方法中，可在允许核酸杂交的条件下使所述含接头的核酸与能够特异性结合于所述接头序列(例如，在所述接头序列为双链核酸的情况下，可以特异性结合于所述接头序列的正义链或者特异性结合于所述接头序列的反义链)的寡核苷酸(其在本申请中也被称为“线性扩增引物”)接触，以形成寡核苷酸杂交体。所述能够特异性结合于所述接头序列的寡核苷酸(“线性扩增引物”)可以包含或不包含经修饰的核苷酸。在某些情形中，所述寡核苷酸不经甲基化修饰处理。
在本申请中，所述能够特异性结合于所述接头序列的寡核苷酸与所述接头序列可以至少部分互补。例如，在所述接头序列为双链核酸的情况下，所述寡核苷酸可以与所述接头序列的正义链至少部分互补。在某些情况下，所述寡核苷酸可以与所述接头序列的反义链至少部分互补。例如，所述能够特异性结合于所述接头序列的寡核苷酸可以基本上与所述接头序列互补(例如，完全互补，或者至少80％互补，例如至少85％互补，至少90％互补，至少91％互补，至少92％互补，至少93％互补，至少94％互补，至少95％互补，至少96％互补，至少97％互补，至少98％互补或者至少99％或以上互补，例如，100％互补)。例如，该寡核苷酸可以是单链的核酸序列，例如单链DNA序列(例如，其可包含约1-100个核苷酸，例如约1-90，1-80，1-70，1-60，1-50，1-40，1-35，1-30，1-25，1-20或1-15个核苷酸，例如至少1 个核苷酸，至少2个核苷酸，至少3个核苷酸，至少4个核苷酸，至少5个核苷酸，至少6个核苷酸，至少7个核苷酸，至少8个核苷酸，至少9个核苷酸，至少10个核苷酸，至少11个核苷酸，至少12个核苷酸，至少13个核苷酸，至少14个核苷酸，至少15个核苷酸，至少16个核苷酸，至少17个核苷酸，至少18个核苷酸，至少19个核苷酸，至少20个核苷酸或更多)。
在某些实施方式中，所述能够特异性结合于所述接头序列的寡核苷酸可包含SEQ ID NO:3所示的核酸序列。
在某些情形中，所述寡核苷酸杂交体可包括一个或多个不与模板(例如，所述接头序列)杂交的核苷酸。例如，所述寡核苷酸杂交体可包括一个或多个错配的核苷酸(例如，非互补核苷酸)以及有时，不杂交的核酸5’和/或3’区域。在某些实施方式中，所述寡核苷酸杂交体可包含标签(例如，质量可区分标签(mass distinguishable tag)、序列标签、发光标签或放射性标签)。在某些实施方式中，所述寡核苷酸杂交体可包含捕获剂(例如，生物素其他捕获剂)。在某些实施方式中，所述寡核苷酸杂交体可包含终止核苷酸。
随后，可以对包含所述寡核苷酸杂交体的目的分子(例如，核酸分子)进行所述线性扩增。在本申请中，所述线性扩增反应是在不支持目的核酸序列(例如，样品中的所述核酸分子)的指数级扩增的条件下进行的。例如，所述线性扩增反应将通常产生约2倍至约10，000倍的核酸序列扩增，例如10倍至约10000倍的核酸序列扩增(例如，约50倍，约100倍，约200倍，约300倍，约400倍，约500倍，约600倍，约700倍，约800倍，约900倍，约1000倍，约1500倍，约2000倍，约3000倍，约5000倍，约8000倍，约10000倍或更多的核酸序列扩增)。
在某些情况下，所述线性扩增可以是基本上等温的。在某些情况下，所述线性扩增可以包括核酸转录反应。例如，所述线性扩增可以包含核酸转录反应、链置换扩增反应(SDA)和/或滚环扩增反应(RCA)。例如，所述线性扩增可以包含非酶促等温链置换和扩增(NISDA)。在某些情况下，所述线性扩增为不针对特定靶标的非靶标特异性扩增。例如，所述线性扩增可使得样品中的核酸分子被基本上无差别地、以基本上一样或类似的比例被扩增。经所述线性扩增后，所获得的线性扩增产物可包含由所述核酸(例如，样品中的核酸)编码的RNA。
例如，该线性扩增反应可不涉及具有PCR以及其他常见扩增技术特有的热循环过程。例如，线性扩增反应可涉及使待扩增的核酸序列(例如，包含所述寡核苷酸杂交体的目的分子)与扩增组合物(例如，线性扩增组合物)接触，所述扩增组合物(例如，线性扩增组合物)支持目标核酸序列的线性扩增但是缺乏使目标核酸序列指数级扩增所需的至少一种组分。
例如，所述扩增组合物(例如，线性扩增组合物)可包括扩增酶，该扩增酶可选自逆转录酶、聚合酶，以及它们的组合。所述聚合酶可选自RNA依赖性的DNA聚合酶、DNA依赖性的DNA聚合酶、DNA依赖性的RNA聚合酶，以及它们的组合。在某些情况下，所述扩增组合物(例如，线性扩增组合物)中包含RNA聚合酶。例如，所述RNA聚合物可选自T7 RNA聚合酶，SP6 RNA聚合酶和T3 RNA聚合酶。
例如，当所述接头序列和/或所述寡核苷酸杂交体中包含SP6启动子序列时，所述扩增组合物(例如，线性扩增组合物)中可包含SP6 RNA聚合酶。当所述接头序列和/或所述寡核苷酸杂交体中包含T7启动子序列时，所述扩增组合物(例如，线性扩增组合物)中可包含T7 RNA聚合酶。当所述接头序列和/或所述寡核苷酸杂交体中包含T3启动子序列时，所述扩增组合物(例如，线性扩增组合物)中可包含T3 RNA聚合酶。
在本申请中，所述线性扩增的反应优选无法支持指数级扩增反应。例如，因为其缺乏指数级扩增所需的一种或多种组分，和/或存在抑制指数级扩增的一种或多种试剂，和/或该反应运行的条件(例如，温度等)不利于指数级扩增等。例如，所述指数级扩增所需的组分和/或所述抑制剂和/或所述反应运行的条件可选自以下组：扩增寡核苷酸(例如，包含DNA聚合酶的5'启动子序列的扩增寡核苷酸、非启动子的扩增寡核苷酸，或者它们的组合)、酶(例如，聚合酶，例如DNA聚合酶)、核酸酶(例如，核酸外切酶、核酸内切酶、裂解酶、核糖核酸酶、磷酸化酶、糖基化酶等)、酶辅因子、螯合剂(例如，EDTA或者EGTA)、核糖核苷三磷酸(rNTP)、脱氧核苷三磷酸(dNTP)、Mg 2+、盐、缓冲液、酶抑制剂、阻断寡核苷酸、pH、温度、盐浓度，以及它们的组合。
在本申请中，经所述线性扩增反应之后获得的产物为线性扩增产物。例如，所述线性扩增产物中可包含由样品中的所述核酸分子转录得到的RNA分子。
指数扩增
在本申请的方法中，可对所述线性扩增产物进行指数扩增，以生成所述核酸的指数扩增产物。
在所述指数扩增中，可使用扩增组合物(例如，指数扩增组合物)，其中的组分可包括但不限于：核苷酸(例如，三磷酸核苷酸)，修饰的核苷酸，寡核苷酸(例如，用于基于聚合酶的扩增的引物寡核苷酸和用于基于连接酶的扩增的寡核苷酸结构单元(building block))，一种或多种盐(例如，含镁盐)，一种或多种缓冲液，一种或多种聚合剂(例如，连接酶，聚合酶)，一种或多种切口酶(例如，切割双链核酸的一条链的酶)和一种或多种核酸酶(例如，外切核酸酶，内切核酸酶，RNA酶)。可以使用任意适于扩增的聚合酶，例如具有或没有外切核酸酶活性的 聚合酶、DNA聚合酶和RNA聚合酶、这些酶的突变体形式。能够使用任意适于结合一个寡核苷酸的5'端与另一个寡核苷酸的3'端的连接酶。扩增条件也能够包括某些反应条件，如等温或温度循环条件。在扩增方法中循环温度的方法是已知的，如通过热循环设备。术语“循环”通常是指利用单引物或多引物的扩增(例如，扩增反应或延伸反应)，其中使用温度循环。在一些实施方式中，扩增条件也能够包括乳化剂(例如，油)，所述乳化剂用于形成多个反应区室，在所述区室中能够扩增单个核酸分子物质。
例如，所述指数扩增反应是在允许该样品或系统中的核酸分子进行指数级扩增的条件下进行的反应。例如，可使所述线性扩增产物与扩增组合物(例如，指数扩增组合物)接触，从而使得在合适的条件下能够发生对所述核酸的指数级扩增。所述指数扩增组合物中通常包含最少量的、所述线性扩增组合物中所缺乏的、指数级扩增所需的一种或多种组分。例如，所述指数扩增组合物中可包含下列组分或条件中的一种或多种：扩增寡核苷酸、逆转录酶、聚合酶、核酸酶、磷酸化酶、酶辅因子、螯合剂、核糖核苷三磷酸(rNTP)、脱氧核苷三磷酸(dNTP)、Mg 2+、最佳pH、最佳温度、盐，以及它们的组合。聚合酶通常选自RNA依赖性的DNA聚合酶、DNA依赖性的DNA聚合酶、DNA依赖性的RNA聚合酶以及它们的组合。在某些实施方案中，所述指数扩增组合物中包含逆转录酶和DNA聚合酶。
在某些情形中，所述指数扩增包括聚合酶链式反应PCR。
在某些情形中，所述指数扩增反应为RT-PCR反应，例如一步法RT-PCR反应，或两步法RT-PCR反应。例如，逆转录可为PCR扩增和下游实验提供cDNA模板，所选择的逆转录酶可对所有样本均具有最高效率，其中包括难转录RNA样本，如降解、抑制剂残留或具有高度二级结构的RNA样本。所述一步法RT-PCR可以包括在单次反应(例如，在单个反应管中)中将第一链cDNA合成(RT)和后续PCR反应结合在一起。两步法RT-PCR可包括两个独立的反应，例如，可首先进行第一链cDNA合成(RT)，然后在通过PCR扩增第一步中所得的cDNA。
在某些情形中，所述指数扩增为不针对特定靶标的非靶标特异性扩增。例如，所获得的扩增产物或扩增产物彼此可具有不同的核酸序列(例如，因为其各自分别基于不同的模板序列扩增得到)。而在特异性扩增中，往往扩增获得的产物均与模板序列相同。例如，通过使用通用引物，所述指数扩增可以对样品或系统中存在的基本上所有核酸分子(例如，序列不同的核酸分子)以相似的比例或倍数进行扩增。在某些实施方式中，所述指数扩增基本上不使用针对特定靶点或针对特定靶序列的特异性扩增引物，例如，其不会仅仅特异性地扩增样品或反应体系中存在的某些特定的核酸分子或基因片段，而基本上不扩增或者以显著更低的 比例扩增样品或反应体系中存在的另外一些核酸分子或基因片段。
然而，所述指数扩增可以是序列特异性的，即其可以是针对样品中多个分子或多种分子共有的序列进行的指数扩增。
因此，在某些实施方式中，通过使用通用引物实现基于聚合酶的扩增。在这样的方法中，将与一个或多个通用引物杂交的杂交区域引入模板核酸中。例如，这类杂交试剂可以掺入(i)与靶核酸杂交并被延伸的引物，(ii)与靶核酸或(i)的产物接合(例如，使用连接酶连接)的寡核苷酸，和/或(iii)具有在基因特异性序列5'端制造的通用序列的引物。使用通用引物的扩增方法能够提供例如仅使用一条或两条扩增引物扩增多种靶核酸的优点(在本申请中也称为“不针对特定靶标的非靶标特异性扩增”)。
在一些实施方式中，所述指数扩增包括重复多个温度循环以扩增靶核酸的量。在一些实施方式中，扩增反应循环2次或更多次。在一些实施方式中，扩增反应循环10次或更多次。在一些实施方式中，扩增反应循环约10、15、20、50、100、200、300次或更多次。在一些实施方式中，扩增反应循环20至50次。在一些实施方式中，扩增反应循环30至45次。
在本申请的方法中，能够扩增单链核酸目标的链(例如，能够以RNA分子作为模板进行所述指数扩增)，并且能够扩增双链核酸目标的一条或两条链。在一些实施方式中，扩增产物(扩增产物)长约10-约10000个核苷酸、例如长约10-约1000个核苷酸、长约10-约500个核苷酸、长约10-约100个核苷酸，并且有时长约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或1000个核苷酸。
甲基化检测
在某些实施方式中，本申请的方法可用于鉴别样品中一种或多种甲基化靶核酸的存在、不存在或其含量。在某些情形中，本申请的方法可用于鉴别样品中一种或多种靶核酸中核苷酸基因座的甲基化状态。
例如，在对样品中的核酸进行线性扩增的过程中，还包括以下步骤：用可以修饰未甲基化的特定核苷酸以生成其他核苷酸的试剂处理所述样品。例如，所述可修饰未甲基化的特定核苷酸以生成其他核苷酸的试剂可以包括可将未甲基化的胞嘧啶修饰生成尿嘧啶的试剂。例如，所述可修饰未甲基化的特定核苷酸以生成其他核苷酸的试剂可以包括亚硫酸氢盐，β-葡萄糖基转移酶，TET酶，吡啶硼烷和/或A3A脱氨酶。
例如，可以使核酸分子(例如DNA)变性，并使用亚硫酸氢盐处理所述核酸分子，其中 没有甲基化修饰的C(胞嘧啶)被转化为U(尿嘧啶)，而经甲基化修饰的5mC仍保持是5mC。相应地，经过所述亚硫酸氢盐处理后，靶区域的特定碱基位置会是U或者C，U代表靶区域没有甲基化修饰(因此被转化为了U)，C代表靶区域有甲基化修饰(因此不被转化为U)。
在某些情形中，所述可修饰未甲基化的特定核苷酸以生成其他核苷酸的试剂可以包括除亚硫酸氢盐之外的试剂(参见图3A-3D)。图3A显示了使用亚硫酸氢盐(Bisulfite)进行处理的过程，亚硫酸氢盐处理可以将C脱氨成U，读取(例如，测序)的时候读出为T，而5mC和5hmC不受影响，读取(例如，测序)的时候依然读出为C。图3B显示了TAPS测序的过程。使用β-葡萄糖基转移酶(β-GT)将5hmC用糖保护，使用TET酶将5mC氧化成为5caC，使用吡啶硼烷(Pyridine borane)将5caC还原成二氢尿嘧啶DHU，读取(例如，测序)的时候读出为T。图3C显示了TAB-Seq方法的过程。使用β-葡萄糖基转移酶(β-GT)将5hmC用糖保护，使用TET酶将5mC氧化成为5caC，亚硫酸氢盐处理可以将C和5caC脱氨成U，读取(例如，测序)的时候读出为T，而5hmC不受影响，读取(例如，测序)的时候依然读出为C。图3D显示了ACE-seq方法的过程。使用β-葡萄糖基转移酶(β-GT)将5hmC用糖保护，读取(例如，测序)的时候读出为C，用A3A脱氨酶将C和5mC脱氨生成U，读取(例如，测序)的时候读出为T。
在本申请的方法的某些实施方式中，步骤a)(e.g.，用于生成所述核酸的线性扩增产物的步骤)可包括：
使源自所述样品的核酸与本申请的所述接头序列连接，以形成所述含接头的核酸，其中所述接头序列包含一种或多种修饰(例如，甲基化修饰，例如5mC，5hmC，5ghmC等)，所述修饰使得所述接头序列经亚硫酸氢盐处理后，其核酸序列不发生改变；用包含亚硫酸氢盐的试剂处理所述含接头的核酸，以形成经转变的核酸；使所述经转变的核酸与所述能够特异性结合于所述接头序列的寡核苷酸接触，以形成所述寡核苷酸杂交体；和使所述寡核苷酸杂交体与扩增组合物在能够发生线性扩增的条件下接触，以生成所述核酸的线性扩增产物。
甲基化模式的变化往往是癌症和其他疾病发展和进展中的早期事件。在许多癌症中，由于异常甲基化，某些基因被不适当地抑制或激活。甲基化模式抑制或激活转录的能力可以被遗传。
因此，本申请的方法可用于检测作为靶序列表观遗传变化的序列变异，例如靶序列中甲基化模式的变化。甲基对胞嘧啶的共价加成主要存在于CpG二核苷酸(微卫星)上。尽管不位于启动子区域的CpG岛的功能仍有待探索，但启动子区域中的CpG岛得到了特别的关注， 因为其甲基化状态调节相关基因的转录和表达。启动子区域的甲基化导致基因表达的沉默。这种沉默是永久性的，并通过有丝分裂过程持续。由于其在基因表达中的重要作用，DNA甲基化对发育过程，印记和X染色体失活以及肿瘤发生、衰老以及寄生DNA的抑制均有影响。甲基化被认为参与许多广泛的肿瘤如肺、乳腺和结肠癌以及白血病的癌发生。甲基化和蛋白质功能障碍(长Q-T综合症)或代谢疾病(暂时性新生儿糖尿病，2型糖尿病)之间也存在关联。
DNA(例如，基因组DNA，cfDNA，或ctDNA)的亚硫酸氢盐处理可用于分析DNA中甲基化胞嘧啶残基的位置。用亚硫酸氢盐处理核酸将胞嘧啶残基脱氨基成尿嘧啶残基，而甲基化的胞嘧啶保持不变。因此，通过将未用亚硫酸氢盐处理的靶核酸的序列与依照本申请的方法用亚硫酸氢盐处理的核酸的序列进行比较，可以推断出甲基化核酸中的甲基化程度以及胞嘧啶被甲基化的位置。类似地，TAPS测序，TAB-Seq，ACE-seq等也可用于分析DNA中甲基化胞嘧啶残基(例如，5mC，5hmC，5ghmC等)的位置。
延伸反应
在本申请的方法中，可在包括终止核苷酸的延伸条件下使所述核酸的指数扩增产物与延伸引物接触，从而生成延伸的寡核苷酸(即进行延伸反应)。本申请的延伸反应可以为单碱基延伸反应。例如，仅使延伸引物进行了一个碱基的延伸(例如，利用终止核苷酸进行的单碱基延伸)后，该延伸反应就终止了。
在本申请的延伸反应中，核酸聚合酶可以模板特异性的方式将一个或多个核苷酸添加到引物(例如，寡核苷酸)的3'末端。
本领域已知适合用于引物延伸反应的条件。通常，将引物退火(即，杂交)至靶核酸以形成引物-模板复合物。引物-模板复合物在合适条件下与DNA聚合酶和一个或多个游离的核苷酸接触，以允许添加一个或多个核苷酸至引物的3'端。在某些实施方式中，引物与希望检测的核酸位点(例如，靶位点，如希望鉴别的甲基化修饰位点)不直接杂交，而是与该位置的相邻位置杂交(例如，该位置的5'端)。在一些实施方式中，引物与靶位点(例如，希望鉴别的甲基化修饰位点)的相邻区域直接杂交。在一些实施方式中，可在引物延伸反应中使用终止核苷酸来终止引物延伸。
在本申请中，“杂交位点”是指扩增产物或靶核酸(例如，样品中的待测核酸分子)上的特定位点。在某些实施方式中，寡核苷酸的末端相邻于或基本上相邻于扩增产物或靶核酸上具有与另一个扩增产物或靶核酸序列不同的位点。当位点和寡核苷酸末端之间没有核苷酸时，寡核苷酸末端与该位点“相邻”。在某些实施方式中，当位点和寡核苷酸末端之间存在1、2、3、4、5、6、7、8、9或10个核苷酸时，寡核苷酸末端与位点“基本相邻”。
延伸反应通常在延伸条件下进行。延伸条件可以/试剂包括但不限于：一种或多种寡核苷酸、延伸核苷酸(例如，核苷酸三磷酸(dNTP))、终止核苷酸(例如，一种或多种双脱氧核苷酸三磷酸(ddNTP)或无环核苷酸)、一种或多种盐(例如，含镁的盐)、一种或多种缓冲液(例如，含有β-NAD、TritonX-100)以及一种或多种聚合剂(例如，DNA聚合酶、RNA聚合酶)。
能够选择和使用任意合适的延伸反应。例如，能够使用延伸反应来区分SNP等位基因或特定的甲基化位点，这通过将脱氧核苷酸和/或终止核苷酸(例如，双脱氧核苷酸，无环核苷酸)掺入到与靶核酸中特定位点相邻的区域杂交的延伸寡核苷酸中。所述引物通常由聚合酶延伸。在一些实施方式中，寡核苷酸由仅仅一个与待测位点互补的脱氧核苷酸或终止核苷酸(例如，双脱氧核苷酸或无环核苷酸)延伸。在一些实施方式中，寡核苷酸通过掺入dNTP延伸并且通过ddNTP或无环核苷酸终止，或在某些实施方式中，寡核苷酸通过掺入ddNTP或无环核苷酸终止而没有dNTP延伸。
在一些实施方式中，可以通过5种终止核苷酸(例如，ddATP、ddUTP、ddTTP、ddGTP、ddCTP)中的任一种延伸寡核苷酸。靶核酸或其对应的扩增产物，可以被用作模板并且可以部分确定延伸反应中哪个终止核苷酸被加入到寡核苷酸上。在某些实施方式中，可以利用其他终止核苷酸(例如，无环核苷酸或终止子)。
在某些实施方式中，延伸可以在等温条件下或在非等温环境下(例如，热循环条件)进行。一种或多种靶核酸可以在延伸反应中延伸，并且可以延伸各靶核酸的一个或多个变体。核酸可以延伸一个或多个核苷酸，并且在一些实施方式中，延伸产物长度为约10个核苷酸至约10000个核苷酸，长度为约10至约1000个核苷酸，长度为约10至约500个核苷酸，长度为约10至约100个核苷酸，并且有时长度为约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或1000个核苷酸。掺入终止核苷酸(例如，ddNTP)、杂交位置或其他因子可以确定寡核苷酸延伸的长度。在某些实施方式中，在同一检测过程中进行扩增和延伸反应。
在一些实施方式中，延伸反应包括重复多个温度循环以扩增反应中延伸产物的量。在一些实施方式中，延伸反应循环2次或更多次。在一些实施方式中，延伸反应循环10次或更多次。在一些实施方式中，延伸反应循环约10，15，20，50，100，200，300，400，500或600次或更多次。在一些实施方式中，延伸反应循环20至50次。在一些实施方式中，延伸反应循环20至100次。在一些实施方式中，延伸反应循环20至300次。在一些实施方式中，延伸反应循环200至300次。在一些实施方式中，延伸反应循环至少50、55、60、65、70、75、 80、85、90、95或100次。
在一些实施方式中，与靶核酸(例如，本申请的指数扩增产物)杂交的寡核苷酸(例如，本申请的延伸引物)在延伸组合物存在的情况下通过一个核苷酸延伸。延伸组合物可以包括一种或多种缓冲液、盐、酶(例如，聚合酶，Klenow等)、水、模板(例如，DNA、RNA、扩增产物等)、引物(例如，寡核苷酸)、三磷酸核苷酸、甘油、大分子排除分子(macromolecular exclusion molecule)和本领域所使用的任何其他添加剂。延伸组合物可以包括终止核苷酸(例如，双脱氧核苷酸(例如，ddNTP)或五环核苷酸)，非终止或延伸核苷酸(例如，dNTP)或终止核苷酸和非终止核苷酸的混合物。延伸组合物基本上由一种或多种特定终止核苷酸组成，可以包含延伸组合物(例如，缓冲液、盐、模板等)的任何其他组分，但是除了指定的那些之外，基本上不包含任何其他终止核苷酸或三磷酸核苷酸(列，dNTP)。例如，基本上由ddTTP和ddCTP组成的延伸组合物不含ddATP，ddGTP或任何其他dNTP。在一些实施方式中，延伸组合物中的核苷酸仅为与靶核酸杂交的终止核苷酸和寡核苷酸，靶核酸或其扩增产物通过一个核苷酸延伸。在一些实施方式中，延伸组合物基本上由终止核苷酸(例如，ddNTP，无环核苷酸)组成。
在一些实施方式中，在延伸组合物中存在(或在一些实施方式中不存在)的终止核苷酸确定将哪个终止核苷酸加入到寡核苷酸中。在一些实施方式中，延伸组合物包含一种或多种终止核苷酸(例如，ddNTP或无环核苷酸)。在具有超过一种终止核苷酸的某些实施方式中，2、3或4种链终止核苷酸是不同的终止核苷酸(即，不是相同的物质，例如，ddA、ddT、ddC、ddG、ddU、acyATP、acyCTP、acyGTP、acyTTP和acy-溴-UTP)。在一些实施方式中，延伸组合物包含一种或多种终止核苷酸和一种或多种非终止核苷酸(例如，dNTP)。在一些实施方式中，延伸组合物包括对应特定变体(例如，第一变体、低量变体或低丰度变体)的终止核苷酸，并因此仅能够延伸特定变体。在一些实施方式中，延伸组合物中包括能够允许延伸第二变体(例如，野生型、高量变体或高丰度变体)的终止核苷酸，从而允许延伸第二变体。在一些实施方式中，本申请的方法包括将杂交的寡核苷酸物质与包含一种或多种终止核苷酸的延伸组合物在延伸条件下接触，从而生成延伸的寡核苷酸物质，其中与第一变体(例如，低丰度变体、较少丰度变体、低量变体)杂交的寡核苷酸由终止核苷酸延伸，并且与第二变体(例如，野生型、高丰度变体、高量变体)杂交的寡核苷酸由终止核苷酸延伸。
例如，所述延伸引物能够与所述靶核酸(例如，希望鉴别的特定位点或靶核酸)特异性结合。在本申请中，所述延伸引物可以包含至少约1个核苷酸，例如，至少约2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、 18个、19个、20个、21个、22个、23个、24个或更多个核苷酸的核酸分子(例如，单链核酸分子，例如单链DNA分子)。所述延伸引物可与其靶序列(例如，希望鉴别的特定位点或靶核酸)基本上完全互补(例如，至少80％互补，例如至少85％互补，至少90％互补，至少91％互补，至少92％互补，至少93％互补，至少94％互补，至少95％互补，至少96％互补，至少97％互补，至少98％互补或者至少99％或以上互补，例如，100％互补)。
在某些实施方式中，所述延伸反应(例如，单碱基延伸反应)中可使用DNA聚合酶，例如NEB公司的Therminator TM DNA聚合酶，Therminator DNA聚合酶是9°N TM DNA聚合酶的突变体，经过改造后的酶可以识别并掺入一些修饰的碱基比如ddNTP。由于该酶的热稳定性特性，可以通过反复变性、退火和延伸充分提高引物的使用效率，提高产量。
也可使用其他的T7DNA聚合酶，例如经基因工程化的T7DNA聚合酶也可以用作单碱基延伸反应。例如，所述DNA聚合酶可以是基因工程改造形式的T7DNA聚合酶(例如，Sequenase)。与野生型酶不同，它可以基本上没有3'→5'核酸外切酶活性。所述DNA聚合酶可包含2个亚单元，其中一个可以是大肠杆菌硫氧还蛋白，另一个可以是噬菌体T7基因5蛋白的基因工程改造版本。该亚单元发生的基因改变(例如，通过体外突变删除28个氨基酸)可清除所有可测量的核酸外切酶活性，但不会改变DNA聚合酶的活性。
例如，所述终止核苷酸可在其戊糖部分的3’碳处被修饰。例如，至少一种所述终止核苷酸可以是双脱氧核苷酸。例如，所述双脱氧核苷酸可选自：ddATP、ddGTP、ddCTP、ddTTP和ddUTP。在某些实施方式中，所述终止核苷酸是无环核苷酸。例如，所述无环核苷酸可选自：acyATP、acyCTP、acyGTP、acyTTP和acy-溴-UTP。
例如，所述终止核苷酸包含：1)对经所述处理后由未甲基化的特定核苷酸修饰生成的所述其他核苷酸具有特异性的终止核苷酸；以及2)对甲基化的所述特定核苷酸具有特异性的终止核苷酸。
检测
在本申请中，所述延伸的寡核苷酸包含可检测标记物。例如，所述可检测标记物可以是质量标记物。例如，所述质量标记物可以是质量可区分标签。在某些情形中，所述终止核苷酸可包含所述质量可区分标签。
例如，在本申请的方法中，可通过质谱检测所述质量标记物。所述质谱可以是，例如，基质辅助激光解吸电离(MALDI)质谱。
所述可检测标记物通常是指可以彼此区分并用于鉴定标记物所连接的核酸的标签。可以选择多种类型的标记物并用于本申请的方法。例如，可以使用寡核苷酸、氨基酸、小型有机 分子、光发射分子、光吸收分子、光散射分子、发光分子、同位素、酶等作为可检测标记物。在某些实施方式中，也能够使用寡核苷酸、氨基酸和/或各种长度、各种荷质比、各种电泳迁移率(毛细管电泳迁移率)和/或各种质量的小分子有机分子作为可检测标记物。因此，可以使用荧光团、放射性同位素、发色剂、发光剂、化学发光剂、光散射剂等作为标记物。标记物的选择取决于所需灵敏度、与核酸偶联的难易、稳定性要求以及可用的设备。
在一些实施方式中，所述可检测标记物与终止核苷酸连接。在一些实施方式中，可以选择和/或设计合适的可检测标记物，以实现质谱中最优的飞行表现并且允许标记物在较高的多重水平上进行区分。在一些实施方式中，标记物是通过电泳或实施PCR检测的荧光标记物或染料。
能够使用任意合适的检测设备以区分样品中的可检测标记物。适于检测质量可区分标签的检测设备包括但不限于某些质谱和凝胶电泳设备。质谱形式的示例包括但不限于：基质辅助激光解吸/电离飞行时间(MALDI-TOF)质谱(MS)、MALDI正交TOF质谱(OTOF MS；二维)、激光解吸质谱(LDMS)、电喷雾(ES)质谱、离子回旋共振(ICR)质谱和傅立叶变换质谱。本申请所述的方法容易适用于质谱形式，其中分析物经挥发和离子化(“离子化MS”，例如，MALDI-TOF MS，LDMS，ESMS，线性TOF，OTOF)。正交离子提取MALDI-TOF和轴向MALDI-TOF能够产生相对高的分辨率，并且因此，相对高水平的多重化。适用于检测光发射、光吸收和/或光散射标记的检测设备包括但不限于某些光检测器和光电检测器(例如，检测荧光、化学发光、吸收和/或光散射标记物)。
可以通过多种方法检测通过本申请的方法获得的延伸产物(例如，本申请的延伸的寡核苷酸)。例如，可以用允许检测信号和/或定量信号的任何类型的化学基团或部分标记延伸引物(UEP)和/或终止核苷酸，包括但不限于，质量标记物、放射性分子、荧光分子、抗体、抗体片段、半抗原、碳水化合物、生物素、生物素衍生物、磷光部分、发光部分、电化学发光部分、在氧化或还原时产生电化学信号的部分，例如铁，钌或锇的复合物、色度部分、具有可检测的电子自旋共振、电容、介电常数或电导率的部分、或其标记物的任何组合。
在本申请中，“质量可区分标签”是指以质量作为区分特征的标记物。在一些实施方式中，所述可检测标记物由核苷酸组成，并且有时所述标记物长约5个核苷酸-约50个核苷酸。在某些实施方式中，所述可检测标记物是核苷酸复合体，其有时长约5个核苷酸-约35个核苷酸。在一些实施方式中，所述可检测标记物是肽，其有时长约5个氨基酸-约100个氨基酸。在某些实施方式中，可检测标记物是有机分子单元的多联体(concatemer)。在一些实施方式中，标记物是三苯甲基分子多联体。在某些实施方式中，质量可区分标签是链终止核苷酸。
能够选择和使用多种质量可区分标签，例如复合体、氨基酸和/或多联体。能够通过质量区分核苷酸串(例如，核酸；复合体)、氨基酸串(例如，肽；多肽；复合体)和/或多联体的不同长度和/或组成，并用作标记物。在质量可区分标签中可以采用任意数量的单元，并且这样的单元的上限和下限部分取决于质量窗和用于检测和区分这样的标记的系统的分辨率。因此，能够部分基于用于检测和区分标记物的质量窗和检测器的分辨率选择质量可区分标签的长度和组成。
在某些实施方式中，可以由核酸产物(例如，延伸的寡核苷酸)中释放可检测标记物。可检测标记物和核酸之间的连接可以是能被转录和切割，被切割并且允许检测释放的一种或多种标记的任意类型。在某些实施方式中，可以从标记物连接的分子的其他部分分离标记物。例如，可以通过核酸酶(例如，核糖核酸酶、内切核酸酶)切割的连接；可以通过化学方法切割的连接；可以通过物理处理切割的连接；和可以通过光切割的光可切接头(例如，邻硝基苄基，6-硝基藜芦氧羰基(6-nitroveratryloxycarbonyl)，2-硝基苄基基团)。当使用发射光的检测系统(例如，涉及光的激发射的基质辅助激光解吸电离(MALDI)质谱)时，光可切割接头具有优势，因为切割和检测结合在一起且在一个步骤中进行。
例如，根据本申请所述的方法，在利用质谱(如MALDI质谱)对核酸分子的甲基化情况进行检测时，未甲基化的核酸分子会在dA对应的位置出现一个峰，甲基化的核酸分子会在dG对应的位置出现一个峰，而多余的延伸引物会显示为另外的峰。因此，dG/(dA+dG)的值可代表包含甲基化修饰的所述核酸分子的比例或含量。
多重检测
本申请的方法允许高通量检测或定量样品中多种靶核酸的存在、不存在或其含量。多重化指同时检测超过一种靶核酸(或者超过一个特定的位点，例如甲基化位点)。与质谱联合进行多重反应的常规方法是已知的(参见例如，WO1997037041A2)。相较于必须对各个靶核酸进行单独的质谱分析，多重化提供的优点在于可在单一质谱中鉴定多种靶核酸和其变体(例如，具有不同序列变异的变体)。在一些实施方式中，本申请的方法能用于快速且准确分析靶序列(例如，DNA甲基化，特别是少量DNA的甲基化)的高通量、高度自动化过程。在一些实施方式中，本申请的方法可在单一反应中高度多重化。当位于基因座的基因型未知时，多重化是适用的，并且在一些实施方式中，位于基因座的基因型是已知的。
在某些实施方式中，多重化的靶核酸的数量包括但不限于，约2-约1，000种，约2-约500种，约2-约100种，例如，约1-5、5-9、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、149-150、150-200、200-250、250-300、300-350、350-400、400-450、450-500种或更多种。
用多重分析获得解析质谱的设计方法可包括引物和寡核苷酸设计方法和反应设计方法。就多重分析中的引物与寡核苷酸设计而言，与单重反应采用相同的引物设计总体方针，例如避免假引发和引物二聚体，只是多重反应涉及更多引物。此外，一个试验的质谱中的分析物峰可以从该试验与之多重化的任意试验的产物中充分解析，包括暂停峰(pausingpeak)和任意其它副产物峰。还有，分析物峰优选落入用户指定的质量窗，例如，在5000-8500Da范围内。在一些实施方式中，相对于特定的靶序列，可以设计延伸寡核苷酸。在这类实施方式中，例如，长度通常是可以用户指定的(例如，17-24个碱基或17-26个碱基)的范围之间，并且通常不包含靶序列中不确定的碱基。有时通过计算序列依赖性解链(或杂交/解离)温度Tm来衡量杂交强度。用于设计延伸引物的方法和软件是已知的，并且包括，例如SpectroDESIGNER(Sequenom)。
在一些实施方式中，本申请提供的多重试验是为单碱基延伸设计的。例如，可首先使寡核苷酸(例如，本申请中延伸的寡核苷酸)与源自靶核酸的扩增产物(例如，本申请的扩增产物，如指数扩增产物)在单碱基位置的5'位置处杂交，所述单碱基位置在靶核酸各变体(例如，存在甲基化修饰和不存在甲基化修饰的靶核酸)之间不同。
疾病、疾病结果和/或治疗方案
本申请的方法和产品可用于疾病或病症的诊断(包括疾病或病症的预后)，例如，通过非侵入式的方法进行所述疾病或病症的诊断。
例如，所述疾病或病症可以是与遗传物质(例如，核酸，例如DNA)的变化(例如，甲基化修饰或SNP)相关(例如，依据该甲基化情况进行判断)的疾病或病症，例如肿瘤或癌症。所述遗传物质可以是源自受试者的样品(例如，组织或体液)的cfDNA，例如ctDNA。
在某些实施方式中，本申请的方法和产品可用于改善、调整或确定适合于特定受试者的治疗方案。
试剂盒和系统
另一方面，本申请提供了一种试剂盒和/或系统，其可用于实施本申请所述的方法。
所述试剂盒中可包括：本申请所述的线性扩增组分，本申请所述的指数扩增组分，以及本申请所述的延伸组分。在某些情形中，所述线性扩增组分、指数扩增组分和所述延伸组分在所述试剂盒中互相不混合。
例如，在所述试剂盒中，所述线性扩增组分，所述指数扩增组分，以及所述延伸组分可各自独立地存在于单独的包装中。
例如，所述试剂盒中可包含下述组分中的一种或多种：a)试剂，其可修饰未甲基化的特定核苷酸以生成其他核苷酸；b)一种或多种核酸聚合酶；c)一种或多种引物；d)MALDI基质化合物；和e)MALDI底物。
在某些情形中，在本申请所述的系统中，所述用于使用所述试剂盒的装置可包括热循环仪和/或质谱仪。
本申请所述的试剂盒通常包含含有一种或多种本申请所述组分的一个或多个容器。试剂盒包括任意数量的单独容器、包、管、小瓶、多孔板等中的一种或多种组分，或者可以在这些容器中以多种组合方式组合组分。试剂盒可以包括例如一种或多种下述组分：(i)一个或多个核苷酸(例如，终止核苷酸和/或非终止核苷酸)；其中的一个或多个可以包含检测标记物；(ii)一种或多种寡核苷酸，其中的一种或多种可以包括检测标记物(例如，扩增引物，一种或多种延伸引物，包含标记物的寡核苷酸)；(iii)一种或多种酶(例如，聚合酶，内切核酸酶，限制酶，外切核酸酶等)；(v)对照组分(例如，对照DNA，引物，合成模板，靶核酸等)；(vi)一种或多种缓冲液和(vii)印刷品(例如，说明书，标记物等)。
试剂盒有时与某些方法联用，并且可以包括用于执行一个或多个方法的说明和/或一种或多种组合物的描述。试剂盒可以用于执行本申请所述的方法。说明和/或描述可以是有形形式(例如，纸等)或电子形式的(例如，有形介质上的计算机可读文件等(例如，光盘))，并且可以包括在试剂盒插入物中。试剂盒还可以包括提供这类说明或描述的互联网位置的书面说明。
在某些实施方式中，本申请的试剂盒包括一种或多种终止核苷酸，所述终止核苷酸在其处于寡核苷酸的3'末端时抑制所述酶在寡核苷酸上的活性。在某些实施方式中，所述终止核苷酸是双脱氧核苷酸。在某些实施方式中，所述双脱氧核苷酸选自：ddATP、ddGTP、ddCTP、ddTTP和ddUTP。在某些实施方式中，所述终止核苷酸是无环核苷酸。在某些实施方式中，所述无环核苷酸选自：acyATP、acyCTP、acyGTP、acyTTP和acy-溴-UTP。在某些实施方式中，所述终止核苷酸包括质量可区分标签。
在某些实施方式中，所述试剂盒包括下述一种或多种：寡核苷酸、聚合酶、逆转录酶、一种或多种缓冲液和一种或多种反应对照。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的装置、方法和系统的工作方式，而不用于限制本申请发明的范围。
实施例
实施例1样品合成
合成双链的参比序列，其分别为：
参比序列1(内参1)：其正向核酸序列如SEQ ID NO:4所示，与所述正向核酸序列互补的反向核酸序列如SEQ ID NO:5所示。在所有的参比序列1样品中，含有5-甲基胞嘧啶(即5mC)的核酸分子的含量为0％。
参比序列2(内参2)：其正向核酸序列如SEQ ID NO:7所示，与所述正向核酸序列互补的反向核酸序列如SEQ ID NO:8所示。在所有的参比序列2样品中，含有5-甲基胞嘧啶(即5mC)的核酸分子的含量为25％。
参比序列3(内参3)：其正向核酸序列如SEQ ID NO:10所示，与所述正向核酸序列互补的反向核酸序列如SEQ ID NO:11所示。在所有的参比序列3样品中，含有5-甲基胞嘧啶(即5mC)的核酸分子的含量为50％。
参比序列4(内参4)：其正向核酸序列如SEQ ID NO:13所示，与所述正向核酸序列互补的反向核酸序列如SEQ ID NO:14所示。在所有的参比序列4样品中，含有5-甲基胞嘧啶(即5mC)的核酸分子的含量为75％。
参比序列5(内参5)：其正向核酸序列如SEQ ID NO:16所示，与所述正向核酸序列互补的反向核酸序列如SEQ ID NO:17所示。在所有的参比序列5样品中，含有5-甲基胞嘧啶(即5mC)的核酸分子的含量为100％。
实施例2末端修复和衔接子连接
2.1末端修复和加A尾
使用KAPA Hyper Prep文库构建试剂盒，对实施例1中的参比序列1-参比序列5分别进行末端修复。简要地，如下所述在PCR管中配制末端修复和加A尾(A tailing)反应混合物：
组分 体积
参比双链DNA 1.32μl
末端修复和加A尾酶混合物 0.06μl
末端修复和加A尾缓冲液 15μl
无核酸酶水 48.62μl
总量 65μl
将上述反应混合物吹打混匀，并进行快速离心。
在热循环仪中进行孵育，采用如下所述设置程序：
热盖25℃ 开启
20℃ 15min
65℃ 15min
4℃ 保持
2.2接头连接
将接头原液进行适当稀释，如下表所示：
10ng起始DNA组：
Figure PCTCN2022143769-appb-000001
1ng起始DNA组(低于1ng DNA的按照1ng处理)：
Figure PCTCN2022143769-appb-000002
将连接缓冲液解冻后颠倒混匀，置于冰上备用。
在实施例2.1中进行过末端修复和加A尾反应的PCR管中，按如下方式配制连接反应混合物：
组分 体积
末端修复和加A尾产物 65μl
连接缓冲液 25μl
DNA连接酶 5μl
接头稀释液 1μl
Tris-Hcl(20mM) 4μl
使用移液器轻轻吹打混匀，短暂离心后将反应液收集至管底。
在热循环仪中进行孵育，采用如下所述设置程序：
温度 时间
25℃ 开启
20℃ 15min
4℃ 保持
使用Zymo Research的DNA Clean&Concentrator-5试剂盒，根据说明书的描述，对实施例2.2中的连接产物进行纯化，以获得含接头的核酸。
实施例3核酸转变
使用Zymo Research的EZ DNA Methylation-Gold试剂盒对实施例2.2中获得的、经纯化的含接头的核酸进行亚硫酸氢盐处理。其中，DNA样品分子中的胞嘧啶(C)被转换为尿嘧啶(U)，5-甲基胞嘧啶(5mC)则保持不变。
实施例4线性扩增和逆转录
4.1线性扩增
使用T7 RNA聚合酶对经转变的核酸分子进行线性扩增。
根据下表，配制线性扩增反应混合物：
Figure PCTCN2022143769-appb-000003
用移液器将各组分轻轻混匀，短暂离心后在37℃下孵育2h。
加入线性扩增引物(SEQ ID NO:3)2μl(100μM)，70℃，置于冰上2min。
将反应混合物分为两管，每管中各包含12μl。
4.2逆转录反应
对实施例4.1中获得的线性扩增产物进行逆转录。使用HiScript III RT SuperMix试剂盒，逆转录的引物为SEQ ID NO:3所示的核酸序列。
4.2.1去除基因组DNA
在不含RNase的离心管中配制如下混合液：
组分 体积
4×gDNA wiper Mix 4μl
实施例4.1的线性扩增产物 12μl
用移液器轻轻吹打混匀，在42℃下孵育2min。
4.2.2配制逆转录反应体系
在4.2.1的反应管中直接加入5×HiScript III qRT SuperMix 4μl，用移液器轻轻吹打混匀。
4.2.3根据如下条件进行逆转录反应
温度 时间
50℃ 15min
85℃ 5sec
使用Zymo Research的DNA Clean&Concentrator-5试剂盒对逆转录反应后获得的cDNA产物进行纯化。
实施例5指数扩增
根据下表配制PCR混合物
组分 体积
10×Taq缓冲液(添加Mg 2+) 5μl
dNTP Mix(每一种10mM) 1μl
PCR引物(SEQ ID NO:3)(10μM) 4μl
实施例4.2中获得的cDNA 39.5μl
Taq DNA聚合酶(5U/μl) 0.5μl
进行PCR反应的程序如下：
Figure PCTCN2022143769-appb-000004
使用AxyPrep PCR清洁试剂盒(AP-PCR-250G)对PCR反应的产物进行纯化。
实施例6单碱基延伸
将实施例5中获得的PCR产物加入无核酸酶的纯水中，于98℃处理10min，随后迅速放在冰上。在每管反应混合物中分别加入1μl延伸引物(参比序列1的延伸引物如SEQ ID NO:6所示，参比序列2的延伸引物如SEQ ID NO:9所示，参比序列3的延伸引物如SEQ ID NO:12所示，参比序列4的延伸引物如SEQ ID NO:15所示，参比序列5的延伸引物如SEQ ID NO:18所示)。
Figure PCTCN2022143769-appb-000005
Figure PCTCN2022143769-appb-000006
进行单碱基延伸反应的程序如下：
Figure PCTCN2022143769-appb-000007
实施例7 MALDI-TOF分析
首先进行脱盐处理。可使用2种方式来进行脱盐。
在一种方式中，使用Zymo Research的Oligo Clean&Concentrator(D4061)，按照说明书进行纯化，最后用纯水洗脱。
在另一种方式中，使用ZipTipμ-C18移液器吸头(ZTC18S960)，具体步骤如下：
1)使用50％乙腈中性溶液，活化Millipore C 18 ZIP TIP，吸排5-10次；
2)用0.1M四乙基醋酸铵四水化合物TEAA(pH＝7.0)平衡C18 ZIP TIP，吸排5-10次；
3)在20μl实施例6的单碱基延伸体系中加入1μl 2M TEAA(pH＝7.0)，用C18 ZIP TIP吸附，吸排5-10次；
4)用0.1％0.1M TEAA(pH＝7.0)洗涤，吸排3次；
5)用超纯水洗涤，吸排3次；
6)吸取4μl含50％乙腈的溶液洗脱核酸段供质谱分析，在空样品管中吸排5-10次；
7)用70％乙腈中性溶液吸排5-10次，洗净ZIP TIP柱。
使用MassARRAY小型分析仪(MALD1-TOF质谱仪)进行质谱分析，得到检测结果。
通常，从大约2ml的全血中可以提取1ng的cfDNA。通过本申请的方法，可有效检测到样品中约1ng或更少的DNA样品及其中的甲基化修饰。对内参1至内参5的检测结果分别如图2A-2E所示，结果总结在图2F中。图2F表示各个内参特异性引物单碱基延伸之前与延伸之后的分子量，以及使用微量核酸质谱计算出来的特定位点的甲基化含量的准确度。可以看到，针对内参1至内参5进行检测的准确度均至少为约65％或更高。
实施例8基于另一种链置换扩增的扩增反应
甲基化NISDA线性扩增技术
可以以接头为双链DNA，接头可以含有至少一个切口酶(Nickase)可以识别保守序列，以及相对应的保护碱基数；或者是不受胞嘧啶核糖核苷甲基化修饰影响活性的核酸内切酶识别序列。同时，接头的切口酶识别序列中，至少有一条链上的C碱基是被5位甲基化保护的。接头的其中一条链的3’端可以有一个额外的T尾不与互补链匹配，以方便与进行末端修复，3’端加A碱基尾后的DNA片段相连。如果接头中含有核酸内切酶识别序列，则在该内切酶对应的切割位置的两侧或者两侧附近的其中一条链上的核苷酸进行硫代磷酸酯化修饰(Phosphorothioate)、或者进行其它可以抵抗内切酶活性的修饰，从而达到使DNA双链中其中一条被内切酶切断，而另外一条保持原来的完整状态的效果。
将具备所述特征的双链接头与DNA片段相连，例如可以使用DNA连接酶系统。
将完成连接的DNA片段，使用亚硫酸盐处理，使未被甲基化的C碱基转化为T碱基，完成处理的产物应为单链DNA。
加入双链接头中其中一条链经过亚硫酸盐处理后的DNA序列的互补单链引物，经退火后与经亚硫酸盐处理的单链DNA形成部分双链结构，该部分双链DNA至少应包含缺口酶或内切酶识别序列及其相应的保护碱基数。
加入至少包含切口酶(或内切酶)和具备链置换功能的DNA聚合酶，启动基于切口DNA的扩增；在该体系中可以加入DNA单链结合蛋白以稳定反应系统。如图4所示，所获得的线性扩增产物用于本申请的核酸检测方法，例如下游PCR反应、测序、或者核酸质谱的检测，可以实现特定位点的甲基化含量的检测准确度。
实施例9基于另一种滚环扩增的线性扩增反应
甲基化RCA线性扩增技术
可以以接头为双链DNA，接头长度可以不低于5bp，并且接头不含C碱基或者含C碱基但被5位甲基化保护。接头的其中一条链的3’端可以有一个额外的T尾不与互补链匹配，以方便与进行末端修复，3’端加A碱基尾后的DNA片段相连。
将具备所述特征的双链接头与DNA片段相连，可以使用DNA连接酶系统。
将完成连接的DNA片段，使用亚硫酸盐处理，使未被甲基化的C碱基转化为T碱基，完成处理的产物应为单链DNA。
加入与双链接头两条链均序列互补的单链引物，退火，使经过C-T转化的带有接头的单链DNA两端均单链引物结合。
加入具有DNA连接功能酶，使单链带有接头的单链DNA两头连接，形成环状DNA。
然后加入具备链置换功能的DNA聚合酶，启动滚环扩增；在该体系中可以加入DNA单链结合蛋白以稳定反应系统。
如图5所示，滚环扩增产物即为线性扩增产物。所获得的线性扩增产物用于本申请的核酸检测方法，例如下游PCR反应、测序、或者核酸质谱的检测，可以实现特定位点的甲基化含量的检测准确度。
实施例10临床样本检测结果
使用结直肠癌患者的血浆6ml，按照下述步骤对其中的septin9的甲基化状态进行检测。
用易毕恩生物科技抽提试剂盒提取血浆中的cfDNA，然后按照本申请记载的检测方法，使用KAPA Hyper Prep文库构建试剂盒获得的cfDNA进行末端修复和T7甲基化接头连接。然后使用DNA Clean&Concentrator-5(Zymo Research)纯化完成接头连接的cfDNA。接下来使用EZ DNA Methylation-Gold(Zymo Research)试剂盒对纯化后的产物进行亚硫酸盐处理。然后对亚硫酸处理产物进行纯化。使用T7 RNA聚合酶和逆转录酶对亚硫酸盐处理后的纯化产物进行线性扩增，具体操作如实施例4所示。
使用专门针对人基因组septin9启动子区域甲基化位点设计的PCR扩增引物7对组合，对线性扩增产物进行PCR扩增，并进行产物清洁和纯化，PCR扩增程序和清洁纯化操作如实施例5
引物序列
Figure PCTCN2022143769-appb-000008
Figure PCTCN2022143769-appb-000009
其中，forward及reverse为对应位点的PCR扩增引物，extension为对应位点的延伸引物。
用于Septin9的PCR扩增引物对数，共七个位点，七对引物，每个反应中引物浓度均为2.5μM。用于Septin9的延伸引物条数，共七个位点，七个引物，每个反应中引物浓度均为0.5μM。
完成PCR扩增后，在上述所示的反应体系中加入专门针对eptin9启动子区域甲基化位点设计的7条延伸引物组合，延伸反应体系按照实施例6所示程序进行。
按照实施例7所示，对上述中的延伸反应产物进行脱盐纯化。
使用核酸飞行时间质谱仪，对脱盐产物进行分析，结果图6所示，患者的7个的septin9甲基化位点的甲基化修饰均被检出。
结果显示，本申请提供的甲基化检测方法，可以在临床检测中实现较高的准确性。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (64)

  1. 一种鉴别样品中一种或多种靶核酸存在、不存在或其含量的方法，所述方法包括：
    a)在使得源自所述样品的核酸能够线性扩增的条件下处理所述样品，以生成所述核酸的线性扩增产物；
    b)对所述线性扩增产物进行指数扩增，以生成所述核酸的指数扩增产物；
    c)在包括终止核苷酸的延伸条件下使所述核酸的指数扩增产物与延伸引物接触，从而生成延伸的寡核苷酸；
    d)分析所述延伸的寡核苷酸，从而鉴别所述样品中所述一种或多种靶核酸存在、不存在或其含量。
  2. 根据权利要求1所述的方法，其中，步骤a)包括：
    使所述样品与能够结合于所述核酸的寡核苷酸接触，以形成寡核苷酸杂交体；和
    使所述寡核苷酸杂交体与扩增组合物在能够发生线性扩增的条件下接触，以生成所述核酸的线性扩增产物。
  3. 根据权利要求1-2中任一项所述的方法，其中步骤a)包括：
    使源自所述样品的核酸与接头序列连接，以形成含接头的核酸；
    使所述含接头的核酸与能够特异性结合于所述接头序列的寡核苷酸接触，以形成寡核苷酸杂交体；和
    使所述寡核苷酸杂交体与扩增组合物在能够发生线性扩增的条件下接触，以生成所述核酸的线性扩增产物。
  4. 根据权利要求3所述的方法，其中所述接头序列包含RNA聚合酶的启动子序列。
  5. 根据权利要求3-4中任一项所述的方法，其中所述接头序列包含SP6启动子序列、T7启动子序列和/或T3启动子序列。
  6. 根据权利要求3-5中任一项所述的方法，其中所述能够特异性结合于所述接头序列的寡核苷酸与所述接头序列至少部分互补。
  7. 根据权利要求4-6中任一项所述的方法，其中所述能够特异性结合于所述接头序列的寡核苷酸包含与所述RNA聚合酶的启动子序列互补的序列。
  8. 根据权利要求5-7中任一项所述的方法，其中所述能够特异性结合于所述接头序列的寡核苷酸包含SP6启动子序列的互补序列、T7启动子序列的互补序列和/或T3启动子序列的互补序列。
  9. 根据权利要求3-8中任一项所述的方法，其中所述接头序列包含一种或多种修饰，所述修饰使得所述接头序列经亚硫酸氢盐处理后，其核酸序列不发生改变。
  10. 根据权利要求3-9中任一项所述的方法，其中所述接头序列包含一种或多种碱基的甲基化修饰。
  11. 根据权利要求10所述的方法，其中所述接头序列中包含的胞嘧啶为经甲基化修饰的胞嘧啶。
  12. 根据权利要求3-11中任一项所述的方法，其中所述接头序列直接或间接连接于所述核酸的3’端。
  13. 根据权利要求3-12中任一项所述的方法，其中所述接头序列的5’端直接或间接连接于所述核酸的3’端。
  14. 根据权利要求3-13中任一项所述的方法，其中使所述核酸与所述接头序列连接包括：
    修复所述核酸的末端，使其成为平末端。
  15. 根据权利要求3-14中任一项所述的方法，其中使所述核酸与所述接头序列连接包括：
    修复所述核酸的末端，使其成为平末端，并且在所述经修复的序列的3’末端添加脱氧腺苷dA。
  16. 根据权利要求1-15中任一项所述的方法，其中所述线性扩增包含核酸转录反应、链置换扩增反应和/或滚环扩增反应。
  17. 根据权利要求1-16中任一项所述的方法，其中所述线性扩增为不针对特定靶标的非靶标特异性扩增。
  18. 根据权利要求1-17中任一项所述的方法，其中所述核酸的线性扩增产物包含由所述核酸编码的RNA。
  19. 根据权利要求1-18中任一项所述的方法，其中所述线性扩增中使用RNA聚合酶。
  20. 根据权利要求19所述的方法，其中所述RNA聚合酶包括T7 RNA聚合酶，SP6 RNA聚合酶，和/或T3 RNA聚合酶。
  21. 根据权利要求1-20中任一项所述的方法，其中，步骤a)还包括：
    用可修饰未甲基化的特定核苷酸以生成其他核苷酸的试剂处理所述样品。
  22. 根据权利要求21所述的方法，其中，所述可修饰未甲基化的特定核苷酸以生成其他核苷酸的试剂包括可将未甲基化的胞嘧啶修饰生成尿嘧啶的试剂。
  23. 根据权利要求21-22中任一项所述的方法，其中，所述可修饰未甲基化的特定核苷酸以生成其他核苷酸的试剂包括亚硫酸氢盐，β-葡萄糖基转移酶，TET酶，吡啶硼烷和/或A3A脱氨酶。
  24. 根据权利要求1-23中任一项所述的方法，其中，步骤a)包括：
    使源自所述样品的核酸与所述接头序列连接，以形成含接头的核酸，其中所述接头序列包含一种或多种修饰，所述修饰使得所述接头序列经亚硫酸氢盐处理后，其核酸序列不发生改变；
    用包含亚硫酸氢盐的试剂处理所述含接头的核酸，以形成经转变的核酸；
    使所述经转变的核酸与能够特异性结合于所述接头序列的寡核苷酸接触，以形成寡核苷酸杂交体；和
    使所述寡核苷酸杂交体与扩增组合物在能够发生线性扩增的条件下接触，以生成所述核酸的线性扩增产物。
  25. 根据权利要求1-24中任一项所述的方法，其用于鉴别样品中一种或多种甲基化靶核酸的存在、不存在或其含量。
  26. 根据权利要求1-25中任一项所述的方法，其用于鉴别样品中一种或多种靶核酸中核苷酸基因座的甲基化状态。
  27. 根据权利要求1-26中任一项所述的方法，其中所述指数扩增包括聚合酶链式反应PCR。
  28. 根据权利要求1-27中任一项所述的方法，其中所述b)包括对所述线性扩增产物进行逆转录，并对所述逆转录产物进行PCR扩增。
  29. 根据权利要求1-28中任一项所述的方法，其中所述指数扩增包括逆转录酶聚合酶链式反应RT-PCR。
  30. 根据权利要求1-29中任一项所述的方法，其中所述指数扩增为不针对特定靶标的非靶标特异性扩增。
  31. 根据权利要求1-30中任一项所述的方法，其中所述指数扩增包括使用DNA聚合酶和/或逆转录酶。
  32. 根据权利要求1-31中任一项所述的方法，其中，所述终止核苷酸在其戊糖部分的3’碳处被修饰。
  33. 根据权利要求1-32中任一项所述的方法，其中，至少一种所述终止核苷酸是双脱氧核苷酸。
  34. 根据权利要求33所述的方法，其中，所述双脱氧核苷酸选自：ddATP、ddGTP、ddCTP、ddTTP和ddUTP。
  35. 根据权利要求1-31中任一项所述的方法，其中，所述终止核苷酸是无环核苷酸。
  36. 根据权利要求35所述的方法，其中，所述无环核苷酸选自：acyATP、acyCTP、acyGTP、acyTTP和acy-溴-UTP。
  37. 根据权利要求1-36中任一项所述的方法，其中，所述方法能够鉴别所述样品中的2种或更多种靶核酸。
  38. 根据权利要求1-37中任一项所述的方法，其中，所述终止核苷酸包含：1)对经所述处理后由未甲基化的特定核苷酸修饰生成的所述其他核苷酸具有特异性的终止核苷酸；以及2)对甲基化的所述特定核苷酸具有特异性的终止核苷酸。
  39. 根据权利要求1-38中任一项所述的方法，其中，所述延伸的寡核苷酸包含可检测标记物。
  40. 根据权利要求39所述的方法，其中，所述可检测标记物是质量标记物。
  41. 根据权利要求40所述的方法，其中，所述质量标记物是质量可区分标签。
  42. 根据权利要求41所述的方法，其中，所述终止核苷酸包含所述质量可区分标签。
  43. 根据权利要求40-42中任一项所述的方法，其中，通过质谱检测所述质量标记物。
  44. 根据权利要求43所述的方法，其中，所述质谱是基质辅助激光解吸电离(MALDI)质谱。
  45. 根据权利要求1-44中任一项所述的方法，其中，源自所述样品的核酸包含cfDNA和/或基因组gDNA。
  46. 根据权利要求1-45中任一项所述的方法，其中，源自所述样品的核酸包含源自肿瘤的ctDNA，和/或来自器官或胎儿的脱落细胞的DNA。
  47. 根据权利要求1-46中任一项所述的方法，其中，源自所述样品的核酸的含量为低于约10ng。
  48. 根据权利要求1-47中任一项所述的方法，其中，源自所述样品的核酸的含量为低于约1ng。
  49. 根据权利要求1-48中任一项所述的方法，其中，所述样品为生物样品，所述生物样品包括：血液，器官组织，福尔马林固定石蜡包埋FFPE样品，尿，粪便，脑脊液，胸腹水，口腔涮洗液，肺泡灌洗液，羊水等体液和组织，和/或体外培养样品。
  50. 根据权利要求1-49中任一项所述的方法，其中，所述样品源自受试者。
  51. 根据权利要求1-50中任一项所述的方法，其中，所述延伸引物能够与所述靶核酸特异性结合。
  52. 根据权利要求1-51中任一项所述的方法，其中，所述延伸引物的核酸序列与所述靶核酸的序列至少部分互补。
  53. 根据权利要求1-52中任一项所述的方法，其用于鉴别所述核酸分子中一个或多个CpG二核苷酸的胞嘧啶核苷酸的甲基化状态。
  54. 根据权利要求53所述的方法，其中所述一个或多个CpG二核苷酸位于基因的启动子区。
  55. 根据权利要求1-54中任一项所述的方法，其为体外或离体方法。
  56. 一种鉴别与疾病、疾病结果和/或治疗方案结果相关的生物标记物的方法，所述方法包括 以下步骤：
    i)根据权利要求1-55中任一项所述的方法，鉴别源自一个或多个样品的一种或多种靶核酸的存在、不存在或含量，所述一个或多个样品源自一个或多个具有已知疾病、疾病结果和/或治疗方案的结果的受试者；
    ii)根据权利要求1-55中任一项所述的方法，鉴别源自一个或多个样品的一种或多种靶核酸的存在、不存在或含量，所述一个或多个样品源自正常受试者；以及
    iii)鉴定步骤i)中所述一种或多种靶核酸的存在、不存在或含量与步骤ii)中所述一种或多种靶核酸的存在、不存在或含量之间的差异，并据此，将所述差异鉴定为与所述疾病、疾病结果和/或治疗方案结果相关的生物标记物。
  57. 一种鉴别与疾病、疾病结果和/或治疗方案结果相关的甲基化的方法，所述方法包括以下步骤：
    i)根据权利要求1-55中任一项所述的方法，鉴别源自一个或多个样品的一种或多种靶核酸中甲基化或未甲基化的核苷酸，所述一个或多个样品源自一个或多个具有已知疾病、疾病结果和/或治疗方案的结果的受试者；
    ii)根据权利要求1-55中任一项所述的方法，鉴别源自一个或多个样品的一种或多种靶核酸中甲基化或未甲基化的核苷酸，所述一个或多个样品源自正常受试者；以及
    iii)鉴定步骤i)中所述一种或多种靶核酸中甲基化或未甲基化的核苷酸与步骤ii)中所述一种或多种靶核酸中甲基化或未甲基化的核苷酸之间的差异，并据此，将所述差异的甲基化或未甲基化的核苷酸鉴别为与所述疾病、疾病结果和/或治疗方案结果相关的甲基化。
  58. 用于根据权利要求1-55中任一项所述的方法来鉴别样品中一种或多种靶核酸存在、不存在或其含量的试剂盒，所述试剂盒包含：
    线性扩增组分；
    指数扩增组分；以及
    延伸组分，所述延伸组分包含终止核苷酸，热稳定性延伸酶，以及延伸引物。
  59. 用于鉴别样品中一种或多种靶核酸存在、不存在或其含量的试剂盒，所述试剂盒包含：
    线性扩增组分；
    指数扩增组分；以及
    延伸组分，所述延伸组分包含终止核苷酸，热稳定性延伸酶，以及延伸引物。
  60. 根据权利要求58-59中任一项所述的试剂盒，其中所述线性扩增组分，所述指数扩增组分，以及所述延伸组分互相不混合。
  61. 根据权利要求58-60中任一项所述的试剂盒，其中所述线性扩增组分，所述指数扩增组分，以及所述延伸组分各自独立地存在于单独的包装中。
  62. 根据权利要求58-61中任一项所述的试剂盒，其还包含下述组分中的一种或多种：
    a)试剂，其可修饰未甲基化的特定核苷酸以生成其他核苷酸；
    b)一种或多种核酸聚合酶；
    c)一种或多种引物；
    d)MALDI基质化合物；和
    e)MALDI底物。
  63. 系统，其包含权利要求58-62中任一项所述的试剂盒，以及任选地包含选自下组的一个或多个部分：用法说明书、试剂和用于使用所述试剂盒的装置。
  64. 根据权利要求63所述的系统，其中所述用于使用所述试剂盒的装置包括热循环仪和/或质谱仪。
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