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WO2020227953A1 - 一种基于自发光的单通道测序方法 - Google Patents

一种基于自发光的单通道测序方法 Download PDF

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Publication number
WO2020227953A1
WO2020227953A1 PCT/CN2019/086974 CN2019086974W WO2020227953A1 WO 2020227953 A1 WO2020227953 A1 WO 2020227953A1 CN 2019086974 W CN2019086974 W CN 2019086974W WO 2020227953 A1 WO2020227953 A1 WO 2020227953A1
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Prior art keywords
luciferase
nucleic acid
molecular label
compound
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PCT/CN2019/086974
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English (en)
French (fr)
Inventor
廖莎
陈曦
陈奥
章文蔚
徐崇钧
陈宏敏
赵杰
傅德丰
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Egi Tech Shenzhen Co Ltd
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Egi Tech Shenzhen Co Ltd
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Priority to JP2021563644A priority Critical patent/JP7485483B2/ja
Priority to PCT/CN2019/086974 priority patent/WO2020227953A1/zh
Priority to KR1020217040845A priority patent/KR102843262B1/ko
Priority to CA3139169A priority patent/CA3139169A1/en
Priority to CN202410524599.2A priority patent/CN118931711A/zh
Priority to AU2019445584A priority patent/AU2019445584B2/en
Priority to US17/608,316 priority patent/US20220205036A1/en
Priority to MX2021013983A priority patent/MX2021013983A/es
Priority to EP19928475.3A priority patent/EP3988670A4/en
Application filed by Egi Tech Shenzhen Co Ltd filed Critical Egi Tech Shenzhen Co Ltd
Priority to BR112021022809A priority patent/BR112021022809A2/pt
Priority to SG11202111778YA priority patent/SG11202111778YA/en
Priority to CN201980095781.8A priority patent/CN113748216B/zh
Publication of WO2020227953A1 publication Critical patent/WO2020227953A1/zh
Anticipated expiration legal-status Critical
Priority to ZA2021/10159A priority patent/ZA202110159B/en
Priority to JP2024073905A priority patent/JP2024099789A/ja
Ceased legal-status Critical Current

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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
    • C12Q1/6869—Methods for sequencing
    • 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
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates

Definitions

  • the invention relates to the field of nucleic acid sequencing.
  • the present invention provides a self-luminescence-based single-channel sequencing method, in which the self-luminescence signal is used to distinguish the sequential incorporation of different nucleotides, thereby realizing the determination of the polynucleotide sequence.
  • DNA sequencing technology includes the first-generation DNA sequencing technology represented by Sanger sequencing method, and the second-generation DNA sequencing technology represented by Illumina Hiseq2500, Roche 454, ABI Solid, BGISEQ-500, etc.
  • Sanger invented the dideoxy terminal termination sequencing method and became the representative of the first generation of sequencing technology.
  • the Sanger sequencing method has the characteristics of simple experimental operation, intuitive and accurate results, and short experimental period. It has a wide range of applications in clinical gene mutation detection and genotyping that require high timeliness of detection results.
  • the disadvantages of the Sanger sequencing method are low throughput and high cost, which limits its application in large-scale gene sequencing.
  • the second-generation sequencing technology came into being.
  • the second-generation DNA sequencing technology has the characteristics of large sequencing throughput, low cost, high degree of automation, and single-molecule sequencing.
  • an experimental process can generate 10-200G base data.
  • the average sequencing cost per base is less than 1/1000 of the sequencing cost of Sanger sequencing, and the sequencing results obtained It can be processed and analyzed directly by the computer. Therefore, the second-generation DNA sequencing technology is very suitable for large-scale sequencing.
  • the currently developed second-generation DNA sequencing technology mainly involves sequencing by ligation (SBL) technology and sequencing by synthesis (SBS) technology.
  • SBL sequencing by ligation
  • SBS sequencing by synthesis
  • Typical examples of these sequencing technologies include the SOLiD sequencing method developed by Applied Biosystems, the combined probe anchor ligation method (cPAL) independently developed by Complete Genomics and the combined probe anchor synthesis method (cPAS) developed by BGI, Illumina The Illumina sequencing method developed by the company and Solexa technology in cooperation and so on.
  • Illumina and Complate Genomics adopt the method of detecting light signals.
  • the sequencing device In order to read the fluorescent signal carried by each base, the sequencing device must be equipped with at least two monochromatic excitation light sources and at least two cameras, which results in expensive manufacturing cost and huge volume of the sequencing device.
  • the second-generation gene sequencing technology has gradually grown from an emerging technology to a mainstream sequencing method, and has gradually become an important detection tool in the clinical field, in the prevention and control of infectious diseases, the diagnosis of genetic diseases, and non-invasive prenatal screening. Investigation and other fields are playing an increasing role.
  • the development of low-cost and miniaturized sequencers has gradually become a development trend in the sequencing field.
  • the three sequencing methods based on four-channel, dual-channel and single-channel have their own merits, but compared with the three, single-channel sequencing has fewer consumables, lower costs, and is easier to achieve miniaturization and portable instrumentation.
  • Such advantages have gradually become the development trend in the sequencing field.
  • products based on monochrome channels on the market mainly include ion torrent series sequencers, 454 sequencers and the latest Iseq100 from Illumina.
  • Illumia's Iseq100 is based on monochromatic fluorescence technology and semiconductor technology to realize a miniaturized sequencer and maintain high sequencing quality.
  • the optical signal is excited by the laser, the instrument is equipped with an additional laser, which increases the volume of the instrument.
  • special processing must be performed on the semiconductor chip to filter out the background generated by the excitation light. This processing will cause high costs and increase the cost of sequencing.
  • the inventor of the present application developed a new sequencing method that uses the signal of the self-luminescence system to distinguish four bases of A, (T/U), C, and G. Therefore, the luminescence signal used to implement the sequencing method of the present invention is derived from bioluminescence or chemiluminescence, so there is no need to configure an additional laser, and there is no need to adopt an additional design to filter the background generated by the laser light source, thereby reducing sequencing costs.
  • the sequencing device used in the sequencing method of the present invention can even be conveniently carried around for immediate/on-site detection.
  • the 3'hydroxyl group of the deoxyribonucleotide used in the sequencing method of the present invention is modified and blocked. During the sequencing process, only one deoxyribonucleotide can be synthesized per reaction, ensuring that only one deoxyribonucleotide can be combined per reaction. Ribonucleotides improve the accuracy of sequencing.
  • the present invention provides a method for sequencing nucleic acid molecules, which includes the following steps:
  • nucleic acid molecule to be sequenced connected to the support, or connect the nucleic acid molecule to be sequenced to the support;
  • the first compound is attached to the first molecular label
  • the second compound is attached with a second molecular label
  • the third compound is connected with the first molecular label and the second molecular label, or part of the third compound is connected with the first molecular label and another part of the third compound is connected with the second molecular label,
  • the fourth compound has no molecular marker attached
  • the primer serves as the initial growing nucleic acid strand, and the nucleic acid molecule to be sequenced together to form a duplex connected to the support;
  • Steps (3)-(7) are optionally repeated to obtain sequence information of the nucleic acid molecule.
  • the self-luminescence detection of the nucleotide to be sequenced is realized through the connection of luciferase and nucleotide derivatives, so that no additional excitation light source is required.
  • the linkage of luciferase and nucleotides is achieved through the specific binding of the label on the luciferase and the corresponding label on the nucleotide derivative.
  • the first nucleotide is linked to a first luciferase
  • the second nucleotide is linked to a second luciferase
  • the third nucleotide is linked to a first luciferase and a second luciferase.
  • the fourth nucleotide is not linked to any luciferase. Then pass the corresponding substrates of the two luciferases to detect the luminescence signals of the four bases; when the substrates of the first luciferase are passed, the first nucleotide and the third nucleotide emit light, when When the substrate of the second luciferase is passed through, the second and third nucleotides emit light, so the base can be identified based on the luminescence of the four nucleotides.
  • the method for sequencing a nucleic acid molecule of the present invention includes the following steps:
  • nucleic acid molecule to be sequenced connected to the support, or connect the nucleic acid molecule to be sequenced to the support;
  • the first compound is attached to the first molecular label
  • the second compound is attached with a second molecular label
  • the third compound is connected with the first molecular label and the second molecular label, or part of the third compound is connected with the first molecular label and another part of the third compound is connected with the second molecular label,
  • the fourth compound has no molecular marker attached
  • the primer serves as the initial growing nucleic acid strand, and the nucleic acid molecule to be sequenced together to form a duplex connected to the support;
  • the two luciferases can be specific sexually binding the first molecular marker and the second molecular marker;
  • Steps (3)-(12) are optionally repeated to obtain sequence information of the nucleic acid molecule.
  • the linking of the two luciferases and nucleotides can also be carried out separately.
  • the first luciferase labeled with the first molecule can be added to make it contact with the nucleotide labeled with the first molecule Carry out the binding reaction, then use the elution buffer to remove the unbound first luciferase, add the first luciferase substrate, and detect the fluorescence signal at the same time; then add the second labeled second luciferase to make It performs a binding reaction with the nucleotides labeled with the second molecule, and then removes the unbound second luciferase with an elution buffer, adds the second luciferase substrate, and detects the fluorescence signal at the same time.
  • the present invention provides a method for sequencing nucleic acid molecules, which includes the following steps:
  • nucleic acid molecule to be sequenced connected to the support, or connect the nucleic acid molecule to be sequenced to the support;
  • the first compound is attached to the first molecular label
  • the second compound is attached with a second molecular label
  • the third compound is connected with the first molecular label and the second molecular label, or part of the third compound is connected with the first molecular label and another part of the third compound is connected with the second molecular label,
  • the fourth compound has no molecular marker attached
  • the primer serves as the initial growing nucleic acid strand, and the nucleic acid molecule to be sequenced together to form a duplex connected to the support;
  • the two luciferases may be the same, which can specifically bind to the first molecular label and the second molecular label, respectively, that is, only one luciferase and substrate are used, and the first labeled
  • the ligation of the first luciferase and the second labeled second luciferase to the nucleotide is performed separately.
  • the first labeled first luciferase can be added to make it react with the nucleotide labeled with the first molecule, and then the unbound first luciferase can be removed with the elution buffer, and luciferase can be added Enzyme substrate, while detecting the fluorescent signal; then add the second labeled second luciferase to make it bind to the second molecule labeled nucleotide, and then use the elution buffer to remove unbound The second luciferase, adding a luciferase substrate, simultaneously detects the fluorescent signal.
  • the present invention provides a method for sequencing nucleic acid molecules, which includes the following steps:
  • nucleic acid molecule to be sequenced connected to the support, or connect the nucleic acid molecule to be sequenced to the support;
  • the first compound is attached to the first molecular label
  • the second compound is attached with a second molecular label
  • the third compound is connected with the first molecular label and the second molecular label, or part of the third compound is connected with the first molecular label and another part of the third compound is connected with the second molecular label,
  • the fourth compound has no molecular marker attached
  • the primer serves as the initial growing nucleic acid strand, and the nucleic acid molecule to be sequenced together to form a duplex connected to the support;
  • the present invention also relates to a kit for sequencing polynucleotides, which comprises:
  • the first compound is attached to the first molecular label
  • the second compound is attached with a second molecular label
  • the third compound is connected with the first molecular label and the second molecular label, or part of the third compound is connected with the first molecular label and another part of the third compound is connected with the second molecular label,
  • the fourth compound is not attached to a molecular marker
  • the two luciferases can respectively specifically bind the first molecular label and the second molecular label, and the two luciferases can be the same or different.
  • the kit of the present invention further comprises: reagents and/or devices for extracting nucleic acid molecules from a sample; reagents for pretreating nucleic acid molecules; and reagents for connecting nucleic acid molecules to be sequenced Support; reagent used to connect the nucleic acid molecule to be sequenced to the support (for example, covalent or non-covalent connection); primer used to initiate the nucleotide polymerization reaction; used to carry out the nucleotide polymerization reaction Polymerase; one or more buffer solutions; one or more washing solutions; or any combination thereof.
  • polynucleotide refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or analogs thereof.
  • Polynucleotides can be single-stranded, double-stranded, or contain both single-stranded and double-stranded sequences.
  • Polynucleotide molecules can be derived from double-stranded DNA (dsDNA) form (for example, genomic DNA, PCR and amplification products, etc.), or can be derived from single-stranded form of DNA (ssDNA) or RNA and can be converted into dsDNA form, And vice versa.
  • dsDNA double-stranded DNA
  • ssDNA single-stranded form of DNA
  • RNA can be converted into dsDNA form, And vice versa.
  • the exact sequence of the polynucleotide molecule can be known or unknown.
  • genes or gene fragments for example, probes, primers, EST or SAGE tags
  • genomic DNA for example, genomic DNA fragments, exons, introns, messenger RNA (mRNA), transport RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, synthetic polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid of any of the above sequences Probes, primers or amplified copies.
  • a polynucleotide may include nucleotides or nucleotide analogs. Nucleotides usually contain a sugar (such as ribose or deoxyribose), a base, and at least one phosphate group. Nucleotides can be abasic (i.e. lack bases). Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleosides Acid and its mixtures.
  • nucleotides include, for example, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine Glycoside triphosphate (TTP), cytidine acid (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenosine acid (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), Deoxythymidine Monophosphate (dTMP), Deoxythymidine Diphosphate (dT
  • Nucleotide analogs containing modified bases can also be used in the methods described herein. Whether it has a natural backbone or a similar structure, exemplary modified bases that can be included in a polynucleotide include, for example, inosine, xathanine, hypoxathanine, isocytosine, isoguanine Purine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2 -Propyl adenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halogenated uracil, 15-halogenated cytosine, 5-propynyluracil, 5-propyne Basecytosine, 6-Azouracil, 6-Azocytosine, 6-Azothymine, 5-uracil, 4-thiouracil, 8-halogenated adenine
  • nucleotides include A, C, G, T or U nucleotides.
  • nucleotide A refers to a nucleotide containing adenine (A) or a modification or analog thereof, such as ATP, dATP.
  • Nucleotide G refers to a nucleotide containing guanine (G) or a modification or analog thereof, such as GTP, dGTP.
  • Nucleotide C refers to a nucleotide containing cytosine (C) or a modification or analog thereof, such as CTP, dCTP.
  • Nucleotide T refers to a nucleotide containing thymine (T) or a modification or analog thereof, such as TTP, dTTP.
  • Nucleotide U refers to a nucleotide containing uracil (U) or a modification or analog thereof, such as UTP, dUTP.
  • the present invention relates to labeling nucleotides with different labels individually or in combination, so that different luciferases can be linked to the nucleotides.
  • the molecular marker for labeling nucleotides and the marker specifically binding thereto may be any pair of molecules that can specifically bind to each other. The specific binding between the paired members realizes the linkage of nucleotides to luciferase.
  • Exemplary pairing members include, but are not limited to: (a) haptens or antigenic compounds combined with corresponding antibodies or binding parts or fragments thereof, such as digoxigenin-digoxin antibody, N3G-N3G antibody, FITC-FITC antibody ; (B) Nucleic acid aptamer and protein; (c) Non-immune binding pair (such as biotin-avidin, biotin-streptavidin, biotin-neutravidin); (d) hormone -Hormone binding protein; (e) receptor-receptor agonist or antagonist; (f) lectin-carbohydrate; (g) enzyme-enzyme cofactor; (h) enzyme-enzyme inhibitor; and (i) A pair of complementary oligonucleotides or polynucleotides capable of forming a nucleic acid duplex.
  • haptens or antigenic compounds combined with corresponding antibodies or binding parts or fragments thereof, such as digoxigenin-digoxin antibody, N3G
  • the first molecular marker and the second molecular marker are small molecular markers selected from biotin, digoxin, N3G or FITC.
  • the two luciferases can respectively specifically bind the first molecular label and the second molecular label.
  • the first molecular marker is biotin
  • the first luciferase can be streptavidin-labeled luciferase
  • the second molecular marker is digoxin
  • the second fluorescence The luciferase may be a luciferase labeled with a digoxin antibody that is different from the first luciferase
  • the second luciferase may also be a luciferase labeled with a digoxin antibody that is the same as the first luciferase.
  • Enzyme The source of the luciferase includes, but is not limited to, firefly, gaussia, Renilla and other organisms.
  • the streptavidin-labeled luciferase may be SA-Gluc: Streptavidin-Gaussia princeps luciferase from Adivity.
  • the luciferase labeled with digoxin antibody may be digoxin antibody-Gluc or digoxin antibody-Nluc.
  • the expression “the first compound is attached to the first molecular label” means that all the first compounds are attached to the first molecular label, or part of the first compound is attached to the first molecular label but the remaining first compounds are not attached molecular marker.
  • the expression “the second compound is connected with the second molecular label” means that all the second compounds are connected with the second molecular label, or part of the second compounds are connected with the second molecular label but the remaining second compounds are not connected with the molecular label.
  • the third compound is connected with the first molecular label and the second molecular label.
  • all the third compounds are connected with the first molecular label and the second molecular label, or part of the third compound is connected with the first molecular label and the second molecular label.
  • Molecular markers but the remaining third compounds are not linked to molecular markers.
  • the nucleotides linked with different luciferases of the present invention are suitable for sequencing by synthesis.
  • Sequencing by synthesis as used herein is a variety of sequencing by synthesis methods well known in the art. Basically, sequencing by synthesis involves first hybridizing the sequenced nucleic acid molecule with a sequencing primer, and then in the presence of a polymerase, polymerizing the sequenced nucleic acid molecule at the 3'end of the sequencing primer as described herein. Nucleotides of different luciferases. After polymerization, nucleotides are identified by detecting the fluorescent signal emitted by the luciferase. After the luciferase is removed from the labeled nucleotides, the next polymer sequencing cycle is performed.
  • the method for determining the sequence of a target polynucleotide can be carried out as follows: denaturing the target polynucleotide sequence, contacting the target polynucleotide with different nucleotides, respectively, so as to form the complement of the target nucleotide, and detecting The incorporation of said nucleotides.
  • the method utilizes polymerization, which allows the polymerase to extend the complementary strand by incorporating the correct nucleotides complementary to the target.
  • the polymerization reaction also requires special primers to initiate polymerization.
  • the incorporation of the nucleotide is carried out by polymerase, and then the incorporation event is measured.
  • polymerase There are many different polymerases, and it is easy for a person skilled in the art to determine the most suitable polymerase.
  • Preferred enzymes include DNA polymerase I, Klenow fragment, DNA polymerase III, T4 or T7 DNA polymerase, Taq polymerase or vent polymerase. It is also possible to use polymerases engineered to have specific properties.
  • the sequencing method is preferably performed on the target polynucleotide arranged on a solid support.
  • a plurality of target polynucleotides can be immobilized on the solid support through a linker molecule, or can be attached to particles such as microspheres, and the particles can also be attached to a solid support material.
  • the polynucleotide can be attached to the solid support by a variety of methods, including the use of biotin-streptavidin interaction.
  • Methods for immobilizing polynucleotides on a solid support are well known in the art and include lithography techniques and spotting each polynucleotide on a specific position on the solid support.
  • Suitable solid supports are well known in the art and include glass slides and beads, ceramic and silicon surfaces, and plastic materials.
  • the support is generally flat, although microbeads (microspheres) can also be used, and the latter can also be attached to other solid supports by known methods.
  • the microspheres can have any suitable size, and their diameter is usually 10-100 nanometers.
  • the polynucleotide is directly attached on a flat surface, preferably on a flat glass surface.
  • the connection is preferably carried out in the form of a covalent bond.
  • the array used is preferably a single molecule array, which includes polynucleotides located in a unique optically resolvable region, for example as described in International Application No. WO00/06770.
  • a primer sequence In order to perform the polymerase reaction, usually a primer sequence must first be annealed to the target polynucleotide.
  • the primer sequence is recognized by the polymerase and serves as the starting site for the subsequent extension of the complementary strand The role of.
  • the primer sequence can be added as an independent component relative to the target polynucleotide.
  • the primer and the target polynucleotide may each be part of a single-stranded molecule, and an intramolecular duplex, that is, a hairpin loop structure, is formed by the primer part and a part of the target.
  • the structure can be immobilized on the solid support at any position of the molecule.
  • Other conditions necessary for carrying out the polymerase reaction are well known to those skilled in the art, and these conditions include temperature, pH, and buffer composition.
  • the labeled nucleotide of the present invention is brought into contact with the target polynucleotide to enable polymerization.
  • the nucleotides can be added sequentially, that is, each type of nucleotide (A, C, G or T/U) is added separately, or added at the same time.
  • the polymerization step is allowed to proceed for a time sufficient to incorporate one nucleotide.
  • the unincorporated nucleotides are removed, for example, by removing the solution phase of the reaction system in the previous step, leaving the duplexes attached to the support.
  • two luciferases containing different luciferases can be added to carry out a binding reaction.
  • the two luciferases can respectively specifically bind to the molecular marker for labeling nucleotides, thereby realizing luciferase and incorporation.
  • the connection of nucleotides Then by adding the corresponding luciferase substrate and detecting the fluorescent signal, the identification of the incorporated nucleotide is realized. It is also possible to add two kinds of luciferases containing the same luciferase to carry out the binding reaction.
  • the luciferases can respectively specifically bind to the molecular markers for labeling nucleotides, thereby realizing the luciferase and the incorporated nuclear The connection of glycidyl acid. Then by adding the corresponding luciferase substrate and detecting the fluorescent signal, the identification of the incorporated nucleotide is realized.
  • the four deoxyribonucleotide analogues are respectively labeled with different small molecule markers biotin (abbreviated as B) and digoxigenin (abbreviated as D), for example, nucleotide A labeled B, nuclear The nucleotide C marks B and D, the nucleotide T marks D, and the nucleotide G is not marked.
  • B biotin
  • D digoxigenin
  • the four labeled deoxynucleotide analogs and the sequencing polymerase mixture are first introduced, and under the action of the polymerase, one deoxyribonucleotide analog is mixed according to the principle of base complementary pairing.
  • one deoxyribonucleotide analog is mixed according to the principle of base complementary pairing.
  • unbound deoxyribonucleotide analogs can be removed.
  • two luciferases containing different luciferases are added.
  • the first luciferase is labeled with streptavidin, which binds to nucleotide A or nucleotide C labeled with small molecule B, and the second fluorescence
  • the enzyme is labeled with digoxigenin antibody, which binds to nucleotide C or nucleotide T labeled with small molecule D.
  • the first luciferase substrate After removing the unbound luciferase with the elution buffer, add the first luciferase substrate, the nucleotide connected to the first luciferase emits light, and the detector detects the signal; the second luciferase is added As the substrate, the nucleotide connected with the second luciferase emits light, and the signal is detected by the detector, and the luminescence shown in the table below is obtained, and the base can be identified.
  • the ligation of two luciferases containing different luciferases and the labeled nucleotides and signal detection can be performed separately.
  • the first luciferase is added, which is labeled with streptavidin and binds to nucleotide A or nucleotide C labeled with small molecule B.
  • the first luciferase substrate is added, the nucleotides connected to the first luciferase emit light, and the signal is detected by a detector.
  • the ligation of the two luciferases containing the same luciferase to the labeled nucleotide and the signal detection are performed separately.
  • the first luciferase is added, which is labeled with streptavidin and binds to nucleotide A or nucleotide C labeled with small molecule B.
  • the luciferase substrate is added, the nucleotides connected to the first luciferase emit light, and the signal is detected by a detector.
  • the reagent that denatures the first luciferase remove the reaction solution and add the second luciferase labeled with digoxigenin antibody, which binds to nucleotide C or nucleotide T labeled with small molecule D, Then use the elution buffer to remove the unbound second luciferase, add the luciferase substrate, connect the base of the second luciferase to emit light, and use the detector to detect the signal, which is shown in the table above The luminescence of the luminescence can be used for base recognition.
  • the way of detecting the fluorescent signal is well known in the art.
  • a device that detects the wavelength of fluorescence Such devices are well known in the art.
  • a device may be a confocal scanning microscope that scans the surface of a solid support with a laser to image the fluorophore directly bound to the nucleic acid molecule being sequenced.
  • a sensitive 2-D detector such as a charge-coupled detector (CCD) can be used to observe each of the signals generated, for example.
  • CCD charge-coupled detector
  • Other techniques such as Scanning Near Field Optical Microscopy (SNOM) can also be used, for example.
  • suitable conditions can be used to remove the label on the nucleotide.
  • the labeled nucleotides of the present invention also have a 3'protecting group.
  • the protecting group and the label are usually two different groups on the 3'-blocked labeled nucleotide, but in other embodiments, the protecting group and the label The substance can also be the same group.
  • protecting group means a group that prevents the polymerase (which incorporates nucleotides containing the group into the polynucleotide chain being synthesized) from being able to contain the group.
  • the incorporation of a group of nucleotides into the polynucleotide chain being synthesized continues to catalyze the incorporation of another nucleotide.
  • Such protecting groups are also referred to herein as 3'-OH protecting groups.
  • Nucleotides containing such protective groups are also referred to herein as 3' blocked nucleotides.
  • the protecting group may be any suitable group that can be added to the nucleotide, as long as the protecting group can prevent additional nucleotide molecules from being added to the polynucleotide chain and at the same time without destroying the polynucleotide chain. In the case of easy removal from the sugar portion of the nucleotide.
  • nucleotides modified with protecting groups need to be resistant to polymerase or other suitable enzymes for incorporating the modified nucleotides into the polynucleotide chain.
  • the ideal protecting group exhibits long-term stability, can be efficiently incorporated by polymerase, prevents secondary or further incorporation of nucleotides, and can under mild conditions that do not damage the structure of the polynucleotide It is preferably removed under aqueous conditions.
  • WO 91/06678 discloses that 3'-OH protecting groups include esters and ethers, -F, -NH2, -OCH3, -N3, -OPO3, -NHCOCH3,2 nitrobenzene carbonate, 2,4-sulfene Acyl dinitro and tetrahydrofuran ether.
  • Metzker et al. disclose the synthesis of eight 3'-modified 2-deoxyribonucleoside 5'-triphosphates (3'-modified dNTPs) And application.
  • WO2002/029003 describes the use of an allyl protecting group to cap the 3'-OH group on the growing DNA strand in the polymerase reaction.
  • various protecting groups reported in International Application Publications WO2014139596 and WO2004/018497 can be used, including, for example, those protecting groups illustrated in Figure 1A of WO2014139596 and those 3'hydroxyl protecting groups defined in the claims (ie Protecting groups), and such as those exemplified in Figures 3 and 4 of WO2004/018497 and those defined in the claims.
  • the above references are all incorporated herein by reference in their entirety.
  • the protecting group can be directly attached to the 3'position or can be attached to the 2'position (the protecting group has sufficient size or charge to block the interaction at the 3'position).
  • the protecting group can be attached at the 3'and 2'positions and can be cleaved to expose the 3'-OH group.
  • the sequencing protocol requires the removal of the protecting group to create a usable 3'-OH site for continuous strand synthesis.
  • the reagents that can remove the protecting group from the modified nucleotide as used herein depend to a large extent on the protecting group used. For example, removal of the ester protecting group from the 3'hydroxyl function is usually accomplished by alkaline hydrolysis. The ease of removing the protecting group varies greatly; generally, the greater the electronegativity of the substituent on the carbonyl carbon, the greater the ease of removal.
  • the highly electronegative trifluoroacetic acid group can be rapidly cleaved from the 3'hydroxyl group at pH 7 in methanol (Cramer et al., 1963), so it is unstable during polymerization at this pH.
  • the phenoxyacetate group is cleaved in less than 1 minute, but a significantly higher pH is required, for example with NH-/methanol (Reese and Steward, 1968).
  • Various hydroxy protecting groups can be selectively cleaved using chemical methods other than alkaline hydrolysis.
  • the 2,4-dinitrophenylthio group can be quickly cleaved by treatment with nucleophiles such as thiophenol and thiosulfate (Letsinger et al., 1964).
  • Allyl ether is cleaved by treatment with Hg(II) in acetone/water (Gigg and Warren, 1968). Use Ag(I) or Hg(II) to remove thiopyranyl ether under neutral conditions (Cohen and Steele, 1966; Cruse et al., 1978). Photochemical deblocking can be used with photochemically cleavable protecting groups. Several protecting groups can be used in this method. The use of o-nitrobenzyl ether as a protective group for the 2'-hydroxyl functionality of ribonucleosides is known and proven (Ohtsuka et al., 1978); it is removed by irradiation at 260 nm.
  • the protecting group of alkyl carbonate o-nitrobenzyl carbonate is also removed by irradiation at pH 7 (Cama and Christensen, 1978). Enzymolysis blocking of the 3'-OH protecting group is also possible. It has been demonstrated that T4 polynucleotide kinase can convert the 3'-phosphate end into a 3'-hydroxyl end, which can then be used as a primer for DNA polymerase I (Henner et al., 1983). This 3'-phosphatase activity is used to remove the 3'protecting group of those dNTP analogs containing phosphate as the protecting group.
  • reagents that can remove protecting groups from 3'-blocked nucleotides include, for example, phosphines (e.g., tris(hydroxymethyl)phosphine (THP)), which can, for example, add azide-containing 3'-OH protecting groups Groups are removed from nucleotides (for this application of phosphine, see, for example, the description in WO2014139596, the entire content of which is incorporated herein by reference).
  • Other reagents that can remove protecting groups from 3'-blocked nucleotides also include, for example, those described on pages 114-116 in the specification of WO2004/018497 for removing 3'-ene as a 3'-OH protecting group.
  • the nucleotide label is preferably removed together with the protecting group after detection.
  • the label may be incorporated with a protecting group, thereby allowing the 3'-blocked nucleotide to be removed along with the protecting group after it has been incorporated into the nucleic acid strand.
  • the label may be attached to the nucleotide separately from the protecting group using a linking group.
  • a linking group may, for example, be attached to the purine or pyrimidine base of the nucleotide.
  • the linking group used is cleavable. The use of a cleavable linking group ensures that the label can be removed after detection, which avoids any signal interference with any labeled nucleotides subsequently incorporated.
  • a non-cleavable linking group can be used, because after the labeled nucleotide is incorporated into the nucleic acid strand, subsequent nucleotide incorporation is not required, so there is no need to remove the label from the nucleotide. Remove.
  • the label and/or linking group may have a size or structure sufficient to block the incorporation of other nucleotides into the polynucleotide chain (that is, the label itself can serve as a protective Group).
  • the blocking may be due to steric hindrance, or may be due to a combination of size, charge, and structure.
  • Cleavable linking groups are well known in the art, and conventional chemical methods can be used to connect the linking group to the nucleotide base and the label.
  • the linking group can be attached to any position of the nucleotide base, provided that Watson-Crick base pairing can still be performed.
  • For purine bases if the linking group is through position 7 of the purine or a preferred deaza purine analog, through 8-modified purine, through N-6 modified adenine or N-2 Modified guanine linking would be preferred.
  • the connection is preferably via cytosine, position 5 on thymine and uracil, and position N-4 on cytidine.
  • nucleoside cleavage site can be located at a position on the linking group, which can ensure that a part of the linking group remains connected to the nucleotide base after cleavage.
  • Suitable linking groups include, but are not limited to, disulfide linking groups, acid-labile linking groups (including dialkoxybenzyl linking groups, Sieber linking groups, indole linking groups, tert-butyl Sieber Linking group), electrophilic cleavable linking group, nucleophilic cleavable linking group, photo-cleavable linking group, linking group cleaved under reducing and oxidizing conditions, safety-catch ) Linking groups, and linking groups that undergo cleavage through elimination mechanisms.
  • Suitable linking groups can be modified with standard chemical protecting groups, as disclosed in the following documents: Greene&Wuts, Protective Groups in Organic Synthesis, John Wiley&Sons. Guillier et al. disclosed other suitable cleavable linking groups for solid phase synthesis (Chem. Rev. 100:2092-2157, 2000).
  • the linking group can be cleaved by any suitable method, including exposure to acids, bases, nucleophiles, electrophiles, free radicals, metals, reducing or oxidizing reagents, light, temperature, enzymes, etc., each of which will be exemplarily described below.
  • a suitable cleavage method for the cleavable linking group Generally, the cleavable linking group can be cleaved under the same conditions as the protecting group, so that only one treatment is required to remove the label and protecting group.
  • Electrophilic cleavage linking groups are typically cleaved by protons and include acid-sensitive cleavage.
  • Suitable electrophilic cleavable linking groups include modified benzyl systems such as trityl, p-oxybenzyl ester, and p-oxybenzyl amide.
  • Other suitable linking groups include tert-butoxycarbonyl (Boc) groups and acetal systems.
  • Boc tert-butoxycarbonyl
  • thiophilic metals such as nickel, silver or mercury in the cleavage of thioacetals or other sulfur-containing protecting groups.
  • Nucleophilic cleavable linking groups include groups that are unstable in water (that is, can be simply cleaved at alkaline pH), such as esters, and groups that are unstable to non-aqueous nucleophiles. Fluoride ions can be used to cleave silicon-oxygen bonds in groups such as triisopropylsilane (TIPS) or tert-butyldimethylsilane (TBDMS). Photodegradable linking groups are widely used in sugar chemistry. Preferably, the light required to activate cleavage does not affect other components in the modified nucleotide.
  • Suitable linking groups include those based on O-nitrobenzyl compounds and nitroveratryl compounds. Linking groups based on benzoin chemistry can also be used (Lee et al., J. Org. Chem. 64:3454-3460, 1999). Various linking groups that are sensitive to reductive cleavage are known. Catalytic hydrogenation using palladium-based catalysts has been used to cleave benzyl and benzyloxycarbonyl groups. Disulfide bond reduction is also known in the art.
  • Safety-catch linkers are those that are cleaved in two steps. In a preferred system, the first step is the generation of reactive nucleophilic centers, and the subsequent second step involves intramolecular cyclization, which results in cleavage.
  • the levulinate linkage can be treated with hydrazine or photochemical methods to release the active amine, which is then cyclized to cleave the ester elsewhere in the molecule (Burgess et al., J. Org. Chem. 62: 5165 -5168, 1997).
  • An elimination reaction can also be used to cleave the linking group.
  • Base-catalyzed elimination of groups such as fluorenylmethoxycarbonyl and cyanoethyl and palladium-catalyzed reductive elimination of allyl systems can be used.
  • the linking group may include a spacer unit.
  • the length of the linking group is not important, as long as the label and the nucleotide are kept at a sufficient distance so as not to interfere with the interaction between the nucleotide and the enzyme.
  • the linking group may consist of a functional group similar to the 3'-OH protecting group. This will allow a single treatment to remove the label and protecting group.
  • a particularly preferred linking group is an azide-containing linking group cleavable by phosphine.
  • the reagents that can remove the label from the modified nucleotide as used herein depend to a large extent on the label used.
  • the protective group removal reagent described above is used to remove the label.
  • the label is attached to the base of the nucleotide through a cleavable linking group, the label is removed using a reagent that cleaves the linking group as described above.
  • the same reagent is used to remove the label and protecting group from the modified nucleotide, for example where the linking group consists of a functional group similar to the 3'-OH protecting group.
  • the present invention also provides a kit for sequencing polynucleotides, which comprises:
  • the first compound is attached to the first molecular label
  • the second compound is attached with a second molecular label
  • the third compound is connected with the first molecular label and the second molecular label, or part of the third compound is connected with the first molecular label and another part of the third compound is connected with the second molecular label,
  • the fourth compound is not attached to a molecular marker
  • the two luciferases can respectively specifically bind to the first molecular label and the second molecular label.
  • the molecular label used to label the four compounds and the label used to label the two luciferases are as defined above.
  • the kit of the present invention further comprises: reagents and/or devices for extracting nucleic acid molecules from a sample; reagents for pretreating nucleic acid molecules; and reagents for connecting nucleic acid molecules to be sequenced Support; reagent used to connect the nucleic acid molecule to be sequenced to the support (for example, covalent or non-covalent connection); primer used to initiate the nucleotide polymerization reaction; used to carry out the nucleotide polymerization reaction Polymerase; one or more buffer solutions; one or more washing solutions; or any combination thereof.
  • the kit of the present invention further includes reagents and/or devices for extracting nucleic acid molecules from the sample.
  • Methods for extracting nucleic acid molecules from samples are well known in the art. Therefore, various reagents and/or devices for extracting nucleic acid molecules, such as reagents for disrupting cells, reagents for precipitating DNA, reagents for washing DNA, can be configured in the kit of the present invention as needed, Reagents for dissolving DNA, reagents for precipitating RNA, reagents for washing RNA, reagents for dissolving RNA, reagents for protein removal, reagents for DNA removal (for example, when the target nucleic acid molecule is RNA ), reagents for removing RNA (for example, when the target nucleic acid molecule is DNA), and any combination thereof.
  • the kit of the present invention further includes reagents for pretreating nucleic acid molecules.
  • the reagents used for pretreatment of nucleic acid molecules are not subject to additional restrictions and can be selected according to actual needs.
  • the reagents used for pretreatment of nucleic acid molecules include, for example, reagents for fragmentation of nucleic acid molecules (such as DNase I), reagents for filling ends of nucleic acid molecules (such as DNA polymerase, such as T4 DNA polymerase, Pfu DNA polymerase, Klenow DNA polymerase), linker molecules, tag molecules, reagents used to connect linker molecules with target nucleic acid molecules (such as ligases, such as T4 DNA ligase), reagents used to repair nucleic acid nicks (such as , DNA polymerases that lose 3'-5' exonuclease activity but show 5'-3' exonuclease activity), reagents used to amplify nucleic acid molecules (for example, DNA polymerase, primers, dNTPs), Reagents for separating and purifying nucleic acid molecules (such as chromatography columns), and any combination thereof.
  • the kit of the present invention further comprises a support for connecting nucleic acid molecules to be sequenced.
  • the support may have any technical features and any combination thereof described in detail for the support above.
  • the support can be made of various suitable materials.
  • materials include, for example, inorganic materials, natural polymers, synthetic polymers, and any combination thereof.
  • Specific examples include, but are not limited to: cellulose, cellulose derivatives (such as nitrocellulose), acrylic resin, glass, silica gel, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, and two Cross-linked polystyrene such as vinylbenzene (see, for example, Merrifield Biochemistry 1964, 3, 1385-1390), polyacrylamide, latex, dextran, rubber, silicon, plastic, natural sponge, metal plastic, cross-linked Dextran (for example, Sephadex TM ), agarose gel (Sepharose TM ), and other supports known to those skilled in the art.
  • cellulose cellulose derivatives (such as nitrocellulose)
  • acrylic resin glass, silica gel, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl
  • the support used to connect the nucleic acid molecules to be sequenced may be a solid support including an inert substrate or matrix (for example, a glass slide, polymer beads, etc.). It has been functionalized, for example, by the application of intermediate materials containing reactive groups that allow the covalent attachment of biomolecules such as polynucleotides.
  • examples of such supports include, but are not limited to, polyacrylamide hydrogels supported on an inert substrate such as glass, in particular the polyacrylamide hydrogels described in WO 2005/065814 and US 2008/0280773, wherein The content of the said patent application is incorporated herein in its entirety by reference.
  • biomolecules e.g., polynucleotides
  • an intermediate material e.g., hydrogel
  • the intermediate material itself can be non-covalently attached to the substrate or matrix (e.g., glass substrate).
  • the support is a glass or silicon wafer with a chemical group of avidin, amino, acrylamide silane or aldehyde group on the surface modified.
  • the support or solid support is not limited to its size, shape and configuration.
  • the support or solid support is a planar structure, such as a slide, chip, microchip, and/or array.
  • the surface of such a support may be in the form of a planar layer.
  • the support or its surface is non-planar, such as the inner or outer surface of a tube or container.
  • the support or solid support includes microspheres or beads.
  • the support used to connect the nucleic acid molecules to be sequenced is an array of beads or pores.
  • the kit of the present invention further includes reagents for connecting (for example, covalently or non-covalently connecting) the nucleic acid molecule to be sequenced to the support.
  • reagents include, for example, reagents that activate or modify nucleic acid molecules (such as their 5'ends), such as phosphoric acid, thiol, amine, carboxylic acid, or aldehyde; reagents that activate or modify the surface of a support, such as amino- Alkoxysilane (for example, aminopropyltrimethoxysilane, aminopropyltriethoxysilane, 4-aminobutyltriethoxysilane, etc.); crosslinking agent, such as succinic anhydride, phenyl diisosulfide Cyanate (Guo et al., 1994), maleic anhydride (Yang et al., 1998), 1-ethyl-3-(3-dimethylaminopropyl
  • the kit of the present invention further comprises a primer for initiating the nucleotide polymerization reaction.
  • the primer is not subject to additional restrictions as long as it can specifically anneal to a region of the target nucleic acid molecule.
  • the length of the primer may be 5-50 bp, such as 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40- 45, 45-50bp.
  • the primer may comprise naturally occurring or non-naturally occurring nucleotides.
  • the primer contains or consists of naturally-occurring nucleotides.
  • the primers comprise modified nucleotides, such as locked nucleic acids (LNA).
  • the primers comprise universal primer sequences.
  • the kit of the present invention further comprises a polymerase for carrying out a nucleotide polymerization reaction.
  • a polymerase for carrying out a nucleotide polymerization reaction.
  • various suitable polymerases can be used.
  • the polymerase can use DNA as a template to synthesize a new DNA strand (eg, DNA polymerase).
  • the polymerase can use RNA as a template to synthesize a new DNA strand (for example, reverse transcriptase).
  • the polymerase can synthesize a new RNA strand (for example, RNA polymerase) using DNA or RNA as a template. Therefore, in certain preferred embodiments, the polymerase is selected from the group consisting of DNA polymerase, RNA polymerase, and reverse transcriptase.
  • the kit of the present invention further contains one or more buffer solutions.
  • buffers include, but are not limited to, a buffer solution for DNase I, a buffer solution for DNA polymerase, a buffer solution for ligase, a buffer solution for eluting nucleic acid molecules, and a buffer solution for dissolving nucleic acid molecules.
  • Buffer solution a buffer solution for nucleotide polymerization reaction (such as PCR), and a buffer solution for ligation reaction.
  • the kit of the present invention may contain any one or more of the above-mentioned buffer solutions.
  • the kit of the present invention further comprises one or more washing solutions.
  • washing solutions include, but are not limited to, phosphate buffer, citrate buffer, Tris-HCl buffer, acetate buffer, carbonate buffer, and the like.
  • the kit of the present invention may contain any one or more of the aforementioned washing solutions.
  • the method of the present invention uses only two molecular markers to realize the labeling of four nucleotides, and realizes self-luminescence by using luciferase. Therefore, the sequencing device used in the sequencing method of the present invention does not need to be equipped with an excitation light source, nor does it need to adopt an additional design to filter the background generated by the laser light source. On the one hand, the manufacturing cost of the sequencing device is greatly reduced, which is helpful for the promotion and application of the sequencing device and the sequencing method; on the other hand, the volume of the sequencing device is significantly reduced, making the sequencing device lighter and easier to carry.
  • thermo protein coupling kit Purchase a thermo protein coupling kit, and follow the instructions to couple the antibody to Nluc (enzyme in the figure refers to Nluc, and antibody in the figure refers to digoxigenin antibody):
  • Polymerization reaction solution 50mM Tris-Hcl, 50mM NaCl, 10mM(NH 4 ) 2 SO 4 , 0.02mg/ml polymerase BG9(BGI), 3mM MgSO 4 , 1mM EDTA, each of the above four dNTPs is 1uM
  • Elution buffer 5XSSC, Tween20 0.05%
  • Enzyme binding reaction solution Dilute the above two enzymes into TBST buffer, the final concentration of the two enzymes is 2ug/ml each;
  • Substrate 1 reaction solution prepare 50mM tris-Hcl 0.5mM Nacl buffer, and dilute 50X Coelenterazine (nanolight) to 1X;
  • Substrate 2 reaction solution prepare 50mM tris-Hcl 0.5mM Nacl buffer, and dilute 50X NLuc FLASH Substrate (nanolight) to 1X;
  • Resection buffer 20mM THPP, 0.5M NaCl, 50mM Tris-HCl, pH 9.0, 0.05% tween20;
  • Luciferase binding add 100ul enzyme binding reaction solution, incubate at 35°C for 30 minutes, so that SA-gluc binds to biotin-labeled dCTP and dATP derivatives, and Ab-Nluc binds to the Digoxin-labeled dCTP and dTTP derivatives, remove the reaction solution, add the eluent, and blow gently
  • Enzyme 1 signal detection Set appropriate microplate reader parameters, add substrate 1 reaction solution, perform enzyme 1 signal detection, and record the highest signal value;
  • Enzyme 2 signal detection remove the substrate reaction solution 1, set the appropriate microplate reader parameters, add the substrate 2 reaction solution, perform enzyme 2 signal detection, and record the highest signal value;
  • Substrate 1 signal (W) Substrate 2 signal (W) cycle1 0.1 210 cycle2 188 0.12 cycle3 173 169 cycle4 206 0.11 cycle5 198 0.13 cycle6 176 171 cycle7 0.13 186 cycle8 203 0.15 cycle9 166 159 cycle10 0.12 0.16
  • cycle1 and cycle7 only have signals when substrate 1 is passed through. It can be concluded that these two cycles are polymerized with biotin-dTTP, and the first and seventh bases of the library to be tested are T Base
  • Cycle2, cycle4, cycle5 and cycle8 only have signals when substrate 2 is passed through. It can be concluded that these four cycles are polymerized with digoxin-dATP, so the second, fourth, fifth and eighth bases of the library to be tested are A Base
  • Cycle3, cycle6, and cycle9 all have signals generated after passing through substrate 1 and substrate 2. It can be concluded that these two cycles are polymerized with biotin-digoxin-dCTP, so the third, sixth, and ninth library to be tested Each base is a C base;
  • Cycle10 does not emit light during the two substrate passes. It can be concluded that this cycle has polymerized coldG, so the tenth base of this library is the G base.
  • the first 10 bases of the tested sequence are: TACAACTACG, which matches 100% with the first 10 bp base sequence TACAACTACG of the library to be tested.
  • Antibody purchased from Abcam, according to the instructions of thermofisher NHS-s-s-biotin, label the antibody with biotin label to prepare antibody-s-s-biotin.
  • Polymerization reaction solution 50mM Tris-Hcl, 50mM NaCl, 10mM(NH 4 ) 2 SO 4 , 0.02mg/ml polymerase BG9(BGI), 3mM MgSO 4 , 1mM EDTA, each of the above four dNTPs is 1uM
  • Elution buffer 5XSSC, Tween20 0.05%
  • Enzyme binding reaction solution Dilute the above two proteins SA-Gluc and antibody-s-s-biotin into TBST buffer respectively, and the final concentration of the two proteins is 2ug/ml each;
  • Substrate reaction solution prepare 50mM Tris-HCl 0.5mM NaCl buffer, and dilute 50X Coelenterazine (nanolight) to 1X;
  • Enzyme inactivation buffer 5mM DTT, 50mM Tris-HCl, pH9.0;
  • Resection buffer 20mM THPP, 50mM Tris-HCl, pH9.0, 0.5M NaCl, 0.05% tween20;
  • Luciferase binding add 100ul luciferase SA-Gluc binding reaction solution, incubate at 35°C for 30 minutes to make SA-gluc bind to biotin-labeled dCTP and dATP derivatives to remove the reaction Add the eluent, gently pipetting several times, then remove the eluent;
  • the first signal detection Set the appropriate microplate reader parameters, add the substrate reaction solution, perform signal detection, and record the highest signal value;
  • Enzyme inactivation remove the substrate reaction solution, add enzyme inactivation buffer, incubate at 35°C for 10 min, remove the reaction solution, and then add elution buffer for elution;
  • Second luciferase binding Add antibody-SS-biotin reaction solution, incubate at 35°C for 30 minutes, make antibody-ss-biotin bind to digoxin-labeled dCTP and dTTP derivatives, and remove the reaction
  • Add elution buffer pipette gently several times to remove the eluate, add 100ul SA-Gluc binding reaction solution, incubate at 35°C for 30min, after removing the reaction solution, add elution buffer to elute After finishing, remove the elution buffer;
  • the second signal detection set the appropriate microplate reader parameters, add the substrate reaction solution, perform signal detection, and record the highest signal value;
  • a.10bp signal detection is as follows:
  • Second detection (w) cycle1 0.6 223 cycle2 312 0.7 cycle3 278 327 cycle4 254 0.3 cycle5 239 0.4 cycle6 243 275 cycle7 0.27 228 cycle8 267 0.5 cycle9 263 279 cycle10 0.7 0.6
  • Cycle1 and cycle7 have signal values only for the first time. It is speculated that these two cycles are polymerized with biotin-dTTP, so the first and seventh bases of the library to be tested are T;
  • Cycle2, cycle4, cycle5 and cycle8 only have signals in the second detection. It can be concluded that these four cycles are aggregated with digoxin-dATP, so the second, fourth, fifth and eighth bases of the library to be tested are A bases ;
  • Cycle3, cycle6 and cycle9 all have signals generated during the first and second detections. It can be concluded that these two cycles are aggregated with biotin-digoxin-dCTP, so the third, sixth and ninth library to be tested
  • the base is a C base
  • Cycle10 does not emit light during the two signal detections. It can be concluded that this cycle has polymerized coldG, so the tenth base of this library is the G base.
  • the first 10 bases of the tested sequence are: TACAACTACG, which matches 100% with the first 10 bp base sequence TACAACTACG of the library to be tested.

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Abstract

本发明提供了一种基于单荧光染料的测序方法，其中利用自发光的信号来分辨不同核苷酸的依次掺入，从而实现多核苷酸序列的测定。

Description

一种基于自发光的单通道测序方法 技术领域
本发明涉及核酸测序领域。特别地，本发明提供了一种基于自发光的单通道测序方法，其中利用自发光的信号来分辨不同核苷酸的依次掺入，从而实现多核苷酸序列的测定。
背景技术
DNA测序技术包括以桑格(Sanger)测序法为代表的第一代DNA测序技术和以Illumina Hiseq2500,Roche 454,ABI Solid，BGISEQ-500等为代表的第二代DNA测序技术。1977年，桑格发明了双脱氧末端终止测序法，成为第一代测序技术的代表。2001年，依托第一代测序技术，完成了人类基因组草图。桑格测序法具有实验操作简单、结果直观准确和实验周期短等特点，在对检测结果时效性要求很高的临床基因突变检测以及基因分型等领域有着广泛的应用。然而，桑格测序法的缺点是通量小、成本高，这限制了其在大规模基因测序中的应用。为克服桑格测序法的缺点，第二代测序技术应运而生。第二代DNA测序技术与第一代DNA测序技术相比，具有测序通量大、成本低、自动化程度高和单分子测序的特点。以Hiseq2500V2的测序技术为例，其一个实验流程可以产生10-200G碱基的数据，平均每个碱基的测序成本不到桑格测序法的测序成本的1/1000，并且所获得的测序结果可通过计算机直接进行处理和分析。因此，第二代DNA测序技术非常适合于大规模测序。
目前已开发的第二代DNA测序技术主要涉及，边连接边测序(sequencing by ligation,SBL)技术和边合成边测序(sequencing by synthesis,SBS)技术。这些测序技术的典型实例包括，Applied Biosystems公司开发的SOLiD测序法，Complete Genomics自主开发的组合探针锚定连接法(cPAL)和华大基因开发的组合探针锚定合成法(cPAS)，Illumina公司和Solexa technology公司合作开发的Illumina测序法等等。在这些测序方式中，Illumina和Complate Genomics采用了检测光信号的方法，为了实现4种碱基(A、T/U、C和G)的鉴别和区分，通常需要使用4种荧光染料来分别对这4种碱基进行标记。在这种情况下，为了读取各个碱基携带的荧光信号，测序装置必须配备至少2种单色激发光源和至少2个相机，这导致测序装置的制造成本昂贵且体积巨大。
在过去10多年，第二代基因测序技术已经逐步从新兴技术成长为主流的测序手段，并逐步成为临床领域的重要检测工具，在传染性疾病防御和控制、遗传病的诊断、无创产前筛查等领域发挥着越来越大的作用。为了进一步扩大测序市场，让测序仪平民化，低成本小型化测序仪的开发逐渐成为了测序领域的发展趋势。作为二代测序技术的经典手段，基于四通道，双通道以及单通道的三种测序方法各有千秋，但三者相比，单通道测序以其耗材更少，成本更低，更易实现仪器小型化便携式等优势，已逐渐成为测序领域的发展趋势。目前市场上基于单色通道的产品主要包括ion torrent系列的测序仪，454测序仪以及illumina公司最新推出的Iseq100。
目前基于单通道的测序技术中，ion torrent系列的仪器因polymer结构测序错误率高而限制了其使用范围，同样罗氏的454仪器也因测序准确率不够高，加上其高额的测序成本已逐渐退出了测序市场。Illumia公司的Iseq100基于单色荧光技术和半导体技术，实现了小型化的测序仪，且保持了较高的测序质量。但是因其光信号通过激光器激发，仪器配置额外的激光器，增加了仪器的体积。另外为了避免激发光产生的背景值，需要对半导体芯片做特殊处理从而滤掉激发光产出的背景，这种处理将造成高额的成本，增加了测序成本。
因此，本领域需要一种不需外界光源激发、无需采用额外的设计过滤激光光源产 生的背景的低成本测序方法。
发明概述
为了解决上述技术问题，本申请的发明人开发了一种新的测序方法，其使用自发光体系的信号来分辨A、(T/U)、C和G四种碱基。由此，用于实施本发明测序方法的发光信号来源于生物发光或化学发光，所以无需配置额外的激光器，也无需采用额外的设计来过滤激光光源产生的背景，降低了测序成本。用于本发明测序方法的测序装置甚至可以方便地随身携带，用于即时/现场检测。此外，本发明的测序方法中使用的脱氧核糖核苷酸3’端羟基被修饰阻断，在测序过程中，每次反应只能合成一个脱氧核糖核苷酸，保证每次反应仅结合一个脱氧核糖核苷酸，提高了测序的准确性。
在一个方面，本发明提供了一种对核酸分子进行测序的方法，其包括以下步骤：
(1)提供连接于支持物上的待测序的核酸分子，或者将待测序的核酸分子连接于支持物上；
(2)添加用于起始核苷酸聚合反应的引物，用于进行核苷酸聚合反应的聚合酶，以及四种化合物，从而形成含有溶液相和固相的反应体系；其中，所述四种化合物分别为核苷酸A、(T/U)、C和G的衍生物，并且具有碱基互补配对能力；并且，所述四种化合物的核糖或脱氧核糖的3'位置处的羟基(-OH)被保护基团保护；并且，
第一化合物连接有第一分子标记，
第二化合物连接有第二分子标记，
第三化合物连接有第一分子标记和第二分子标记，或部分第三化合物连接有第一分子标记且另外一部分第三化合物连接有第二分子标记，
第四化合物没有连接分子标记；
(3)使引物退火至待测序的核酸分子上，所述引物作为起始的生长的核酸链，与所述待测序的核酸分子一起形成连接于支持物上的双链体；
(4)在允许聚合酶进行核苷酸聚合反应的条件下，使用聚合酶进行核苷酸聚合反应，从而将所述四种化合物中的一种并入生长的核酸链的3'端；
(5)使上一步骤中的双链体与两种不同的荧光素酶接触并进行结合反应，所述两种荧光素酶分别能够特异性结合所述第一分子标记和第二分子标记，然后使所述荧光素酶在底物存在的情况下发生荧光反应，检测发出的荧光信号；
(6)去除各个核苷酸的分子标记；
(7)任选地重复步骤(3)-(7)，从而获得所述核酸分子的序列信息。
在本发明的实施方案中，通过荧光素酶与核苷酸衍生物的连接，实现了待测序核苷酸的自发光检测，从而不需要额外的激发光源。在具体实施方案中，通过荧光素酶上的标记物与核苷酸衍生物上的对应的标记物的特异性结合，实现了荧光素酶与核苷酸的连接。在具体的实施方案中，第一核苷酸连接上第一荧光素酶，第二核苷酸连接上第二荧光素酶，第三核苷酸连接上第一荧光素酶和第二荧光素酶，第四核苷酸不连接任何荧光素酶。然后分别通入两种荧光素酶的对应底物来检测四种碱基的发光信号；当通入第一荧光素酶的底物时，第一核苷酸和第三核苷酸发光，当通入第二荧光素酶的底物时，第二核苷酸和第三核苷酸发光，因而根据四种核苷酸的发光情况可以进行碱基的识别。
因此，在一个示例性的实施方案中，本发明的对核酸分子进行测序的方法包括以下步骤：
(1)提供连接于支持物上的待测序的核酸分子，或者将待测序的核酸分子连接于支持物上；
(2)添加用于起始核苷酸聚合反应的引物，用于进行核苷酸聚合反应的聚合酶， 以及四种化合物，从而形成含有溶液相和固相的反应体系；其中，所述四种化合物分别为核苷酸A、(T/U)、C和G的衍生物，并且具有碱基互补配对能力；并且，所述四种化合物的核糖或脱氧核糖的3'位置处的羟基(-OH)被保护基团保护；并且，
第一化合物连接有第一分子标记，
第二化合物连接有第二分子标记，
第三化合物连接有第一分子标记和第二分子标记，或部分第三化合物连接有第一分子标记且另外一部分第三化合物连接有第二分子标记，
第四化合物没有连接分子标记；
(3)使引物退火至待测序的核酸分子上，所述引物作为起始的生长的核酸链，与所述待测序的核酸分子一起形成连接于支持物上的双链体；
(4)在允许聚合酶进行核苷酸聚合反应的条件下，使用聚合酶进行核苷酸聚合反应，从而将所述四种化合物中的一种并入生长的核酸链的3'端；
(5)移除前一步骤的反应体系的溶液相，保留连接于支持物上的双链体，并加入两种不同的荧光素酶，进行结合反应，所述两种荧光素酶分别能够特异性结合所述第一分子标记和第二分子标记；
(6)用洗脱缓冲液去除未结合的荧光素酶；
(7)加入第一荧光素酶的底物，同时检测荧光信号；
(8)移除上一步反应的溶液；
(9)加入第二荧光素酶的底物，同时检测荧光信号；
(10)移除上一步反应的溶液；
(11)去除各个核苷酸的分子标记和3’保护基团；
(12)任选地移除上一步反应的溶液；
(13)任选地重复步骤(3)-(12)，从而获得所述核酸分子的序列信息。
在具体的实施方案中，两种荧光素酶与核苷酸的连接也可以分开进行，例如可以先加入经第一标记的第一荧光素酶，使其与经第一分子标记的核苷酸进行结合反应，然后用洗脱缓冲液去除未结合的第一荧光素酶，加入第一荧光素酶的底物，同时检测荧光信号；然后再加入经第二标记的第二荧光素酶，使其与经第二分子标记的核苷酸进行结合反应，然后用洗脱缓冲液去除未结合的第二荧光素酶，加入第二荧光素酶的底物，同时检测荧光信号。具体而言，本发明提供了一种对核酸分子进行测序的方法，其包括以下步骤：
(1)提供连接于支持物上的待测序的核酸分子，或者将待测序的核酸分子连接于支持物上；
(2)添加用于起始核苷酸聚合反应的引物，用于进行核苷酸聚合反应的聚合酶，以及四种化合物，从而形成含有溶液相和固相的反应体系；其中，所述四种化合物分别为核苷酸A、(T/U)、C和G的衍生物，并且具有碱基互补配对能力；并且，所述四种化合物的核糖或脱氧核糖的3'位置处的羟基(-OH)被保护基团保护；并且，
第一化合物连接有第一分子标记，
第二化合物连接有第二分子标记，
第三化合物连接有第一分子标记和第二分子标记，或部分第三化合物连接有第一分子标记且另外一部分第三化合物连接有第二分子标记，
第四化合物没有连接分子标记；
(3)使引物退火至待测序的核酸分子上，所述引物作为起始的生长的核酸链，与所述待测序的核酸分子一起形成连接于支持物上的双链体；
(4)在允许聚合酶进行核苷酸聚合反应的条件下，使用聚合酶进行核苷酸聚合反应，从而将所述四种化合物中的一种并入生长的核酸链的3'端；
(5)移除前一步骤的反应体系的溶液相，保留连接于支持物上的双链体，并加入第一荧光素酶，进行结合反应，所述第一荧光素酶能够特异性结合所述第一分子标记；
(6)用洗脱缓冲液去除未结合的第一荧光素酶；
(7)加入第一荧光素酶的底物，同时检测荧光信号；
(8)移除上一步反应的溶液；
(9)加入第二荧光素酶,进行结合反应，所述第二荧光素酶能够特异性结合所述第二分子标记；
(10)用洗脱缓冲液去除未结合的第二荧光素酶；
(11)加入第二荧光素酶的底物，同时检测荧光信号；
(12)移除上一步反应的溶液；
(13)任选地去除各个核苷酸的分子标记和3’保护基团；
(14)任选地重复步骤(3)-(13)或(3)-(11)一次或多次，从而获得所述核酸分子的序列信息。
在具体的实施方案中，两种荧光素酶可以相同，其分别能够特异性结合所述第一分子标记和第二分子标记，即仅使用一种荧光素酶和底物，经第一标记的第一荧光素酶和经第二标记的第二荧光素酶与核苷酸的连接分开进行。例如可以先加入经第一标记的第一荧光素酶，使其与经第一分子标记的核苷酸进行结合反应，然后用洗脱缓冲液去除未结合的第一荧光素酶，加入荧光素酶的底物，同时检测荧光信号；然后再加入经第二标记的第二荧光素酶，使其与经第二分子标记的核苷酸进行结合反应，然后用洗脱缓冲液去除未结合的第二荧光素酶，加入荧光素酶的底物，同时检测荧光信号。具体而言，本发明提供了一种对核酸分子进行测序的方法，其包括以下步骤：
(1)提供连接于支持物上的待测序的核酸分子，或者将待测序的核酸分子连接于支持物上；
(2)添加用于起始核苷酸聚合反应的引物，用于进行核苷酸聚合反应的聚合酶，以及四种化合物，从而形成含有溶液相和固相的反应体系；其中，所述四种化合物分别为核苷酸A、(T/U)、C和G的衍生物，并且具有碱基互补配对能力；并且，所述四种化合物的核糖或脱氧核糖的3'位置处的羟基(-OH)被保护基团保护；并且，
第一化合物连接有第一分子标记，
第二化合物连接有第二分子标记，
第三化合物连接有第一分子标记和第二分子标记，或部分第三化合物连接有第一分子标记且另外一部分第三化合物连接有第二分子标记，
第四化合物没有连接分子标记；
(3)使引物退火至待测序的核酸分子上，所述引物作为起始的生长的核酸链，与所述待测序的核酸分子一起形成连接于支持物上的双链体；
(4)在允许聚合酶进行核苷酸聚合反应的条件下，使用聚合酶进行核苷酸聚合反应，从而将所述四种化合物中的一种并入生长的核酸链的3'端；
(5)移除前一步骤的反应体系的溶液相，保留连接于支持物上的双链体，并加入第一荧光素酶，进行结合反应，所述第一荧光素酶能够特异性结合所述第一分子标记；
(6)用洗脱缓冲液去除未结合的第一荧光素酶；
(7)加入荧光素酶的底物，同时检测荧光信号；
(8)移除上一步反应的溶液；
(9)加入使荧光素酶变性的试剂；
(10)移除上一步反应的溶液；
(11)加入第二荧光素酶,进行结合反应，所述第二荧光素酶能够特异性结合所述第二分子标记；
(12)用洗脱缓冲液去除未结合的第二荧光素酶；
(13)加入荧光素酶的底物，同时检测荧光信号；
(14)任选地移除上一步反应的溶液；
(15)任选地去除各个核苷酸的分子标记和3’保护基团；
(16)任选地重复步骤(3)-(15)或(3)-(13)一次或多次，从而获得所述核酸分子的序列信息。
在另一个方面，本发明还涉及用于对多核苷酸进行测序的试剂盒，其包含：
(a)四种化合物，所述四种化合物分别为核苷酸A、(T/U)、C和G的衍生物，并且具有碱基互补配对能力；并且，所述四种化合物的核糖或脱氧核糖的3'位置处的羟基(-OH)被保护基团保护；并且，
第一化合物连接有第一分子标记，
第二化合物连接有第二分子标记，
第三化合物连接有第一分子标记和第二分子标记，或部分第三化合物连接有第一分子标记且另外一部分第三化合物连接有第二分子标记，
第四化合物没有连接分子标记；和
(b)两种荧光素酶，两种荧光素酶分别能够特异性结合所述第一分子标记和第二分子标记，两种荧光素酶可以相同或不同。
在某些优选的实施方案中，本发明的试剂盒还包含：用于从样品中提取核酸分子的试剂和/或装置；用于预处理核酸分子的试剂；用于连接待测序的核酸分子的支持物；用于将待测序的核酸分子与支持物连接(例如，共价或非共价连接)的试剂；用于起始核苷酸聚合反应的引物；用于进行核苷酸聚合反应的聚合酶；一种或多种缓冲溶液；一种或多种洗涤溶液；或其任何组合。
发明详述
除非另外定义，否则本文使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常理解的含义相同的含义。本文提及的所有专利、申请和其他出版物均通过引用整体并入本文。如果本文中提出的定义与通过引用并入本文的专利、申请和其他出版物中所述的定义相抵触或不一致，则以本文所述的定义为准。
如本文所用，术语“多核苷酸”是指脱氧核糖核酸(DNA)、核糖核酸(RNA)或其类似物。多核苷酸可以是单链的、双链的或含有单链和双链序列两者。多核苷酸分子可以来源于双链DNA(dsDNA)形式(例如，基因组DNA、PCR和扩增产物等)，或者可以来源于单链形式的DNA(ssDNA)或RNA并且其可以转化为dsDNA形式，并且反之亦然。多核苷酸分子的准确序列可以是已知的或未知的。以下是多核苷酸的示例性实例：基因或基因片段(例如，探针、引物、EST或SAGE标签)、基因组DNA、基因组DNA片段、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、核糖酶、cDNA、重组多核苷酸、合成多核苷酸、分枝多核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、任何上述序列的核酸探针、引物或扩增拷贝。
多核苷酸可以包括核苷酸或核苷酸类似物。核苷酸通常含有糖(如核糖或脱氧核糖)、碱基和至少一个磷酸基。核苷酸可以是无碱基的(即，缺少碱基)。核苷酸包括脱氧核糖核苷酸、修饰的脱氧核糖核苷酸、核糖核苷酸、修饰的核糖核苷酸、肽核苷酸、修饰的肽核苷酸、修饰磷酸盐糖主链核苷酸及其混合物。核苷酸的实例包括(例如)腺苷一磷酸(AMP)、腺苷二磷酸(ADP)、腺苷三磷酸(ATP)、胸苷一磷酸(TMP)、胸苷二磷酸(TDP)、胸苷三磷酸(TTP)、胞苷酸(CMP)、胞苷二磷酸(CDP)、胞苷三磷酸(CTP)、鸟苷一磷酸(GMP)、鸟苷二磷酸(GDP)、鸟苷三磷酸(GTP)、尿苷一磷酸(UMP)、尿苷 二磷酸(UDP)、尿苷三磷酸(UTP)、脱氧腺苷酸(dAMP)、脱氧腺苷二磷酸(dADP)、脱氧腺苷三磷酸(dATP)、脱氧胸腺嘧啶核苷一磷酸(dTMP)、脱氧胸腺嘧啶核苷二磷酸(dTDP)、脱氧胸苷三磷酸(dTTP)、去氧胞二磷(dCDP)、脱氧胞苷三磷酸(dCTP)、脱氧鸟苷一磷酸(dGMP)、脱氧鸟苷二磷酸(dGDP)、脱氧鸟苷三磷酸(dGTP)、脱氧尿苷一磷酸(dUMP)、脱氧尿苷二磷酸(dUDP)和脱氧尿苷三磷酸(dUTP)。还可以在本文所述的方法中使用包含修饰的碱基的核苷酸类似物。无论是具有天然主链还是类似结构，可以包含在多核苷酸中的示例性修饰的碱基包括(例如)肌苷、黄嘌呤(xathanine)、次黄嘌呤(hypoxathanine)、异胞嘧啶、异鸟嘌呤、2-氨基嘌呤、5-甲基胞嘧啶、5-羟甲基胞嘧啶、2-氨基腺嘌呤、6-甲基腺嘌呤、6-甲基鸟嘌呤、2-丙基鸟嘌呤、2-丙基腺嘌呤、2-硫脲嘧啶、2-硫胸腺嘧啶、2-硫胞嘧啶、15-卤代脲嘧啶、15-卤代胞嘧啶、5-丙炔基尿嘧啶、5-丙炔基胞嘧啶、6-偶氮尿嘧啶、6-偶氮胞嘧啶、6-偶氮胸腺嘧啶、5-尿嘧啶、4-硫尿嘧啶、8-卤代腺嘌呤或鸟嘌呤、8-氨基腺嘌呤或鸟嘌呤、8-硫腺嘌呤或鸟嘌呤、8-硫烷基腺嘌呤或鸟嘌呤、8-羟基腺嘌呤或鸟嘌呤、5-卤素取代的尿嘧啶或胞嘧啶、7-甲基鸟嘌呤、7-甲基腺嘌呤、8-氮杂鸟嘌呤、8-氮杂腺嘌呤、7-去氮鸟嘌呤、7-去氮腺嘌呤、3-去氮鸟嘌呤、3-去氮腺嘌呤等。如本领域中已知的，某些核苷酸类似物不能引入多核苷酸，例如，核苷酸类似物，如腺苷5’-磷酰硫酸。
通常而言，核苷酸包括核苷酸A、C、G、T或U。如本文所用，术语“核苷酸A”是指含有腺嘌呤(A)或其修饰物或类似物的核苷酸，例如ATP、dATP。“核苷酸G”是指含有鸟嘌呤(G)或其修饰物或类似物的核苷酸，例如GTP、dGTP。“核苷酸C”是指含有胞嘧啶(C)或其修饰物或类似物的核苷酸，例如CTP、dCTP。“核苷酸T”是指含有胸腺嘧啶(T)或其修饰物或类似物的核苷酸，例如TTP、dTTP。“核苷酸U”是指含有尿嘧啶(U)或其修饰物或类似物的核苷酸，例如UTP、dUTP。
核苷酸的标记
本发明涉及用不同的标记物单独或组合地标记核苷酸，从而使得可以在核苷酸上连接不同的荧光素酶。如本文所用，用于标记核苷酸的所述分子标记和与其特异性结合的标记可以是任何能够彼此特异性结合的分子配对。配对成员之间的特异性结合实现核苷酸与荧光素酶的连接。示例性的配对成员包括但不限于：(a)与相应抗体或其结合部分或片段组合的半抗原或抗原性化合物，例如地高辛-地高辛抗体，N3G-N3G抗体，FITC-FITC抗体；(b)核酸适配体和蛋白质；(c)非免疫结合对(例如生物素-抗生物素蛋白、生物素-链霉亲和素、生物素-中性抗生蛋白)；(d)激素-激素结合蛋白；(e)受体-受体激动剂或拮抗剂；(f)凝集素-碳水化合物；(g)酶-酶辅因子；(h)酶-酶抑制剂；和(i)能够形成核酸双链体的互补的寡核苷酸或多核苷酸对。
在具体的实施方案中，所述第一分子标记和第二分子标记是小分子标记物，其选自生物素、地高辛、N3G或FITC。两种荧光素酶分别能够特异性结合所述第一分子标记和第二分子标记。例如，在一个具体实施方案中，第一分子标记是生物素，则第一荧光素酶可以是经链霉亲和素标记的荧光素酶；第二分子标记是地高辛，则第二荧光素酶可以是经地高辛抗体标记的与所述第一荧光素酶不同的荧光素酶，第二荧光素酶也可以是经地高辛抗体标记的与第一荧光素酶相同的荧光素酶。所述荧光素酶来源包括但不限于firefly，gaussia，Renilla等生物。例如，经链霉亲和素标记的荧光素酶可以是Adivity公司的SA-Gluc:Streptavidin-Gaussia princeps luciferase。经地高辛抗体标记的荧光素酶可以是digoxin抗体-Gluc或digoxin抗体-Nluc。
如本文中所使用，表述“第一化合物连接有第一分子标记”是指全部第一化合物都连接有第一分子标记，或者部分第一化合物连接有第一分子标记但其余第一化合物没有连接分子标记。同理，表述“第二化合物连接有第二分子标记”是指全部第二化合物都连接有第二分子标记，或者部分第二化合物连接有第二分子标记但其余第二化合物没有连接分子标记。表述“第三化合物连接有第一分子标记和第二分子标记”是指全部 第三化合物都连接有第一分子标记和第二分子标记，或者部分第三化合物连接有第一分子标记和第二分子标记但其余第三化合物没有连接分子标记。
多核苷酸的测序
优选地，本发明的连接有不同的荧光素酶的核苷酸适用于合成法测序。如本文所用的合成法测序是本领域熟知的各种合成法测序方法。基本地，合成法测序涉及首先将被测序的核酸分子与测序引物杂交，随后在聚合酶的存在下，以被测序的核酸分子为模板在测序引物的3’端聚合如本文所述的连接有不同荧光素酶的核苷酸。聚合之后，通过检测所述荧光素酶发出的荧光信号来鉴定核苷酸。在从经标记的核苷酸上除去荧光素酶之后，进行下一个聚合测序循环。
用于测定靶多核苷酸序列的方法可以这样进行：使靶多核苷酸序列变性，使靶多核苷酸分别与不同的核苷酸接触，以便形成所述靶核苷酸的互补体，并且检测所述核苷酸的掺入。所述方法利用了聚合，使得聚合酶通过掺入互补于所述靶的正确的核苷酸，以延伸所述互补链。所述聚合反应还需要特殊引物来启动聚合作用。
对每一轮反应来说，所述核苷酸的掺入是通过聚合酶进行的，并随后测定所述掺入事件。存在很多不同的聚合酶，并且对本领域普通技术人员来说容易确定最适合的聚合酶。优选的酶包括DNA聚合酶I、Klenow片段、DNA聚合酶III、T4或T7DNA聚合酶、Taq聚合酶或vent聚合酶。还可以使用通过工程方法改造成具有特定性质的聚合酶。
所述测序方法优选对排列在固体支持物上的靶多核苷酸进行。可以通过接头分子将多个靶多核苷酸固定在所述固体支持物上，或者可以连接在诸如微球体的颗粒上，所述颗粒还可以连接在固体支持材料上。
可以通过多种方法将所述多核苷酸连接在所述固体支持物上，包括使用生物素-链霉亲和素相互作用。用于将多核苷酸固定在固体支持物上的方法为本领域所公知，并且包括石板印刷技术以及将每一种多核苷酸点样在固体支持物的特定位置上。合适的固体支持物为本领域所公知，并且包括玻璃载玻片和珠、陶瓷和硅表面和塑料材料。所述支持物通常是平面，尽管也可以使用微珠(微球体)，并且还可以通过已知方法将后者连接在其他固体支持物上。所述微球体可以具有任何合适的大小，其直径通常为10-100纳米。在优选实施方案中，将所述多核苷酸直接连接在平面上，优选连接在平的玻璃表面上。连接优选通过共价键的形式进行。所使用的阵列优选是单分子阵列，它包括位于独特的光学可分辨区域的多核苷酸，例如在国际申请号WO00/06770中所描述的。
进行聚合的必须条件对本领域技术人员来说是熟知的。为了进行所述聚合酶反应，通常首先必须使引物序列与所述靶多核苷酸退火，所述引物序列是由所述聚合酶识别的，并且起着所述互补链随后延伸的起始位点的作用。所述引物序列可以相对所述靶多核苷酸作为独立的成分添加。另外，所述引物和靶多核苷酸可以分别是一个单链分子的一部分，由所述引物部分与所述靶的一部分形成分子内双链体，即发卡环结构。可以在所述分子的任何位点，将该结构固定在所述固体支持物上。进行所述聚合酶反应所必需的其他条件，对本领域技术人员来说是熟知的，这些条件包括温度、pH、缓冲液组成。
随后，使本发明的经标记的核苷酸与所述靶多核苷酸接触，以便能够进行聚合。所述核苷酸可以依次添加，即分别添加每一种类型的核苷酸(A，C，G或T/U)，或同时添加。
使所述聚合步骤进行足以掺入一个核苷酸的时间。
然后除去未掺入的核苷酸，例如，通过移除前一步骤的反应体系的溶液相，保留连接于支持物上的双链体。
随后可以加入含有不同荧光素酶的两种荧光素酶，进行结合反应，所述两种荧光 素酶分别能够特异性结合用于标记核苷酸的分子标记，由此实现荧光素酶与掺入的核苷酸的连接。然后通过加入对应的荧光素酶的底物并检测荧光信号而实现掺入的核苷酸的鉴别。也可以加入含有相同荧光素酶的两种荧光素酶，进行结合反应，所述荧光素酶分别能够特异性结合用于标记核苷酸的分子标记，由此实现荧光素酶与掺入的核苷酸的连接。然后通过加入对应的荧光素酶的底物并检测荧光信号而实现掺入的核苷酸的鉴别。
在一个具体实施方案中，四种脱氧核糖核苷酸类似物分别被标记了不同的小分子标记物生物素(简称B)和地高辛(简称D)，例如核苷酸A标记B，核苷酸C标记B和D，核苷酸T标记D，核苷酸G不做标记。上述被标记不同小分子的四种脱氧核糖核苷酸类似物的3’端羟基均作阻断处理，保证每次测序反应仅结合一个脱氧核糖核苷酸。测序反应过程中，先通入所述四种经标记的脱氧核苷酸类似物及测序聚合酶混合物，在聚合酶的作用下，按照碱基互补配对原则，一个脱氧核糖核苷酸类似物掺入到生长的核酸链的3'端。通过移除前一步骤的反应体系的溶液相，保留连接于支持物上的双链体，可去除未结合的脱氧核糖核苷酸类似物。然后，加入含有不同荧光素酶的两种荧光素酶，第一荧光素酶经链霉亲和素标记，其与标记了小分子B的核苷酸A或核苷酸C结合，第二荧光素酶经地高辛抗体标记，其与标记了小分子D的核苷酸C或核苷酸T结合。使用洗脱缓冲液去除未结合的荧光素酶之后，加入第一荧光素酶的底物，连接有第一荧光素酶的核苷酸发光，采用检测器检测信号；加入第二荧光素酶的底物，连接有第二荧光素酶的核苷酸发光，采用检测器检测信号，由此得出下表所示的发光情况，即可进行碱基的识别。
Figure PCTCN2019086974-appb-000001
在一个具体实施方案中，含有不同荧光素酶的两种荧光素酶与经标记的核苷酸的连接以及信号检测可以分开进行。先加入第一荧光素酶，其经链霉亲和素标记，与标记了小分子B的核苷酸A或核苷酸C结合。使用洗脱缓冲液去除未结合的第一荧光素酶之后，加入第一荧光素酶的底物，连接有第一荧光素酶的核苷酸发光，采用检测器检测信号。移除反应溶液后加入经地高辛抗体标记的第二荧光素酶，其与标记了小分子D的核苷酸C或核苷酸T结合，然后用洗脱缓冲液去除未结合的第二荧光素酶，加入第二荧光素酶的底物，连接有第二荧光素酶的碱基发光，采用检测器检测信号，由此得出上表所示的发光情况，即可进行碱基的识别。
在另一个具体实施方案中，含有相同荧光素酶的两种荧光素酶与经标记的核苷酸的连接以及信号检测分开进行。先加入第一荧光素酶，其经链霉亲和素标记，与标记了小分子B的核苷酸A或核苷酸C结合。使用洗脱缓冲液去除未结合的第一荧光素酶之后，加入荧光素酶的底物，连接有第一荧光素酶的核苷酸发光，采用检测器检测信号。然后加入使第一荧光素酶变性的试剂，移除反应溶液后加入经地高辛抗体标记的第二荧光素酶，其与标记了小分子D的核苷酸C或核苷酸T结合，然后用洗脱缓冲液去除未结合的第二荧光素酶，加入荧光素酶的底物，连接有第二荧光素酶的碱基发光，采用检测器检测信号，由此得出上表所示的发光情况，即可进行碱基的识别。
荧光信号的检测
检测荧光信号的方式是本领域熟知的。例如，可以通过检测荧光的波长的装置来 实现。这样的装置是本领域熟知的。例如，这样的装置可以是共焦扫描显微镜，其用激光扫描固体支持物的表面，以便使直接结合被测序的核酸分子上的荧光团成像。另外，可以例如用灵敏的2-D探测器，如电荷偶连的探测器(CCD)观察所产生的每一种信号。还可以例如使用诸如扫描近场光学显微方法(SNOM)的其他技术。
标记物的去除
在检测之后，可以用合适条件除去核苷酸上的标记。
在具体的实施方案中，本发明的经标记的核苷酸还具有3’保护基团。在本发明的一些实施方案中，保护基团和标记物通常是3’阻断的经标记的核苷酸上的两种不同的基团，但在另一些实施方案中，保护基团和标记物也可以是同一基团。
如本文所用，术语“保护基团”意指这样的基团，其阻止聚合酶(其将含有该基团的核苷酸掺入到正在合成的多核苷酸链上的)在将含有该基团的核苷酸掺入到正在合成的多核苷酸链上后继续催化另一核苷酸的掺入。这样的保护基团在本文中也被称为3’-OH保护基团。包含这样的保护基团的核苷酸在本文中也被称为3’阻断的核苷酸。保护基团可以是能够被添加到核苷酸上任何合适的基团，只要该保护基团能防止另外的核苷酸分子被加入至多核苷酸链中且同时在不破坏该多核苷酸链的情况下易于从核苷酸的糖部分除去。此外，经保护基团修饰的核苷酸需要耐受聚合酶或用于将该修饰的核苷酸掺入多核苷酸链内的其他适合的酶。因此，理想的保护基团表现出长期的稳定性，可被聚合酶高效地掺入，阻止核苷酸的二次掺入或进一步掺入，并且能够在不破坏多核苷酸结构的温和条件下优选地在水性条件下被除去。
现有技术已描述了多种符合上述描述的保护基团。例如，WO 91/06678公开3'-OH保护基团包括酯和醚,-F,-NH2,-OCH3,-N3,-OPO3,-NHCOCH3,2硝基苯碳酸酯,2,4-次磺酰二硝基和四氢呋喃醚。Metzker等人(Nucleic Acids Research,22(20):4259-4267,1994)公开了八种3’-修饰的2-脱氧核糖核苷5’-三磷酸酯(3’-修饰的dNTP)的合成和应用。WO2002/029003描述了在聚合酶反应中使用烯丙基保护基团对DNA生长链上的3’-OH基团加帽。优选地，可以使用国际申请公开WO2014139596和WO2004/018497中报导的各种保护基团，包括例如WO2014139596的图1A中示例的那些保护基团和权利要求书中限定的那些3’羟基保护基(即保护基团)，和例如WO2004/018497的图3和4中示例的那些保护基团和权利要求书中限定的那些保护基团。上述参考文献均通过引用整体并入本文。
本领域技术人员将会理解如何将合适的保护基团连接在核糖环上，以便阻断与3’-OH的相互作用。所述保护基团可以直接连接在3’位置上，或者可以连接在2’位置上(所述保护基团具有足够的大小或电荷，以便阻断3’位置上的相互作用)。另外，所述保护基团可以连接在3’和2’位置，并且可以被裂解，以便暴露出3’-OH基团。
在成功地将3'阻断的核苷酸掺入生长的核酸链后，测序方案需要除去保护基团以产生用于连续链合成的可用的3'-OH位点。如本文所用的可从经修饰的核苷酸上除去保护基团的试剂在很大程度上取决于所使用的保护基团。例如，从3'羟基官能团除去酯保护基团通常通过碱水解来实现。除去保护基团的容易程度差异很大；通常，羰基碳上取代基的电负性越大，除去的容易度越大。例如，高电负性的三氟乙酸基团在甲醇中在pH7下能够从3'羟基快速裂解(Cramer等人，1963)，因此其在该pH下的聚合期间是不稳定的。苯氧基乙酸酯基团在少于1分钟内裂解，但是需要显著更高的pH，例如用NH-/甲醇实现(Reese和Steward，1968)。使用除碱水解以外的化学方法可以选择性地切割各种各样的羟基保护基团。通过用亲核试剂例如苯硫酚和硫代硫酸盐处理可迅速裂解2,4-二硝基苯硫基(Letsinger等，1964)。烯丙基醚通过用丙酮/水中的Hg(II)处理而裂解(Gigg and Warren，1968)。使用Ag(I)或Hg(II)在中性条件下除去四氢噻喃基醚(Cohen and Steele，1966；Cruse等人，1978)。光化学去阻断可以与可光化学裂解的保护基团一起使用。有几种保护基团可用于这种方 法。使用邻硝基苄醚作为核糖核苷的2'-羟基官能性的保护基团是已知且被证明的(Ohtsuka等，1978)；其通过在260nm的照射进行除去。碳酸烷基邻硝基苄基碳酸酯保护基也通过在pH7的照射下被除去(Cama and Christensen，1978)。3'-OH保护基团的酶解解阻断也是可能的。已经证明T4多核苷酸激酶可以将3'-磷酸酯末端转化成3'-羟基末端，然后可以用作DNA聚合酶I的引物(Henner等，1983)。该3'-磷酸酶活性用于除去含有磷酸酯作为保护基团的那些dNTP类似物的3'保护基团。
可从3'阻断的核苷酸上除去保护基团的其它试剂包括例如膦(例如三(羟甲基)膦(THP))，其可以例如将含叠氮化物的3’-OH保护基团从核苷酸上除去(关于膦的此应用可参见例如WO2014139596中的记载，其全部内容通过引用并入本文)。可从3'阻断的核苷酸上除去保护基团的其它试剂还包括例如WO2004/018497的说明书中第114-116页描述的用于除去作为3’-OH保护基团的3’-烯丙基、3,4-二甲氧基苄氧基甲基或氟甲氧甲基的相应试剂。
在本发明的实施方案中，核苷酸的标记物优选在检测后与保护基团一起被除去。
在某些实施方案中，标记物可以被掺入保护基团，从而允许在将3'阻断的核苷酸掺入核酸链后其能够与保护基团一起被除去。
在其他实施方案中，标记物可以利用连接基团与保护基团分开地连接在核苷酸上。这样的标记物可以例如连接到核苷酸的嘌呤或嘧啶碱基上。在某些实施方案中，所用的连接基团是可裂解的。可裂解的连接基团的使用能确保所述标记可以在检测之后除去，这避免了与后续掺入的任何经标记的核苷酸的任何信号干扰。在另一些实施方案中，可以使用不可裂解的连接基团，因为在经标记的核苷酸掺入核酸链之后，不需要后续的核苷酸掺入，因此不需要将标记从核苷酸中除去。
在另外的实施方案中，标记物和/或连接基团可以具有足以发挥阻断其他核苷酸掺入到多核苷酸链上的大小或结构(也就是说，所述标记本身可用作保护基团)。所述阻断可能是由于空间位阻造成的，或者可能是由于大小、电荷和结构的组合造成的。
可裂解的连接基团为本领域所公知，并且可以采用常规化学方法，以便将连接基团连接在核苷酸碱基和标记物上。连接基团可以连接在核苷酸碱基的任何位置上，其前提是，仍然能进行Watson-Crick碱基配对。对于嘌呤碱基来说，如果所述连接基团是通过所述嘌呤或优选的脱氮嘌呤类似物的7号位置，通过8-修饰的嘌呤，通过N-6修饰的腺嘌呤或N-2修饰的鸟嘌呤连接的话将是优选的。对于嘧啶来说，连接优选是通过胞嘧啶，胸腺嘧啶和尿嘧啶上的5号位置和胞苷上的N-4位置连接的。
使用术语“可裂解的连接基团”并非意味着需要除去整个连接基团(例如，从核苷酸碱基中除去)。当标记物与碱基相连接时，核苷裂解位点可位于连接基团上的位置，该位置能够确保在裂解后一部分的连接基团仍与所述核苷酸碱基保持连接。
合适的连接基团包括但不局限于二硫连接基团，酸不稳定性连接基团(包括二烷氧基苄基连接基团，Sieber连接基团，吲哚连接基团，叔丁基Sieber连接基团)，亲电可裂解的连接基团，亲核可裂解的连接基团，光可裂解的连接基团，在还原条件、氧化条件下裂解的连接基团，保险栓(safety-catch)连接基团，以及通过消除机制进行裂解的连接基团。合适的连接基团可以用标准化学保护基团改良，正如在以下文献中所披露的：Greene&Wuts，Protective Groups in Organic Synthesis，John Wiley&Sons。Guillier等披露了用于固相合成的其他合适的可裂解的连接基团(Chem.Rev.100：2092-2157，2000)。
所述连接基团可以通过任何合适的方法裂解，包括接触酸，碱，亲核试剂，亲电试剂，自由基，金属，还原或氧化试剂，光照，温度，酶等，下文将示例性描述各种可裂解的连接基团的合适裂解方式。通常地，所述可裂解的连接基团可以在与所述保护基团相同的条件下裂解，以使得仅需要一次处理即可除去所述标记物和保护基团。
亲电裂解的连接基团典型地被质子所裂解，并包括对酸敏感的裂解。合适的亲电 裂解的连接基团包括修饰的苄基系统，诸如三苯甲基、对烃氧基苄基酯和对烃氧基苄基酰胺。其他适合的连接基团包括叔丁氧羰基(Boc)基团和缩醛系统。为制备合适的连接分子，还可以考虑在硫缩醛或其他含硫保护基的裂解中使用诸如镍、银或汞的亲硫金属。亲核裂解的连接基团包括在水中不稳定的基团(即，能够在碱性pH值下简单地裂解)，例如酯类，以及对非水性亲核试剂不稳定的基团。氟离子可用于裂解诸如三异丙基硅烷(TIPS)或叔丁基二甲基硅烷(TBDMS)的基团中的硅氧键。可光解的连接基团在糖化学中被广泛使用。优选地，激活裂解所需的光不影响修饰的核苷酸中的其他组分。例如，如果使用荧光团作为标记，优选地，该荧光团吸收与裂解所述连接分子所需的光不同波长的光。适合的连接基团包括那些基于O-硝基苄基化合物和硝基藜芦基化合物的连接基团。也可以使用基于安息香化学的连接基团(Lee等人,J.Org.Chem.64:3454-3460,1999)。已知多种对还原裂解敏感的连接基团。使用基于钯催化剂的催化氢化已用于裂解苄基和苄氧羰基基团。二硫键还原也为本领域所知。基于氧化的方法为本领域所公知。这些方法包括对烃氧基苄基的氧化以及硫和硒连接基团的氧化。使用碘溶液(aqueous iodine)来使二硫化物和其他基于硫或硒的连接基团裂解也在本发明的范围内。安全拉手型连接基团(safety-catchlinker)为那些在两步中裂解的连接基团。在优选的系统中，第一步是反应性亲核中心的产生，随后的第二步涉及分子内环化，这导致裂解。例如，可以用肼或光化学方法处理乙酰丙酸酯连接来释放活性的胺，然后所述胺被环化以使分子中其他位置的酯裂解(Burgess等人,J.Org.Chem.62:5165-5168,1997)。也可以使用消除反应裂解连接基团。可以使用诸如芴甲氧羰基和氰基乙基的基团的碱催化的消除以及烯丙基系统的钯催化的还原消除。
在某些实施方案中，连接基团可包含间隔单元。连接基团的长度并不重要，只要所述标记物与核苷酸保持足够的距离，以免干扰核苷酸与酶之间的相互作用。
在某些实施方案中，连接基团可由与3’-OH保护基团类似的官能团组成。这会使得仅需要单一处理就除去标记物和保护基团。特别优选的连接基团是可通过膦裂解的含叠氮化物的连接基团。
如本文所用的可从经修饰的核苷酸上除去标记物的试剂在很大程度上取决于所使用的标记物。例如，在标记物掺入保护基团的情况下，使用上文所述的除去保护基团的试剂除去标记物。或者，在标记物通过可裂解的连接基团连接至核苷酸的碱基时，使用如上文所述的裂解连接基团的试剂除去标记物。在优选的实施方案中，使用相同的试剂来从经修饰的核苷酸上除去标记物和保护基团，例如在连接基团由与3’-OH保护基团类似的官能团组成的情况下。
试剂盒
本发明还提供了用于对多核苷酸进行测序的试剂盒，其包含：
(a)四种化合物，所述四种化合物分别为核苷酸A、(T/U)、C和G的衍生物，并且具有碱基互补配对能力；并且，所述四种化合物的核糖或脱氧核糖的3'位置处的羟基(-OH)被保护基团保护；并且，
第一化合物连接有第一分子标记，
第二化合物连接有第二分子标记，
第三化合物连接有第一分子标记和第二分子标记，或部分第三化合物连接有第一分子标记且另外一部分第三化合物连接有第二分子标记，
第四化合物没有连接分子标记；和
(b)两种不同的荧光素酶，两种荧光素酶分别能够特异性结合所述第一分子标记和第二分子标记。
在具体的实施方案中，用于标记四种化合物的分子标记和用于标记两种荧光素酶的标记如上文所定义。
在某些优选的实施方案中，本发明的试剂盒还包含：用于从样品中提取核酸分子的试剂和/或装置；用于预处理核酸分子的试剂；用于连接待测序的核酸分子的支持物；用于将待测序的核酸分子与支持物连接(例如，共价或非共价连接)的试剂；用于起始核苷酸聚合反应的引物；用于进行核苷酸聚合反应的聚合酶；一种或多种缓冲溶液；一种或多种洗涤溶液；或其任何组合。
在某些优选的实施方案中，本发明的试剂盒还包含，用于从样品中提取核酸分子的试剂和/或装置。用于从样品中提取核酸分子的方法是本领域熟知的。因此，可根据需要，在本发明的试剂盒中配置各种用于提取核酸分子的试剂和/或装置，例如用于破碎细胞的试剂，用于沉淀DNA的试剂，用于洗涤DNA的试剂，用于溶解DNA的试剂，用于沉淀RNA的试剂，用于洗涤RNA的试剂，用于溶解RNA的试剂，用于去除蛋白的试剂，用于去除DNA的试剂(例如当目的核酸分子为RNA时)，用于去除RNA的试剂(例如当目的核酸分子为DNA时)，及其任何组合。
在某些优选的实施方案中，本发明的试剂盒还包含，用于预处理核酸分子的试剂。在本发明的试剂盒中，用于预处理核酸分子的试剂不受额外限制，并且可根据实际需要选择。所述用于预处理核酸分子的试剂包括例如，用于核酸分子片段化的试剂(例如DNA酶I)，用于补齐核酸分子末端的试剂(例如DNA聚合酶，例如T4 DNA聚合酶，Pfu DNA聚合酶，Klenow DNA聚合酶)，接头分子，标签分子，用于将接头分子与目的核酸分子相连接的试剂(例如连接酶，例如T4 DNA连接酶)，用于修复核酸切口的试剂(例如，丧失3'-5'核酸外切酶活性但显示5'-3'核酸外切酶活性的DNA聚合酶)，用于扩增核酸分子的试剂(例如，DNA聚合酶，引物，dNTP)，用于分离和纯化核酸分子的试剂(例如层析柱)，以及其任何组合。
在某些优选的实施方案中，本发明的试剂盒还包含用于连接待测序的核酸分子的支持物。所述支持物可以具有上文中针对支持物所详细描述的任何技术特征以及其任何组合。
例如，在本发明中，所述支持物可以由各种合适的材料制成。此类材料包括例如：无机物、天然聚合物、合成聚合物，以及其任何组合。具体的例子包括但不限于：纤维素、纤维素衍生物(例如硝化纤维素)、丙烯酸树脂、玻璃、硅胶、聚苯乙烯、明胶、聚乙烯吡咯烷酮、乙烯基和丙烯酰胺的共聚物、与二乙烯基苯等交联的聚苯乙缔(参见例如，Merrifield Biochemistry 1964,3,1385-1390)、聚丙烯酰胺、乳胶、葡聚糖、橡胶、硅、塑料、天然海绵、金属塑料、交联的葡聚糖(例如，Sephadex TM)、琼脂糖凝胶(Sepharose TM)，以及本领域技术人员已知的其他支持物。
在某些优选的实施方案中，用于连接待测序的核酸分子的支持物可以是包括惰性基底或基质(例如，载玻片、聚合物珠等)的固体支持物，所述惰性基底或基质已例如通过应用含有活性基团的中间材料而被功能化，所述活性基团允许共价连接诸如多核苷酸的生物分子。此类支持物的实例包括但不限于，负载于诸如玻璃的惰性基底上的聚丙酰胺水凝胶，特别是WO 2005/065814和US 2008/0280773中描述的聚丙烯酰胺水凝胶，其中，所述专利申请的内容通过引用以其全文并入本文。在此类实施方案中，生物分子(例如多核苷酸)可被直接地共价地连接至中间材料(例如水凝胶)，而中间材料其自身可被非共价地连接至基底或基质(例如，玻璃基底)。在某些优选的实施方案中，所述支持物为表面修饰了一层亲和素、氨基、丙烯酰胺硅烷或醛基化学基团的玻片或硅片。
在本发明中，支持物或固体支持物不受限于其大小、形状和构造。在一些实施方案中，支持物或固体支持物是平面结构，例如载片、芯片、微芯片和/或阵列。此类支持物的表面可以是平面层的形式。在一些实施方案中，支持物或其表面是非平面的，例如管或容器的内表面或外表面。在一些实施方案中，支持物或固体支持物包括微球或珠。在某些优选的实施方案中，用于连接待测序的核酸分子的支持物为珠或孔的阵 列。
在某些优选的实施方案中，本发明的试剂盒还包含用于将待测序的核酸分子与支持物连接(例如，共价或非共价连接)的试剂。此类试剂包括例如对核酸分子(例如其5'端)进行活化或修饰的试剂，例如磷酸、硫醇、胺、羧酸或醛；对支持物的表面进行活化或修饰的试剂，例如氨基-烷氧基硅烷(例如氨基丙基三甲氧基硅烷、氨基丙基三乙氧基硅烷、4-氨基丁基三乙氧基硅烷等)；交联剂，例如琥珀酰酐、苯基二异硫氰酸盐(Guo等人，1994)、马来酸酐(Yang等人，1998)、1-乙基-3-(3-二甲基氨基丙基)-碳二亚胺盐酸盐(EDC)、间-马来酰亚胺基苯甲酸-N-羟基琥珀酰亚胺酯(MBS)、N-琥珀酰亚胺基[4-碘代乙酰基]氨基苯甲酸(SIAB)、4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺(SMCC)、N-γ-马来酰亚胺基丁酰氧基-琥珀酰亚胺酯(GMBS)、4-(p-马来酰亚胺基苯基)丁酸琥珀酰亚胺(SMPB)；以及其任何组合。
在某些优选的实施方案中，本发明的试剂盒还包含用于起始核苷酸聚合反应的引物。在本发明中，引物不受额外的限制，只要它能够特异性地退火到目标核酸分子的一个区域上。在一些示例性实施方案中，所述引物的长度可以为5-50bp，例如5-10、10-15、15-20、20-25、25-30、30-35、35-40、40-45、45-50bp。在一些示例性实施方案中，所述引物可包含天然存在或非天然存在的核苷酸。在一些示例性实施方案中，所述引物包含天然存在的核苷酸或者由天然存在的核苷酸组成。在一些示例性实施方案中，所述引物包含经修饰的核苷酸，例如锁核酸(LNA)。在某些优选的实施方案中，所述引物包含通用引物序列。
在某些优选的实施方案中，本发明的试剂盒还包含用于进行核苷酸聚合反应的聚合酶。在本发明中，可使用各种合适的聚合酶。在一些示例性实施方案中，所述聚合酶能够以DNA为模板合成新的DNA链(例如DNA聚合酶)。在一些示例性实施方案中，所述聚合酶能够以RNA为模板合成新的DNA链(例如反转录酶)。在一些示例性实施方案中，所述聚合酶能够以DNA或RNA为模板合成新的RNA链(例如RNA聚合酶)。因此，在某些优选的实施方案中，所述聚合酶选自DNA聚合酶，RNA聚合酶，和反转录酶。
在某些优选的实施方案中，本发明的试剂盒还包含一种或多种缓冲溶液。此类缓冲液包括但不限于，用于DNA酶I的缓冲溶液，用于DNA聚合酶的缓冲溶液，用于连接酶的缓冲溶液，用于洗脱核酸分子的缓冲溶液，用于溶解核酸分子的缓冲溶液，用于进行核苷酸聚合反应(例如PCR)的缓冲溶液，和用于进行连接反应的缓冲溶液。本发明的试剂盒可包含上述缓冲溶液的任一种或多种。
在某些优选的实施方案中，本发明的试剂盒还包含一种或多种洗涤溶液。此类洗涤溶液的实例包括但不限于，磷酸盐缓冲液，柠檬酸盐缓冲液，Tris-HCl缓冲液，醋酸盐缓冲液，碳酸盐缓冲液等等。本发明的试剂盒可包含上述洗涤溶液的任一种或多种。
发明的有益效果
与现有技术相比，本发明的技术方案具有以下有益效果：
(1)本发明的方法仅使用两种分子标记即实现对四种核苷酸的标记，并且通过使用荧光素酶实现了自发光。因此，用于本发明测序方法的测序装置无需配备激发光源，也无需采用额外的设计过滤激光光源产生的背景。这一方面大大降低了测序装置的制造成本，有助于测序装置和测序方法的推广和应用；另一方面显著减小了测序装置的体积，使测序装置更加轻便，便于携带。
(2)本发明的测序方法中使用的脱氧核糖核苷酸3’端羟基被修饰阻断，在测序过程中，每次反应只能合成一个脱氧核糖核苷酸，保证每次反应仅结合一个脱氧核糖核苷酸，提高了测序的准确性。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公开的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
实施例
实施例1
1.测序文库构建
(1)设计如下DNA序列：GATATCTGCAGGCATAGAATGAATATTATTGAATCAATAATTAAAGTCGGAGGCCAAGCGGTCTTAGGAAGACAACAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTTTACAACTACGATAATGGGCTGGATACATGGAATGATTATAGATATATTAAGGAATAATGTTAATTAATGCCTAAATTAATTAATCTAAGGGGGTTAATACTTCAGCCTGTGATATC，为建库方便，在序列的两端添加了oligo序列(加粗字体)，并在中间部分插入了BGISEQ-500的接头序列(阴影部分)，斜体标粗部分是待测序列的前10bp碱基。上述序列由金斯瑞生物科技公司合成，为了序列的无限使用将合成的序列插在pUC57载体上，并转化至E.coli菌体中。
(2)培养适合量的含有已知文库的E.coli细菌，并提取质粒，设计如下一对引物：GATATCTGCAGGCAT(引物1)，GATATCACAGGCTGA(引物2)，按如下体系(表一)和流程(表二)将已知序列扩增出来，PCR产物使用磁珠纯化的方式进行。纯化好的PCR产物加入split oligo(ATGCCTGCAGATATCGATATCACAGGCTGA)按照BGISEQ-500SE50环化建库试剂盒(华大智造)及流程进行环化建库，待用；
表一(酶来源于BGI自产)
Figure PCTCN2019086974-appb-000002
表二：
Figure PCTCN2019086974-appb-000003
2.文库序列的扩增
购买Thermo fisher公司的Streptavidin包被的96孔板，将1uM 5’端biotin修饰的引物GCCATGTCGTTCTGTGAGCCAAGG 100ul于其中一个孔常温孵育30min，去掉反应液体，加入6ng上文第1节中所构建的文库和20ul BGISEQ-500试剂盒(华大智造)中的DNB制备缓冲液I，60℃与上述biotin修饰的引物进行引物杂交5min后，加入40ul BGISEQ-500测序试剂盒(华大智造)中的DNB聚合酶I和4ul DNB 聚合酶II，30℃反应60min，加热至65℃终止反应，并小心去除反应液。加入100ul 5uM的测序引物GCTCACAGAACGACATGGCTACGATCCGACTT，常温杂交30min，小心去掉反应液；
3.测序
(1)Acme Bioscience公司外包合成如下所示的4种dNTP：
Figure PCTCN2019086974-appb-000004
Figure PCTCN2019086974-appb-000005
(2)两种荧光素酶的准备:
a.SA-Gluc(购买于adivity公司)
b.抗体(购买于Abcam公司)与Nluc偶联(购买于avidity)制备Ab-Nluc
购买thermo的蛋白偶联试剂盒，根据说明书按照如下程序进行抗体与Nluc的偶联(图中enzyme指Nluc，图中抗体是指地高辛抗体)：
Figure PCTCN2019086974-appb-000006
(3)试剂准备:
配制测序反应中所需要的其他试剂
聚合反应液：50mM Tris-Hcl，50mM NaCl，10mM(NH 4) 2SO 4，0.02mg/ml聚合酶BG9(BGI)，3mM MgSO 4,1mM EDTA,上述四种dNTP各1uM
洗脱缓冲液：5XSSC，Tween20 0.05％；
酶结合反应液：将上述两种酶稀释至TBST缓冲液中，两种酶的终浓度各2ug/ml；
底物1反应液：配制50mM tris-Hcl 0.5mM Nacl缓冲液，将50X Coelenterazine(nanolight)稀释成1X；
底物2反应液：配制50mM tris-Hcl 0.5mM Nacl缓冲液，将50X NLuc FLASH Substrate(nanolight)稀释成1X；
切除缓冲液：20mM THPP，0.5M NaCl，50mM Tris-HCl,pH 9.0,0.05％tween20；
(4)测序反应：
测序流程：
a.聚合：在扩增好文库的孔中，加入100ul聚合酶反应液，将酶标仪温度升高至55℃，反应3min，使四种dNTP聚合到扩增好的文库上，小心去除反应液后，加入 100ul洗脱反应液，轻轻吹打几次，去除洗脱反应液；
b.荧光素酶结合：加入100ul酶结合反应液，在35℃的条件下，孵育30min，使SA-gluc结合至带有biotin标记的dCTP和dATP的衍生物上，Ab-Nluc结合至带有digoxin标记的dCTP和dTTP的衍生物上，去除反应液，加入洗脱液，轻轻吹
打几次后，去除洗脱液；
c.酶1信号检测：设置适合的酶标仪参数，加入底物1反应液，进行酶1信号检测，记录最高信号值；
d.酶2信号检测：移除底物反应液1，设置适合的酶标仪参数，加入底物2反应液，进行酶2信号检测，记录最高信号值；
e.切除；移除底物2反应液，加入200ul洗脱缓冲液，轻轻吹打几次后，去掉洗脱缓冲液，加入100ul切除反应液，55℃，反应3min后，去除切除反应液；加入200ul洗脱缓冲液清洗，重复清洗三次；
f.重复a-e步骤，进行下个循环测序；总共进行10bp测序
(5)测序结果
a.两次发光的信号值如下：
  底物1信号(W) 底物2信号(W)
cycle1 0.1 210
cycle2 188 0.12
cycle3 173 169
cycle4 206 0.11
cycle5 198 0.13
cycle6 176 171
cycle7 0.13 186
cycle8 203 0.15
cycle9 166 159
cycle10 0.12 0.16
b.测序结果分析：
根据发光的信号值，cycle1及cycle7仅在通入底物1时有信号，可推出这两个cycle是聚合了biotin-dTTP，待测文库的第一个碱基和第七个碱基为T碱基；
cycle2，cycle4，cycle5及cycle8仅在通入底物2时有信号，可推出这四个cycle是聚合了digoxin-dATP，因而待测文库的第二，四，五及第八个碱基为A碱基；
cycle3，cycle6及cycle9在通入底物1和底物2后，均有信号产生，可推出这两个cycle是聚合了biotin-digoxin-dCTP，因而待测文库的第三、第六及第九个碱基是C碱基；
Cycle10在两次底物通入时均不发光，可推出这个cycle聚合了coldG，因而此文库的第十个碱基是G碱基。
综上所述，所测序列的前10个碱基为：TACAACTACG，与待测文库前10bp的碱基序列TACAACTACG匹配度100％。
实施例2
测序文库构建及文库序列扩增如实施例1所示。
测序：
(1)四种dNTP如实施例1所示
(2)荧光素酶及相关蛋白的准备
a.SA-Gluc(购买于adivity公司)
b.抗体(购买于Abcam公司),按照thermo fisher NHS-s-s-biotin的使用说明书将抗体上标记biotin标签，制备成抗体-s-s-biotin.
(3)试剂准备
配制测序反应中所需要的其他试剂
聚合反应液：50mM Tris-Hcl，50mM NaCl，10mM(NH 4) 2SO 4，0.02mg/ml聚合酶BG9(BGI)，3mM MgSO 4,1mM EDTA,上述四种dNTP各1uM
洗脱缓冲液：5XSSC，Tween20 0.05％；
酶结合反应液：将上述两种蛋白SA-Gluc和抗体-s-s-biotin分别稀释至TBST缓冲液中，两种蛋白的终浓度各2ug/ml；
底物反应液：配制50mM Tris-HCl 0.5mM NaCl缓冲液，将50X Coelenterazine(nanolight)稀释成1X；
酶失活缓冲液：5mM DTT,50mM Tris-HCl,pH9.0；
切除缓冲液：20mM THPP，50mM Tris-HCl，pH9.0,0.5M NaCl,0.05％tween20；
(4)测序反应
a.聚合：在扩增好文库的孔中，加入100ul聚合酶反应液，将酶标仪温度升高至55℃，反应3min，使四种dNTP聚合到扩增好的文库上，小心去除反应液后，加入100ul洗脱反应液，轻轻吹打几次，去除洗脱反应液；
b.荧光素酶结合：加入100ul荧光素酶SA-Gluc结合反应液，在35℃的条件下，孵育30min，使SA-gluc结合至带有biotin标记的dCTP和dATP的衍生物上，去除反应液，加入洗脱液，轻轻吹打几次后，去除洗脱液；
c.第一次信号检测：设置适合的酶标仪参数，加入底物反应液，进行信号检测，记录最高信号值；
d.酶失活：移除底物反应液，加入酶失活缓冲液，35℃孵育10min，去除反应液，再加入洗脱缓冲液进行洗脱；
e.第二次荧光素酶结合：加入抗体-S-S-biotin反应液，在35℃的条件下孵育30min，使抗体-s-s-biotin结合至带有digoxin标记的dCTP和dTTP衍生物上，去除反应液，加入洗脱缓冲液，轻轻吹打几次，去除洗脱液，加入100ul SA-Gluc结合反应液，在35℃的条件下孵育30min，去除反应液后，加入洗脱缓冲液，洗脱完毕后，去除洗脱缓冲液；
f.第二次信号检测：设置适合的酶标仪参数，加入底物反应液，进行信号检测，记录最高信号值；
g.切除；移除底物2反应液，加入200ul洗脱缓冲液，轻轻吹打几次后，去掉洗脱缓冲液，加入100ul切除反应液，55℃，反应3min后，去除切除反应液；加入200ul洗脱缓冲液清洗，重复清洗三次；
h.重复a-g步骤，进行下个循环测序；总共进行10bp测序
(5)测序结果
a.10bp信号检测如下：
  第一次检测(w) 第二次检测(w)
cycle1 0.6 223
cycle2 312 0.7
cycle3 278 327
cycle4 254 0.3
cycle5 239 0.4
cycle6 243 275
cycle7 0.27 228
cycle8 267 0.5
cycle9 263 279
cycle10 0.7 0.6
b.测序结果分析：
cycle1和cycle7，仅在第一次有信号值，推测这两个cycle聚合上了biotin-dTTP，因而待测文库的第一个及第七个碱基是T；
cycle2，cycle4，cycle5及cycle8仅在第二次检测时有信号，可推出这四cycle是聚合了digoxin-dATP，因而待测文库的第二，四，五及第八个碱基为A碱基；
cycle3，cycle6及cycle9在第一次和第二次检测时，均有信号产生，可推出这两个cycle是聚合了biotin-digoxin-dCTP，因而待测文库的第三、第六及第九个碱基是C碱基；
Cycle10在两次信号检测时均不发光，可推出这个cycle聚合了coldG，因而此文库的第十个碱基是G碱基。
综上所述，所测序列的前10个碱基为：TACAACTACG，与待测文库前10bp的碱基序列TACAACTACG匹配度100％。

Claims (12)

  1. 一种对核酸分子进行测序的方法，其包括以下步骤：
    (1)提供连接于支持物上的待测序的核酸分子，或者将待测序的核酸分子连接于支持物上；
    (2)添加用于起始核苷酸聚合反应的引物，用于进行核苷酸聚合反应的聚合酶，以及四种化合物，从而形成含有溶液相和固相的反应体系；其中，所述四种化合物分别为核苷酸A、(T/U)、C和G的衍生物，并且具有碱基互补配对能力；并且，所述四种化合物的核糖或脱氧核糖的3'位置处的羟基(-OH)被保护基团保护；并且，
    第一化合物连接有第一分子标记，
    第二化合物连接有第二分子标记，
    第三化合物连接有第一分子标记和第二分子标记，或部分第三化合物连接有第一分子标记且另外一部分第三化合物连接有第二分子标记，
    第四化合物没有连接分子标记；
    (3)使引物退火至待测序的核酸分子上，所述引物作为起始的生长的核酸链，与所述待测序的核酸分子一起形成连接于支持物上的双链体；
    (4)在允许聚合酶进行核苷酸聚合反应的条件下，使用聚合酶进行核苷酸聚合反应，从而将所述四种化合物中的一种并入生长的核酸链的3'端；
    (5)使上一步骤中的双链体与两种不同的荧光素酶接触并进行结合反应，所述两种荧光素酶分别能够特异性结合所述第一分子标记和第二分子标记，然后使所述荧光素酶在底物存在的情况下发生荧光反应，检测发出的荧光信号；
    (6)去除各个核苷酸的分子标记；
    (7)任选地重复步骤(3)-(6)或(3)-(5)一次或多次，从而获得所述核酸分子的序列信息。
  2. 根据权利要求1所述的方法，其中步骤(5)中所述双链体与两种不同的荧光素酶在一步反应中接触并进行结合反应；或者步骤(5)中所述双链体与两种荧光素酶依次接触并进行结合反应。
  3. 根据权利要求1或2所述的方法，其包括以下步骤：
    (1)提供连接于支持物上的待测序的核酸分子，或者将待测序的核酸分子连接于支持物上；
    (2)添加用于起始核苷酸聚合反应的引物，用于进行核苷酸聚合反应的聚合酶，以及四种化合物，从而形成含有溶液相和固相的反应体系；其中，所述四种化合物分别为核苷酸A、(T/U)、C和G的衍生物，并且具有碱基互补配对能力；并且，所述四种化合物的核糖或脱氧核糖的3'位置处的羟基(-OH)被保护基团保护；并且，
    第一化合物连接有第一分子标记，
    第二化合物连接有第二分子标记，
    第三化合物连接有第一分子标记和第二分子标记，或部分第三化合物连接有第一分子标记且另外一部分第三化合物连接有第二分子标记，
    第四化合物没有连接分子标记；
    (3)使引物退火至待测序的核酸分子上，所述引物作为起始的生长的核酸链，与所述待测序的核酸分子一起形成连接于支持物上的双链体；
    (4)在允许聚合酶进行核苷酸聚合反应的条件下，使用聚合酶进行核苷酸聚合反应，从而将所述四种化合物中的一种并入生长的核酸链的3'端；
    (5)移除前一步骤的反应体系的溶液相，保留连接于支持物上的双链体，并加入 两种不同的荧光素酶，进行结合反应，所述两种荧光素酶分别能够特异性结合所述第一分子标记和第二分子标记；
    (6)用洗脱缓冲液去除未结合的荧光素酶；
    (7)加入第一荧光素酶的底物，同时检测荧光信号；
    (8)移除上一步反应的溶液；
    (9)加入第二荧光素酶的底物，同时检测荧光信号；
    (10)移除上一步反应的溶液；
    (11)去除各个核苷酸的分子标记和3’保护基团；
    (12)任选地移除上一步反应的溶液；
    (13)任选地重复步骤(3)-(12)或(3)-(9)一次或多次，从而获得所述核酸分子的序列信息。
  4. 根据权利要求1或2所述的方法，其包括以下步骤：
    (1)提供连接于支持物上的待测序的核酸分子，或者将待测序的核酸分子连接于支持物上；
    (2)添加用于起始核苷酸聚合反应的引物，用于进行核苷酸聚合反应的聚合酶，以及四种化合物，从而形成含有溶液相和固相的反应体系；其中，所述四种化合物分别为核苷酸A、(T/U)、C和G的衍生物，并且具有碱基互补配对能力；并且，所述四种化合物的核糖或脱氧核糖的3'位置处的羟基(-OH)被保护基团保护；并且，
    第一化合物连接有第一分子标记，
    第二化合物连接有第二分子标记，
    第三化合物连接有第一分子标记和第二分子标记，或部分第三化合物连接有第一分子标记且另外一部分第三化合物连接有第二分子标记，
    第四化合物没有连接分子标记；
    (3)使引物退火至待测序的核酸分子上，所述引物作为起始的生长的核酸链，与所述待测序的核酸分子一起形成连接于支持物上的双链体；
    (4)在允许聚合酶进行核苷酸聚合反应的条件下，使用聚合酶进行核苷酸聚合反应，从而将所述四种化合物中的一种并入生长的核酸链的3'端；
    (5)移除前一步骤的反应体系的溶液相，保留连接于支持物上的双链体，并加入第一荧光素酶，进行结合反应，所述第一荧光素酶能够特异性结合所述第一分子标记；
    (6)用洗脱缓冲液去除未结合的第一荧光素酶；
    (7)加入第一荧光素酶的底物，同时检测荧光信号；
    (8)移除上一步反应的溶液；
    (9)加入第二荧光素酶,进行结合反应，所述第二荧光素酶能够特异性结合所述第二分子标记；
    (10)用洗脱缓冲液去除未结合的第二荧光素酶；
    (11)加入第二荧光素酶的底物，同时检测荧光信号；
    (12)通入洗脱缓冲液，移除上一步反应的溶液；
    (13)任选地去除各个核苷酸的分子标记；
    (14)任选地重复步骤(3)-(13)或(3)-(11)一次或多次，从而获得所述核酸分子的序列信息。
  5. 根据权利要求3或4所述的方法，其所述的第一荧光素酶和第二荧光素酶含有不同的荧光素酶。
  6. 根据权利要求1或2所述的方法，其包括以下步骤：
    (1)提供连接于支持物上的待测序的核酸分子，或者将待测序的核酸分子连接于支持物上；
    (2)添加用于起始核苷酸聚合反应的引物，用于进行核苷酸聚合反应的聚合酶，以及四种化合物，从而形成含有溶液相和固相的反应体系；其中，所述四种化合物分别为核苷酸A、(T/U)、C和G的衍生物，并且具有碱基互补配对能力；并且，所述四种化合物的核糖或脱氧核糖的3'位置处的羟基(-OH)被保护基团保护；并且，
    第一化合物连接有第一分子标记，
    第二化合物连接有第二分子标记，
    第三化合物连接有第一分子标记和第二分子标记，或部分第三化合物连接有第一分子标记且另外一部分第三化合物连接有第二分子标记，
    第四化合物没有连接分子标记；
    (3)使引物退火至待测序的核酸分子上，所述引物作为起始的生长的核酸链，与所述待测序的核酸分子一起形成连接于支持物上的双链体；
    (4)在允许聚合酶进行核苷酸聚合反应的条件下，使用聚合酶进行核苷酸聚合反应，从而将所述四种化合物中的一种并入生长的核酸链的3'端；
    (5)移除前一步骤的反应体系的溶液相，保留连接于支持物上的双链体，并加入第一荧光素酶，进行结合反应，所述第一荧光素酶能够特异性结合所述第一分子标记；
    (6)用洗脱缓冲液去除未结合的第一荧光素酶；
    (7)加入荧光素酶的底物，同时检测荧光信号；
    (8)移除上一步反应的溶液；
    (9)加入使荧光素酶变性的试剂；
    (10)移除上一步反应的溶液；
    (11)加入第二荧光素酶,进行结合反应，所述第二荧光素酶能够特异性结合所述第二分子标记；
    (12)用洗脱缓冲液去除未结合的第二荧光素酶；
    (13)加入荧光素酶的底物，同时检测荧光信号；
    (14)任选地移除上一步反应的溶液；
    (15)任选地去除各个核苷酸的分子标记和3’保护基团；
    (16)任选地重复步骤(3)-(15)或(3)-(13)一次或多次，从而获得所述核酸分子的序列信息。
  7. 根据权利要求6所述的方法，其所述的第一荧光素酶和第二荧光素酶可以含有相同的荧光素酶。
  8. 根据权利要求1-7任一项所述的方法，其中所述第一分子标记和第二分子标记选自生物素、地高辛、N3G或FITC，所述第一荧光素酶和第二荧光素酶分别经链霉亲和素、地高辛抗体、N3G抗体或FITC抗体标记。
  9. 用于对多核苷酸进行测序的试剂盒，其包含：
    (a)四种化合物，所述四种化合物分别为核苷酸A、(T/U)、C和G的衍生物，并且具有碱基互补配对能力；并且，所述四种化合物的核糖或脱氧核糖的3'位置处的羟基(-OH)被保护基团保护；并且，
    第一化合物连接有第一分子标记，
    第二化合物连接有第二分子标记，
    第三化合物连接有第一分子标记和第二分子标记，或部分第三化合物连接有第一分子标记且另外一部分第三化合物连接有第二分子标记，
    第四化合物没有连接分子标记；和
    (b)两种不同的荧光素酶，两种荧光素酶分别能够特异性结合所述第一分子标记和第二分子标记。
  10. 根据权利要求9所述的试剂盒，其所述的两种不同的荧光素酶可以含有相同的荧光素酶，也可以含有不同的荧光素酶。
  11. 根据权利要求9或10所述的试剂盒，其中所述第一分子标记和第二分子标记选自生物素、地高辛、N3G或FITC，所述第一荧光素酶和第二荧光素酶分别经链霉亲和素、地高辛抗体、N3G抗体或FITC抗体标记。
  12. 根据权利要求9-11任一项所述的试剂盒，其还包含：用于从样品中提取核酸分子的试剂和/或装置；用于预处理核酸分子的试剂；用于连接待测序的核酸分子的支持物；用于将待测序的核酸分子与支持物连接(例如，共价或非共价连接)的试剂；用于起始核苷酸聚合反应的引物；用于进行核苷酸聚合反应的聚合酶；一种或多种缓冲溶液；一种或多种洗涤溶液；或其任何组合。
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