A method for analyzing the sequence of a target polynucleotide
By performing the polymerase reaction during signal acquisition in sequencing-by-synthesis technology, and using a mixture of labeled and unlabeled nucleotides with protecting groups, the problem of incomplete multicopy synthesis is solved, resulting in more efficient sequencing and longer read lengths.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- MGI TECH CO LTD
- Filing Date
- 2021-09-07
- Publication Date
- 2026-06-23
AI Technical Summary
Existing sequencing-by-synthesis technology is prone to incomplete synthesis in multi-copy sequencing, leading to signal distortion, affecting sequencing accuracy and read length, and making it difficult to meet the needs of rapid high-throughput sequencing.
By performing polymerase reactions simultaneously with signal acquisition, using a mixture of labeled and unlabeled nucleotides containing protecting groups, conducting multiple polymerization reactions, and removing the protecting groups after each round of reactions, the signal acquisition solution was optimized to improve polymerization efficiency.
Without increasing the overall reaction time, it improved polymerization efficiency and sequencing accuracy, and extended sequencing read length.
Smart Images
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Abstract
Description
Invention Field
[0001] This invention relates to a method for analyzing the sequence of target polynucleotides, which involves multiple polymerizations of a nucleotide mixture and a polymerase to achieve efficient and complete polymerization, and simultaneously detecting the marker during the polymerization reaction. Furthermore, this invention also relates to a kit for analyzing or sequencing polynucleotides. Background of the Invention
[0003] High-throughput sequencing, with sequencing by synthesis (SBS) being the mainstream commercial method, primarily utilizes DNA polymerase and nucleotides with reversible termination and fluorescent labels to identify DNA sequences. It can sequence hundreds of thousands to millions of DNA molecules simultaneously, offering advantages such as high throughput, fast detection speed, flexibility, and low cost.
[0004] Currently, the specific process of sequencing-by-synthesis involves generating a large amount of DNA template using methods such as amplification or rolling circle replication, then anchoring specific sequencing primers, and simultaneously adding DNA polymerase and fluorescently labeled nucleotides to the reaction system; or simultaneously adding a mixture of DNA polymerase, fluorescently labeled nucleotides, and unmodified nucleotides to the reaction system. The 3'-OH groups of these dNTPs are protected, so only one dNTP can be added at a time. Each time a dNTP is added, the DNA replication reaction stops, followed by fluorescence signal excitation and signal collection. Then, chemical reagents are added to quench the fluorescence signal and remove the 3'-OH protecting group of the dNTP, allowing the next round of sequencing to proceed.
[0005] However, this sequencing technology has extremely high requirements for synthesis efficiency. Incomplete synthesis in multiple copies can cause signal distortion, affecting sequencing accuracy and read length. This is one of the key technical challenges currently facing next-generation sequencing. As demands for faster sequencing times and higher throughput increase, the limited efficiency of polymerases, especially their ability to polymerize fluorescent nucleotides, leads to a decrease in polymerization efficiency, impacting sequencing quality. If this polymerization efficiency cannot keep pace with sequencing speed, incomplete polymerization in multiple copies results in signal distortion during sequencing, thus limiting read length and accuracy.
[0006] Therefore, there is a need to provide a method for sequencing polynucleotides that achieves more efficient sequencing results without increasing the overall reaction time, so as to improve the read length and accuracy of sequencing. Summary of the Invention
[0007] This invention provides a scheme for polymerizing nucleotides while acquiring signals, thereby improving polymerization efficiency and achieving higher polymerization efficiency without changing the overall reaction time.
[0008] Therefore, in a first aspect, this application provides a method for analyzing the sequence of a target polynucleotide, comprising:
[0009] (a) Provide target polynucleotides;
[0010] (b) Under conditions that allow hybridization or annealing, the target polynucleotide is contacted with a primer to form a partial double strand comprising the target polynucleotide and a primer used as a growth chain;
[0011] (c) Under conditions allowing the polymerase to perform nucleotide polymerization, the partially duplexed strand is contacted with a mixture of polymerase and a first nucleotide to extend the growth chain, wherein the first nucleotide mixture contains at least one labeled nucleotide.
[0012] Each nucleotide in the first nucleotide mixture contains a protective group (e.g., a protective group attached by a 2' or 3' oxygen atom) in its ribose or deoxyribose portion that can block the extension of the nucleic acid chain.
[0013] (d) Under conditions that allow the polymerase to perform nucleotide polymerization, the product of the previous step is contacted with the polymerase and a mixture of the second nucleotides to extend the growth chain, wherein the second nucleotide mixture contains at least one (e.g., one, two, three or four) unlabeled nucleotides, or irreversible blocking nucleotides, or a combination of the unlabeled nucleotides and irreversible blocking nucleotides.
[0014] Each unlabeled nucleotide in the second nucleotide mixture contains a protective group (e.g., a protective group attached by a 2' or 3' oxygen atom) in its ribose or deoxyribose portion that can block the extension of the nucleic acid chain.
[0015] Furthermore, a photographic polymerization solution is provided and contacted with a second nucleotide mixture, and the presence of a marker in the product of step (c) is detected by the photographic polymerization reaction;
[0016] (e) Remove protecting groups and markers contained in the product of the previous step;
[0017] (f) Optionally repeat steps (c)-(e) once or more;
[0018] Thus, the sequence information of the target polynucleotide is obtained.
[0019] In some embodiments, the photographic polymerization solution comprises the following reagents: nucleic acid polymerase, buffer reagent, surfactant, and reagents for reducing nucleic acid damage under laser photography.
[0020] In some embodiments, the photographic polymerization solution comprises the following reagents: nucleic acid polymerase, buffer reagent, reagent for nucleic acid hybridization (e.g., sodium chloride), and Mg... 2+ Salts, surfactants, and reagents for reducing nucleic acid damage under laser imaging.
[0021] In some embodiments, in step (c), the extension is a template extension using a target polynucleotide. In some embodiments, the extension is an extension of one nucleotide.
[0022] In some embodiments, the first nucleotide mixture comprises a first nucleotide labeled with a first marker, a second nucleotide labeled with a second marker, a third nucleotide labeled with a third marker, and a fourth nucleotide labeled with a fourth marker or an unlabeled fourth nucleotide, or a first nucleotide labeled with a first marker, a second nucleotide labeled with a second marker, a third nucleotide co-labeled with the first and second markers, and an unlabeled fourth nucleotide.
[0023] In some embodiments, in step (d), the second nucleotide mixture comprises an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.
[0024] In some embodiments, the second nucleotide mixture also contains an unlabeled fourth nucleotide.
[0025] In some embodiments, the second nucleotide mixture contains at least one irreversible blocking nucleotide.
[0026] In some embodiments, in step (d), the second nucleotide mixture comprises an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and a first irreversible blocking nucleotide, a second irreversible blocking nucleotide, a third irreversible blocking nucleotide, and a fourth irreversible blocking nucleotide.
[0027] In some embodiments, the second nucleotide mixture also includes an unlabeled fourth nucleotide.
[0028] In some implementations, the first marker, the second marker, the third marker, and the fourth marker may be independently the same or different.
[0029] In some implementations, the first marker, the second marker, the third marker, and the fourth marker are different.
[0030] In some embodiments, the first, second, third, and fourth markers are luminescent markers (e.g., fluorescent markers).
[0031] In some embodiments, the first, second, third, and fourth markers are each independently selected from coumarin, AlexaFluor, Bodipy, fluorescein, tetramethylrhodamine, phenoxazine, acridine, Cy5, Cy3, AF532, Texas red, and their derivatives.
[0032] In some embodiments, the target polynucleotide comprises DNA, RNA, or any combination thereof. In some embodiments, the extended product of the nucleic acid molecule is DNA.
[0033] In some embodiments, the target polynucleotide is obtained from samples derived from eukaryotes (e.g., animals, plants, fungi), prokaryotes (e.g., bacteria, actinomycetes), viruses, bacteriophages, or any combination thereof.
[0034] In some embodiments, the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are each independently selected from A, T, C, G, and U.
[0035] In some implementations, the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are different.
[0036] In some embodiments, the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are A, T, C, and G, respectively.
[0037] In some embodiments, the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are each independently selected from A, T, C, G, and U.
[0038] In some implementations, the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are all different.
[0039] In some embodiments, the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are A, T, C, and G, respectively.
[0040] In some embodiments, the irreversible blocking nucleotide is a dideoxynucleotide.
[0041] In some embodiments, when the first nucleotide mixture comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and a fourth nucleotide labeled with a fourth label; the second nucleotide mixture comprises an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and an unlabeled fourth nucleotide; or,
[0042] When the first nucleotide mixture contains a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and an unlabeled fourth nucleotide, or a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide co-labeled with the first and second labels, and an unlabeled fourth nucleotide, the second nucleotide mixture contains an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.
[0043] In some implementations, the light signal is detected by taking a picture while the polymerization reaction in step (d) is being carried out.
[0044] In some embodiments, the buffer reagent is selected from Trizma base, tris(hydroxymethyl)methylglycine (TRICINE), N,N-dihydroxyethylglycine (BICINE), tris(hydroxymethyl)propanesulfonic acid (TAPS), or any combination thereof.
[0045] In some embodiments, the reagent used for nucleic acid hybridization is selected from sodium chloride, potassium chloride, potassium acetate, or any combination thereof.
[0046] In some embodiments, the Mg-containing 2+ The salt is selected from magnesium sulfate, magnesium chloride, magnesium nitrate, magnesium chromate, or any combination thereof.
[0047] In some embodiments, the surfactant is selected from 10% Tween 20, Tween 80, lauryl polyoxyethylene ether (Brij-35), polyethylene glycol octylphenyl ether (Trinton X-100), ethyl phenyl polyethylene glycol (NP-40), sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide (CTAB), hexadecyltrimethylammonium chloride (CTAC), or any combination thereof.
[0048] In some embodiments, the reagent used to reduce nucleic acid damage under laser imaging is selected from sodium ascorbate, dithiothreitol (DTT), 6-hydroxy-2,5,7,8-tetramethyltryptane-2-carboxylic acid (Trolox), ascorbic acid derivatives (e.g., sodium ascorbate phosphate, magnesium ascorbate phosphate, ascorbate glucoside, ascorbate palmitate, tetraethyldecanol ascorbate, ascorbate methylsilanol pectate, 3-O-ethyl ascorbic acid), reduced glutathione (GSH), N,N'-dimethylthiourea (DMTU), ammonium pyrrolidine dithiocarbamate (APDTC), spermidine, spermidine trihydrochloride, uric acid, sodium pyruvate, L-cysteine, β-mercaptoethylamine, cystamine, or any combination thereof.
[0049] In some embodiments, the reagent used to reduce nucleic acid damage under laser imaging is selected from sodium ascorbate or reduced glutathione.
[0050] In one embodiment, the reagent for reducing nucleic acid damage under laser imaging is selected from reduced glutathione (GSH) and dithiothreitol (DTT) and combinations thereof.
[0051] In one embodiment, the reagent used to reduce nucleic acid damage under laser imaging is reduced glutathione (GSH).
[0052] In a second aspect, this application provides a kit comprising:
[0053] (a) A first nucleotide mixture, the first nucleotide mixture comprising at least one nucleotide labeled with a marker,
[0054] Each nucleotide in the first nucleotide mixture contains a protective group (e.g., a protective group attached by a 2' or 3' oxygen atom) in its ribose or deoxyribose portion that can block the extension of the nucleic acid chain.
[0055] (b) A second nucleotide mixture comprising at least one (e.g., one, two, three or four) unlabeled nucleotide, or irreversible blocking nucleotide, or a combination of said unlabeled nucleotide and irreversible blocking nucleotide;
[0056] Each unlabeled nucleotide in the second nucleotide mixture contains a protective group (e.g., a protective group attached by a 2' or 3' oxygen atom) in its ribose or deoxyribose portion that can block the extension of the nucleic acid chain.
[0057] (c) Photographing the polymer solution.
[0058] In some embodiments, the photographic polymerization solution contains the following reagents: nucleic acid polymerase, buffer reagent, surfactant, and reagents for reducing nucleic acid damage under laser photography.
[0059] In some embodiments, the photographic polymerization solution comprises the following reagents: nucleic acid polymerase, buffer reagent, reagent for nucleic acid hybridization (e.g., sodium chloride), and Mg... 2+ Salts, surfactants, are reagents used to reduce nucleic acid damage under laser imaging.
[0060] In some embodiments, the kit further includes one or more of the following: primers that are wholly or partially complementary to the polynucleotide, nucleic acid amplification buffer, working buffer of an enzyme (e.g., nucleic acid polymerase), water, or any combination thereof.
[0061] In some embodiments, the kit may further include one or more of the following: sequencing slides, reagents for removing protecting groups and markers from nucleotides.
[0062] In some implementations, the kit is used for the analysis of polynucleotides.
[0063] In some implementations, the kit is used for sequencing polynucleotides.
[0064] In some embodiments, the first nucleotide mixture comprises a first nucleotide labeled with a first marker, a second nucleotide labeled with a second marker, a third nucleotide labeled with a third marker, and a fourth nucleotide labeled with a fourth marker or an unlabeled fourth nucleotide, or a first nucleotide labeled with a first marker, a second nucleotide labeled with a second marker, a third nucleotide co-labeled with the first and second markers, and an unlabeled fourth nucleotide.
[0065] In some embodiments, when the first nucleotide mixture comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and a fourth nucleotide labeled with a fourth label; the second nucleotide mixture comprises an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and an unlabeled fourth nucleotide; or,
[0066] When the first nucleotide mixture contains a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and an unlabeled fourth nucleotide, or a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide co-labeled with the first and second labels, and an unlabeled fourth nucleotide, the second nucleotide mixture contains an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.
[0067] In some embodiments, the second nucleotide mixture comprises an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and a first irreversible blocking nucleotide, a second irreversible blocking nucleotide, a third irreversible blocking nucleotide, and a fourth irreversible blocking nucleotide.
[0068] In some embodiments, the second nucleotide mixture further comprises an unlabeled fourth nucleotide.
[0069] In some implementations, the first marker, the second marker, the third marker, and the fourth marker may each be independently the same or different.
[0070] In some implementations, the first marker, the second marker, the third marker, and the fourth marker are different.
[0071] In some embodiments, the first, second, third, and fourth markers are luminescent markers (e.g., fluorescent markers).
[0072] In some embodiments, the first, second, third, and fourth markers are each independently selected from coumarin, AlexaFluor, Bodipy, fluorescein, tetramethylrhodamine, phenoxazine, acridine, Cy5, Cy3, AF532, Texas red, and their derivatives.
[0073] In some embodiments, the target polynucleotide comprises DNA, RNA, or any combination thereof. In some embodiments, the extended product of the nucleic acid molecule is DNA.
[0074] In some embodiments, the target polynucleotide is obtained from samples derived from eukaryotes (e.g., animals, plants, fungi), prokaryotes (e.g., bacteria, actinomycetes), viruses, bacteriophages, or any combination thereof.
[0075] In some embodiments, the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are each independently selected from A, T, C, G, and U.
[0076] In some implementations, the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are different.
[0077] In some embodiments, the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are A, T, C, and G, respectively.
[0078] In some embodiments, the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are each independently selected from A, T, C, G, and U.
[0079] In some implementations, the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are all different.
[0080] In some embodiments, the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are A, T, C, and G, respectively.
[0081] In some embodiments, the irreversible blocking nucleotide is a dideoxynucleotide.
[0082] In some implementations, the light signal is detected by taking a picture while the polymerization reaction in step (d) is being carried out.
[0083] In some embodiments, the buffer reagent is selected from Trizma base, tris(hydroxymethyl)methylglycine (TRICINE), N,N-dihydroxyethylglycine (BICINE), tris(hydroxymethyl)propanesulfonic acid (TAPS), or any combination thereof.
[0084] In some embodiments, the reagent used for nucleic acid hybridization is selected from sodium chloride, potassium chloride, or any combination thereof.
[0085] The Mg-containing component described in some embodiments 2+ The salt is selected from magnesium sulfate, magnesium chloride, magnesium nitrate, magnesium chromate, or any combination thereof.
[0086] In some embodiments, the surfactant is selected from 10% Tween 20, Tween 80, lauryl polyoxyethylene ether (Brij-35), polyethylene glycol octylphenyl ether (Trinton X-100), ethyl phenyl polyethylene glycol (NP-40), sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide (CTAB), hexadecyltrimethylammonium chloride (CTAC), or any combination thereof.
[0087] In some embodiments, the reagent used to reduce nucleic acid damage under laser imaging is selected from sodium ascorbate, dithiothreitol (DTT), 6-hydroxy-2,5,7,8-tetramethyltryptane-2-carboxylic acid (Trolox), ascorbic acid derivatives (e.g., sodium ascorbate phosphate, magnesium ascorbate phosphate, ascorbate glucoside, ascorbate palmitate, tetraethyldecanol ascorbate, ascorbate methylsilanol pectate, 3-O-ethyl ascorbic acid), reduced glutathione (GSH), N,N'-dimethylthiourea (DMTU), ammonium pyrrolidine dithiocarbamate (APDTC), spermidine, spermidine trihydrochloride, uric acid, sodium pyruvate, L-cysteine, β-mercaptoethylamine, cystamine, or any combination thereof.
[0088] In some embodiments, the reagent used to reduce nucleic acid damage under laser imaging is sodium ascorbate or reduced glutathione (GSH).
[0089] In one embodiment, the reagent for reducing nucleic acid damage under laser imaging is selected from reduced glutathione (GSH) and dithiothreitol (DTT) and combinations thereof.
[0090] In one embodiment, the reagent used to reduce nucleic acid damage under laser imaging is reduced glutathione (GSH).
[0091] Terminology Definition
[0092] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents, applications, and other publications mentioned herein are incorporated herein by reference in their entirety. If any definition presented herein conflicts with or is inconsistent with the definitions set forth in the patents, applications, and other publications incorporated herein by reference, the definitions set forth herein shall prevail.
[0093] As used herein, the term "polynucleotide" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or analogues 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) (e.g., genomic DNA, PCR and amplification products, etc.), or from single-stranded DNA (ssDNA) or RNA and can be converted to dsDNA, and vice versa. The exact sequence of a polynucleotide molecule can be known or unknown. Exemplary examples of polynucleotides include: genes or gene fragments (e.g., probes, primers, EST, or SAGE tags), genomic DNA, genomic DNA fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonuclease, cDNA, recombinant polynucleotides, synthetic polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers, or amplified copies of any of the above sequences.
[0094] Polynucleotides can include nucleotides or nucleotide analogues. Nucleotides typically contain a sugar (such as ribose or deoxyribose), a base, and at least one phosphate group. Nucleotides can be baseless (i.e., lacking a base). Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include, for example, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (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), and deoxyadenosine monophosphate (dAMP). Deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP). Nucleotide analogs containing modified bases may also be used in the methods described herein. Exemplary modified bases that can be included in polynucleotides, whether having a natural backbone or a similar structure, include, for example, inosine, xathanine, hypoxanthine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymidine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5 -Propynouracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymidine, 5-uracil, 4-thionuracil, 8-halogenated adenine or guanine, 8-aminoadenine or guanine, 8-thionated adenine or guanine, 8-thioalkyladenine or guanine, 8-hydroxyadenine or guanine, 5-halogenated uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazoguanine, 7-deazoadenine, 3-deazoguanine, 3-deazoadenine, etc. As is known in the art, certain nucleotide analogs cannot be introduced into polynucleotides, for example, nucleotide analogs such as adenosine 5'-phosphorylsulfate.
[0095] Generally, nucleotides include nucleotides A, C, G, T, or U. As used herein, the term "nucleotide A" refers to a nucleotide containing adenine (A) or its modifications or analogues, such as ATP or dATP. "Nucleotide G" refers to a nucleotide containing guanine (G) or its modifications or analogues, such as GTP or dGTP. "Nucleotide C" refers to a nucleotide containing cytosine (C) or its modifications or analogues, such as CTP or dCTP. "Nucleotide T" refers to a nucleotide containing thymine (T) or its modifications or analogues, such as TTP or dTTP. "Nucleotide U" refers to a nucleotide containing uracil (U) or its modifications or analogues, such as UTP or dUTP.
[0096] As used herein, the term "dideoxynucleotide" refers to a nucleotide deoxygenated at the 2' and 3' carbons of the ribose, also known as 2',3'-dideoxynucleotide. Generally, dideoxynucleotides include dideoxynucleotides A, C, G, T, or U. The term "dideoxynucleotide A" refers to a dideoxynucleotide containing adenine (A) or an analogue, such as ddATP. "Dideoxynucleotide G" refers to a dideoxynucleotide containing guanine (G) or an analogue, such as ddGTP. "Dideoxynucleotide C" refers to a dideoxynucleotide containing cytosine (C) or an analogue, such as ddCTP. "Dideoxynucleotide T" refers to a dideoxynucleotide containing thymine (T) or an analogue, such as ddTP. "Dideoxynucleotide U" refers to a dideoxynucleotide containing uracil (U) or an analogue, such as ddUTP. As used in this article, ddNTP is one of dideoxyadenosine triphosphate (ddATP), dideoxyguanosine triphosphate (ddGTP), dideoxycytidine triphosphate (ddCTP), dideoxyuridine triphosphate (ddUTP), dideoxythymidine triphosphate (ddTTP), or a combination of two or more of them, wherein dideoxyuridine triphosphate and dideoxythymidine triphosphate do not occur simultaneously.
[0097] As used in this article, the term "marker" refers to a group that can emit a light-emitting signal under certain conditions.
[0098] As used herein, the term "luminescent label" refers to any substance that, when excited by a suitable excitation wavelength, emits fluorescence at a specific emission wavelength. Such luminescent labels can be chemiluminescent labels, for example, selected from biochemiluminescent labels that elicit different luminescence kinetics and any combination thereof, such as luciferases that elicit different luminescence kinetics and any combination thereof; such luminescent labels can be, for example, fluorophores, such as those selected from coumarin, AlexaFluor, Bodipy, luciferin, tetramethylrhodamine, phenoxazine, acridine, Cy5, Cy3, EF700, AF532, Texas Red and its derivatives, etc.
[0099] As used herein, the term "protecting group" refers to a group that prevents polymerase (which incorporates a nucleotide containing that group into the polynucleotide chain being synthesized) from catalyzing the incorporation of another nucleotide after the incorporation of the nucleotide containing that group into the polynucleotide chain being synthesized. Such a protecting group is also referred to herein as a 3'-OH protecting group. A nucleotide containing such a protecting group is also referred to herein as a 3'-blocking nucleotide. The protecting group can be any suitable group that can be added to a nucleotide, as long as the protecting group prevents the incorporation of additional nucleotide molecules into the polynucleotide chain and is readily removable from the sugar moiety of the nucleotide without damaging the polynucleotide chain. Furthermore, the nucleotide modified with the protecting group needs to be resistant to polymerase or other suitable enzymes for incorporating the modified nucleotide into the polynucleotide chain. Therefore, an ideal protecting group exhibits long-term stability, can be efficiently incorporated by polymerase, prevents secondary or further incorporation of nucleotides, and can be removed under mild conditions, preferably aqueous conditions, without damaging the polynucleotide structure.
[0100] The prior art has described a variety of protecting groups that conform to the above descriptions. For example, WO 91 / 06678 discloses 3'-OH protecting groups including esters and ethers, -F, -NH2, -OCH3, -N3, -OPO3, -NHCOCH3, 2-nitrophenyl carbonate, 2,4-sulfenyldinitro, and tetrahydrofuran ether. Metzker et al. (Nucleic Acids Research, 22(20):4259-4267, 1994) disclosed the synthesis and application of eight 3'-modified 2-deoxyribonucleoside 5'-triphosphates (3'-modified dNTPs). WO2002 / 029003 describes the use of allyl protecting groups to cap 3'-OH groups on DNA growth chains in polymerase reactions. Preferably, various protecting groups reported in international applications WO2014139596 and WO2004 / 018497 can be used, including, for example, those in WO2014139596. Figure 1The protecting groups exemplified in A and the 3' hydroxyl protecting groups (i.e., protecting groups) defined in the claims, and, for example, those in WO2004 / 018497 Figure 3 and 4 The protecting groups exemplified in the examples and those defined in the claims. All of the above references are incorporated herein by reference in their entirety.
[0101] As used herein, the term "reversible blocking group" refers to a group that, when incorporated into a synthesizing polynucleotide chain, prevents the polymerase from proceeding to the next round of polymerization, thus terminating the polymerization reaction. In this case, in each round of polymerization, exactly one base is incorporated into the growing nucleic acid chain. Furthermore, the group can be removed, allowing the growing nucleic acid chain to proceed to the next round of polymerization, introducing another base. Examples of reversible blocking groups include H in the 3'-OH group being replaced by the following groups: ester, ether, -F, -NH2, -OCH3, -N3, -OPO3, -NHCOCH3, 2-nitrophenyl carbonate, 2,4-sulfenyldinitro and tetrahydrofuran ether -CH2-CH=CH2, SS, or blocking the base with a large sterically hindered group and a fluorescent group, with fluorescence linked by SS.
[0102] As used herein, the term "irreversible blocking nucleotide" refers to a nucleotide that, once incorporated into a synthesizing polynucleotide chain, prevents subsequent nucleotides from being incorporated into the chain due to its blocking effect. This blocking effect is irreversible. Irreversible blocking nucleotides typically contain dideoxynucleotides, or nucleotides whose 3'-OH is replaced by groups such as methoxy (3'-OMe, i.e., 3'-OCH3), azide (3'-N3), and ethoxy (3'-OEt, i.e., 3'-OCH2CH3).
[0103] Beneficial technical effects of the present invention
[0104] In sequencing, this invention provides a scheme for polymerizing nucleotides simultaneously with signal acquisition to achieve higher polymerization efficiency. The main method involves optimizing the solution during signal acquisition so that an additional round of polymerization reaction occurs during signal acquisition. The polymerized nucleotides are nucleotides without fluorescent modification but with blocking groups, preferably nucleotides modified with reversible blocking groups, followed by irreversible blocking nucleotides, or a mixture of both. This improves polymerization efficiency, significantly shortens the overall sequencing reaction time, and achieves even higher polymerization efficiency. Attached Figure Description
[0105] Figure 1 The flowcharts for simultaneous synthesis and sequencing of E. coli DNA are shown in the control and experimental groups.
[0106] Figure 2 The results show the Q30 (%) percentage for each cycle in both the control group 1 and the experimental group. The horizontal axis represents the number of sequencing cycles, and the vertical axis represents the Q30 (%) percentage for each cycle.
[0107] Figure 3 The results show the sequencing error rate (%) percentage for each cycle in both the control group 1 and the experimental group. The horizontal axis represents the number of sequencing cycles, and the vertical axis represents the sequencing error rate (%) percentage for each cycle.
[0108] Figure 4 The results show the percentage of lag (%) for each cycle in both the control group 1 and the experimental group. The horizontal axis represents the number of sequencing cycles, and the vertical axis represents the percentage of lag (%) for each cycle.
[0109] Figure 5 The results show the Q30 (%) percentage for each cycle in control group 2 and experimental group 4. The horizontal axis represents the sequencing cycle number, and the vertical axis represents the Q30 (%) percentage for each cycle.
[0110] Figure 6 The results show the percentage of sequencing error rate (%) for each cycle in control group 2 and experimental group 4. The horizontal axis represents the number of sequencing cycles, and the vertical axis represents the percentage of sequencing error rate (%) for each cycle.
[0111] Figure 7 The results show the percentage of lag (%) for each cycle in control group 2 and experimental group 4. The horizontal axis represents the number of sequencing cycles, and the vertical axis represents the percentage of lag (%) for each cycle. Example
[0112] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).
[0113] Unless otherwise specified, the molecular biology experimental methods used in this invention are substantially the same as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Susubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Those skilled in the art will appreciate that the examples are described by way of illustration and are not intended to limit the scope of the invention.
[0114] 1. Key equipment used in this embodiment: MGISEQ-2000RS sequencer, MGIDL-200H loader, MGISEQ-2000RS sequencing slides, MGISEQ-2000RS high-throughput sequencing reagent kit, MGISEQ-200RS sequencer, MGISEQ-200RS high-throughput sequencing reagent kit, MGISEQ-200RS sequencing slides.
[0115] 2. The key reagents used in this embodiment are shown in Table 1 below:
[0116] Table 1. Reagents used
[0117] Reagent Name brand Item number MGISEQ-2000RS High-Throughput Sequencing Kit MGI 1000012552 MGISEQ-200RS High-Throughput Sequencing Kit MGI 1000019841 Cold dATP BGI 01CATP000-10ml Cold dTTP BGI 01CTTP000-10ml Cold dGTP BGI 01CGTP000-10ml Cold dCTP BGI 01CCTP000-10ml ddATP BGI 01DATP000-1ml ddTTP BGI 01DTTP000-1ml ddGTP BGI 01DGTP000-1ml ddCTP BGI 01DCTP000-1ml
[0118] Example 1
[0119] In this embodiment, various reagents are prepared in advance.
[0120] 1) Preparation of nucleotide mixture 1
[0121] As shown in Table 2 below, nucleotide mixture 1 has only reversible blocking groups, where A, T, G, and C are adenine nucleotide, thymine nucleotide, guanine nucleotide, and cytosine nucleotide, respectively.
[0122] Table 2: Nucleotide Mixture 1
[0123] Reagent Name Final concentration (nmol / L) Cold dATP 200 Cold dTTP 200 Cold dGTP 200 Cold dCTP 200
[0124] Among them, Cold dATP refers to adenine nucleotides modified only with reversible blocking groups, Cold dTTP refers to thymine nucleotides modified only with reversible blocking groups, Cold dGTP refers to guanine nucleotides modified only with reversible blocking groups, and Cold dCTP refers to cytosine nucleotides modified only with reversible blocking groups.
[0125] 2) Preparation of nucleotide mixture 2
[0126] As shown in Table 3 below, all nucleotide mixtures 2 are dideoxynucleotides, where ddATP refers to adenosine triphosphate dideoxynucleotide, ddTTP refers to thymine triphosphate dideoxynucleotide, ddGTP refers to guanine triphosphate dideoxynucleotide, and ddCTP refers to cytosine triphosphate dideoxynucleotide.
[0127] Table 3: Nucleotide Mixture 2
[0128] Reagent Name Final concentration (nmol / L) ddATP 200 ddTTP 200 ddGTP 200 ddTTP 200
[0129] 3) Preparation of nucleotide mixture 3
[0130] As shown in Table 4 below, nucleotide mixture 3 has only reversible blocking groups, where A, T, and C are adenine nucleotide, thymine nucleotide, and cytosine nucleotide, respectively.
[0131] Table 4: Nucleotide Mixture 3
[0132] Reagent Name Final concentration (nmol / L) Cold dATP 200 Cold dTTP 200 Cold dCTP 200
[0133] Among them, Cold dATP refers to adenine nucleotides modified only with reversible blocking groups, Cold dTTP refers to thymine nucleotides modified only with reversible blocking groups, and Cold dCTP refers to cytosine nucleotides modified only with reversible blocking groups.
[0134] 4) Preparation of photographic polymerization solution 1
[0135] Prepare the photographic polymerization solution 1 according to Table 5, and adjust the pH to 8.7 with NaOH and HCl. Mix well and set aside.
[0136] Table 5: Photopolymerization solution 1
[0137] Reagent Name Working concentration Trizma base 0.05M Sodium chloride 0.01M Magnesium sulfate 3mM 10% Tween-20 0.05％ Reduced glutathione 0.1M Nucleotide Mixture 1 3uM DNA polymerase 0.01mg / ml Add water to a final volume of 50ml
[0138] 5) Preparation of polymer solution 2 for photography
[0139] Prepare the photographic polymerization 2 according to Table 6, and adjust the pH to 8.7 with NaOH and HCl, then mix well and set aside.
[0140] Table 6: Photograph Polymerization Solution 2
[0141]
[0142]
[0143] 6) Preparation of photographic polymerization solution 3
[0144] Prepare the photographic polymerization solution 3 according to Table 7, and adjust the pH to 8.7 with NaOH and HCl. Mix well and set aside.
[0145] Table 7: Photopolymerization solution 3
[0146] Reagent Name Working concentration Trizma base 0.05M Sodium chloride 0.01M Magnesium sulfate 3mM 10% Tween-20 0.05％ Reduced glutathione 0.1M Nucleotide Mixture 1 2uM Nucleotide Mixture 2 1uM DNA polymerase 0.01mg / ml Add water to a final volume of 50ml
[0147] 7) Preparation of photographic solution
[0148] Prepare the photographic solution according to Table 8, and adjust the pH to 8.2 with NaOH and HCl. Mix well and set aside.
[0149] Table 8: Photographic Solution
[0150]
[0151]
[0152] 8) Preparation of photographic polymerization solution 4
[0153] Prepare the photographic polymerization solution 4 according to Table 9, and adjust the pH to 8.7 with NaOH and HCl. Mix well and set aside.
[0154] Table 9: Photopolymerization Solution 4
[0155] Reagent Name Working concentration Trizma base 0.05M Sodium chloride 0.01M Magnesium sulfate 3mM 10% Tween-20 0.05％ Reduced glutathione 0.1M Nucleotide mixture 3 3uM DNA polymerase 0.01mg / ml Add water to a final volume of 50ml
[0156] Example 2
[0157] All experiments below used E. coli single-stranded circular DNA as a template and the MGISEQ-2000RS high-throughput sequencing kit to prepare DNA nanospheres and load them onto the chip for subsequent sequencing.
[0158] The fluorescently modified and reversibly blocking nucleotide mixtures mentioned in this embodiment include: dATP-1, which refers to an adenine nucleotide with both reversible blocking group modification and Cy5 fluorescence modification; dTTP-1, which refers to a thymine nucleotide with both reversible blocking group modification and ROX fluorescence modification; dGTP-1, which refers to a guanine nucleotide with both reversible blocking group modification and Cy3 fluorescence modification; and dCTP-1, which refers to a cytosine nucleotide with both reversible blocking group modification and EF700 fluorescence modification. (The fluorescently modified and reversibly blocking nucleotide mixtures can vary depending on the platform; for example, a simple reversible blocking group nucleotide or other modification types can be added to this nucleotide mixture.)
[0159] Control group 1: Using the MGISEQ-2000RS high-throughput sequencing kit, the reagent in well #10 of the kit was removed and replaced with the imaging solution prepared above, and the sequencing was performed according to the instructions. Figure 1 The experimental procedure involved SE50 sequencing on the MGISEQ-2000RS sequencing platform. In short, the process involved sequentially polymerizing a mixture of fluorescently modified and reversibly blocked nucleotides on the MGISEQ-2000RS platform, followed by elution of free nucleotides using elution reagents, signal acquisition in imaging solution, removal of protecting groups using excision reagents, and washing with elution reagents. The Q30 decrease and sequencing error rate curves for each cycle were then analyzed to evaluate sequencing quality.
[0160] Experimental Group 1: Using the MGISEQ-2000RS high-throughput sequencing kit, a mixture of fluorescently modified and reversibly blocked nucleotides was sequentially polymerized on the MGISEQ-2000RS sequencing platform. The free nucleotides were then eluted with elution reagents. The reagent in well #10 of the kit was then removed and replaced with the imaging polymerization solution 1 of this invention. This allows for polymerization leveling during signal acquisition. The polymerized nucleotide mixture at this point contains reversibly blocked nucleotides. SE50 sequencing was performed on the MGISEQ-2000RS sequencing platform following the same experimental procedure as the control group. The Q30 decrease rate for each cycle and the sequencing error rate curve for each cycle were then analyzed to evaluate sequencing quality.
[0161] Experimental Group 2: Using the MGISEQ-2000RS high-throughput sequencing kit, a mixture of fluorescently modified and reversibly blocked nucleotides was sequentially polymerized on the MGISEQ-2000RS sequencing platform. Then, the free nucleotides were eluted with elution reagents. The reagent in well #10 of the kit was removed and replaced with the imaging polymerization solution 2 of this invention. This allows for polymerization leveling during signal acquisition. The polymerized nucleotide mixture at this time is dideoxynucleotide. SE50 sequencing was performed on the MGISEQ-2000RS sequencing platform following the same experimental procedure as the control group. The Q30 decrease rate and sequencing error rate curve for each cycle were then analyzed to evaluate the sequencing quality.
[0162] Experimental Group 3: Using the MGISEQ-2000RS high-throughput sequencing kit, a mixture of fluorescently modified and reversibly blocked nucleotides was sequentially polymerized on the MGISEQ-2000RS sequencing platform. The free nucleotides were then eluted with elution reagents. The reagent in well #10 of the kit was removed and replaced with the imaging polymerization solution 3 of this invention. This allows for polymerization leveling during signal acquisition. The polymerized nucleotide mixture at this point is a mixture of reversibly blocked nucleotides and dideoxynucleotides. SE50 sequencing was performed on the MGISEQ-2000RS sequencing platform following the same experimental procedure as the control group. The Q30 decrease rate for each cycle and the sequencing error rate curve for each cycle were then analyzed to evaluate sequencing quality.
[0163] Result: as follows Figures 2-4 And as shown in Tables 10-12, Figure 2 The results show the Q30 (%) percentage for each sequencing cycle under the above conditions, where the horizontal axis represents the number of sequencing cycles and the vertical axis represents the Q30 (%) percentage for each cycle. Figure 3 The results show the percentage of sequencing error rate (%) under the above conditions, where the horizontal axis represents the number of sequencing cycles and the vertical axis represents the percentage of sequencing error rate (%) for each cycle. Figure 4The results show the percentage of lag (%) for each sequencing cycle under the above conditions. Lag is an indicator used to measure whether the sequencing reaction is complete. It refers to the proportion of some copies that are reflected in the N-1 position when sequencing reaches the N position. The horizontal axis is the number of sequencing cycles, and the vertical axis is the percentage of lag (%) for each cycle.
[0164] The smaller the decrease in Q30 (%) in each cycle, the lower the percentage of sequencing error rate (%), and the lower the increase in lag (%), indicating better and more stable sequencing quality. Table 10 shows that the control group had the largest decrease in Q30 (%) over 50 cycles, worse than all experimental groups. Experimental group 1 was slightly better than experimental group 3, and experimental group 3 was slightly better than experimental group 2. Table 11 shows that the error rate of the control group was significantly higher than that of all experimental groups. Specifically, experimental group 1 was slightly better than experimental group 3, and experimental group 3 was slightly better than experimental group 2. Table 12 shows that the increase in lag (%) over 50 cycles in the control group was significantly higher than that in all experimental groups, while there was no significant difference between the experimental groups.
[0165] Table 10. Percentage of Q30 in each cycle
[0166]
[0167]
[0168]
[0169] Table 11. Error rate (%) for each loop
[0170]
[0171]
[0172] Table 12. Lag (%) for each cycle
[0173]
[0174]
[0175] Example 3
[0176] All experiments below used E. coli single-stranded circular DNA as a template and the MGISEQ-200RS high-throughput sequencing kit to prepare DNA nanospheres and load them onto the chip for subsequent sequencing.
[0177] The fluorescently modified and reversibly blocking nucleotide mixtures mentioned in this embodiment include: dATP-1, which refers to an adenine nucleotide with both reversible blocking group modification and Cy3 and Cy5 fluorescence modification; dTTP-1, which refers to a thymine nucleotide with both reversible blocking group modification and Cy5 fluorescence modification; dGTP-1, which refers to a guanine nucleotide with only reversible blocking group modification; and dCTP-1, which refers to a cytosine nucleotide with both reversible blocking group modification and Cy3 fluorescence modification. (The fluorescently modified and reversibly blocking nucleotide mixtures can vary depending on the platform; for example, a nucleotide with a simple reversible blocking group or other modification types can be added to this nucleotide mixture.)
[0178] Control group 2: Using the MGISEQ-200RS high-throughput sequencing kit, the reagent in well #18 of the kit was removed and replaced with the imaging solution prepared above, and the sequencing was performed according to... Figure 1 The experimental procedure involved SE50 sequencing on the MGISEQ-200RS sequencing platform. In short, the process involved sequentially polymerizing a mixture of fluorescently modified and reversibly blocked nucleotides on the MGISEQ-200RS platform, followed by elution of free nucleotides using elution reagents, signal acquisition in imaging solution, removal of protecting groups using excision reagents, and washing with elution reagents. The Q30 decrease and sequencing error rate curves for each cycle were then analyzed to evaluate sequencing quality.
[0179] Experimental Group 4: Using the MGISEQ-200RS high-throughput sequencing kit, a mixture of fluorescently modified and reversibly blocked nucleotides (same as control group 2) was sequentially polymerized on the MGISEQ-200RS sequencing platform. Then, the free nucleotides were eluted with elution reagents. The reagent in well #18 of the kit was then removed and replaced with the imaging polymerization solution 4 of this invention. This allows for polymerization and leveling during signal acquisition. At this point, the polymerized nucleotide mixture is a reversibly blocked nucleotide. SE50 sequencing was performed on the MGISEQ-200RS sequencing platform following the same experimental procedure as control group 2. The Q30 decrease rate and sequencing error rate curve for each cycle were then analyzed to evaluate the sequencing quality.
[0180] Result: as follows Figures 5-7 And as shown in Tables 13-15, Figure 5 The results show the Q30 (%) percentage for each sequencing cycle under the above conditions, where the horizontal axis represents the number of sequencing cycles and the vertical axis represents the Q30 (%) percentage for each cycle. Figure 6 The results show the percentage of sequencing error rate (%) under the above conditions, where the horizontal axis represents the number of sequencing cycles and the vertical axis represents the percentage of sequencing error rate (%) for each cycle. Figure 7The results show the percentage of lag (%) for each sequencing cycle under the above conditions. Lag is an indicator used to measure whether the sequencing reaction is complete. It refers to the proportion of some copies that are reflected in the N-1 position when sequencing reaches the N position. The horizontal axis is the number of sequencing cycles, and the vertical axis is the percentage of lag (%) for each cycle.
[0181] The smaller the decrease in Q30 (%) in each cycle, the lower the percentage of sequencing error rate (%), and the lower the increase in lag (%), indicating better and more stable sequencing quality. Table 13 shows that the decrease in Q30 (%) in the control group over 50 cycles was worse than that in experimental group four. Table 14 shows that the error rate in the control group was significantly higher than that in experimental group four. Table 15 shows that the increase in lag (%) in the control group over 50 cycles was significantly higher than that in experimental group four.
[0182] Table 13. Percentage of Q30 in each cycle
[0183]
[0184]
[0185] Table 14. Error rate (%) for each loop
[0186]
[0187]
[0188]
[0189] Table 15. Lag (%) for each cycle
[0190]
[0191]
[0192]
Claims
1. A method for analyzing the sequence of a target polynucleotide, comprising: (a) Provide target polynucleotides; (b) Under conditions that allow hybridization or annealing, the target polynucleotide is contacted with a primer to form a partial double strand comprising the target polynucleotide and a primer used as a growth chain; (c) Under conditions allowing the polymerase to perform nucleotide polymerization, the partially duplexed strand is contacted with a mixture of polymerase and a first nucleotide to extend the growth chain, wherein the first nucleotide mixture contains at least one labeled nucleotide. Each nucleotide in the first nucleotide mixture contains a protective group in its ribose or deoxyribose portion that can block the elongation of the nucleic acid chain. (d) Under conditions that allow the polymerase to perform nucleotide polymerization, the product of the previous step is contacted with the polymerase and a mixture of the second nucleotides to extend the growth chain, wherein the second nucleotide mixture contains at least one unlabeled nucleotide, or an irreversible blocking nucleotide, or a combination of the unlabeled nucleotide and the irreversible blocking nucleotide. Each unlabeled nucleotide in the second nucleotide mixture contains a protective group in its ribose or deoxyribose portion that can block the elongation of the nucleic acid chain; Furthermore, a photographic polymerization solution is provided and contacted with a second nucleotide mixture, and the presence of a marker in the product of step (c) is detected by the photographic polymerization reaction; The photographic polymerization solution contains the following reagents: nucleic acid polymerase, buffer reagent, surfactant, and reagents for reducing nucleic acid damage under laser photography; (e) Remove protecting groups and markers contained in the product of the previous step; (f) Optionally repeat steps (c)-(e) once or more; Thus, the sequence information of the target polynucleotide is obtained.
2. The method of claim 1, wherein, The protecting group is a protecting group attached by a 2' or 3' oxygen atom.
3. The method of claim 1, wherein, The reagents used to reduce nucleic acid damage under laser imaging are selected from: sodium ascorbate, dithiothreitol (DTT), 6-hydroxy-2,5,7,8-tetramethyltryptane-2-carboxylic acid (Trolox), reduced glutathione (GSH), N,N'-dimethylthiourea (DMTU), ammonium pyrrolidine dithiocarboxylate (APDTC), amitidine, amitidine trihydrochloride, uric acid, sodium pyruvate, L-cysteine, β-mercaptoethylamine, cystamine, or any combination thereof.
4. The method of claim 1, wherein, In step (c), the extension is a target polynucleotide extension.
5. The method of claim 1, wherein the first nucleotide mixture comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and a fourth nucleotide labeled with a fourth label or an unlabeled fourth nucleotide, or a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide co-labeled with the first and second labels, and an unlabeled fourth nucleotide.
6. The method of claim 5, wherein, The first marker, the second marker, the third marker, and the fourth marker are each independently identical or different.
7. The method of claim 5, wherein the first marker, the second marker, the third marker, and the fourth marker are luminescent markers.
8. The method of claim 5, wherein the first marker, the second marker, the third marker, and the fourth marker are each independently selected from coumarin, AlexaFluor, Bodipy, fluorescein, tetramethylrhodamine, phenoxazine, acridine, Cy5, Cy3, and Texas red.
9. The method of claim 1, wherein, In step (d), the second nucleotide mixture comprises an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.
10. The method of claim 9, wherein, The second nucleotide mixture also contains an unlabeled fourth nucleotide.
11. The method of claim 1, wherein, The second nucleotide mixture contains at least one irreversible blocking nucleotide.
12. The method of claim 1 or 5, wherein, In step (d), the second nucleotide mixture comprises an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and a first irreversible blocking nucleotide, a second irreversible blocking nucleotide, a third irreversible blocking nucleotide, and a fourth irreversible blocking nucleotide.
13. The method of claim 12, wherein, The second nucleotide mixture also includes an unlabeled fourth nucleotide.
14. The method of claim 1, wherein, The target polynucleotide may be DNA, RNA, or any combination thereof.
15. The method of claim 1, wherein, The target polynucleotide was obtained from samples derived from eukaryotes, prokaryotes, viruses, or any combination thereof.
16. The method of claim 5, wherein, The first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are A, T, C, and G, respectively.
17. The method of claim 12, wherein, The first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are A, T, C, and G, respectively.
18. The method of claim 17, wherein, The irreversible blocking nucleotide is a dideoxynucleotide.
19. The method of claim 1, wherein when the first nucleotide mixture comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and a fourth nucleotide labeled with a fourth label; the second nucleotide mixture comprises an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and an unlabeled fourth nucleotide; or, When the first nucleotide mixture contains a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and an unlabeled fourth nucleotide, or a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide co-labeled with the first and second labels, and an unlabeled fourth nucleotide, the second nucleotide mixture contains an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.
20. A reagent kit comprising: (a) A first nucleotide mixture, the first nucleotide mixture comprising at least one nucleotide labeled with a marker, Each nucleotide in the first nucleotide mixture contains a protective group in its ribose or deoxyribose portion that can block the elongation of the nucleic acid chain. (b) A second nucleotide mixture comprising at least one unlabeled nucleotide, or an irreversible blocking nucleotide, or a combination of the unlabeled nucleotide and the irreversible blocking nucleotide; Each unlabeled nucleotide in the second nucleotide mixture contains a protective group in its ribose or deoxyribose portion that can block the elongation of the nucleic acid chain; (c) Photographing the polymerization solution; in, The photographic polymerization solution contains the following reagents: nucleic acid polymerase, buffer reagent, surfactant, and reagents for reducing nucleic acid damage under laser photography.
21. The kit of claim 20, wherein, The protecting group is a protecting group attached by a 2' or 3' oxygen atom.
22. The kit of claim 20, wherein, The reagents used to reduce nucleic acid damage under laser imaging are selected from: sodium ascorbate, dithiothreitol (DTT), 6-hydroxy-2,5,7,8-tetramethyltryptane-2-carboxylic acid (Trolox), reduced glutathione (GSH), N,N'-dimethylthiourea (DMTU), ammonium pyrrolidine dithiocarboxylate (APDTC), amitidine, amitidine trihydrochloride, uric acid, sodium pyruvate, L-cysteine, β-mercaptoethylamine, cystamine, or any combination thereof.
23. The kit of claim 20, wherein, The kit also includes one or more of the following: nucleic acid amplification buffer, enzyme working buffer, water, or any combination thereof.
24. The kit of claim 20, wherein, The kit also includes one or more of the following: sequencing slides, reagents for removing protecting groups and markers from nucleotides.
25. The kit of claim 20, wherein, The first nucleotide mixture comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and a fourth nucleotide labeled with a fourth label or an unlabeled fourth nucleotide, or a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide co-labeled with the first and second labels, and an unlabeled fourth nucleotide.
26. The kit of claim 20, wherein the first nucleotide mixture comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and a fourth nucleotide labeled with a fourth label; and the second nucleotide mixture comprises an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and an unlabeled fourth nucleotide; or, When the first nucleotide mixture contains a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and an unlabeled fourth nucleotide, or a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide co-labeled with the first and second labels, and an unlabeled fourth nucleotide, the second nucleotide mixture contains an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.
27. The kit of claim 20, wherein, The second nucleotide mixture comprises an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and a first irreversible blocking nucleotide, a second irreversible blocking nucleotide, a third irreversible blocking nucleotide, and a fourth irreversible blocking nucleotide.
28. The kit of claim 27, wherein the second nucleotide mixture further comprises an unlabeled fourth nucleotide.
29. The kit of claim 25 or 26, wherein, The first marker, the second marker, the third marker, and the fourth marker are each independently identical or different.
30. The kit of claim 25 or 26, wherein the first marker, the second marker, the third marker, and the fourth marker are luminescent markers.
31. The kit of claim 25 or 26, wherein the first, second, third, and fourth markers are each independently selected from coumarin, AlexaFluor, Bodipy, fluorescein, tetramethylrhodamine, phenoxazine, acridine, Cy5, Cy3, and Texas red.
32. The kit of claim 25 or 26, wherein the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are A, T, C, and G, respectively.
33. The kit of claim 27, wherein the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are A, T, C, and G, respectively.
34. The kit of claim 33, wherein the irreversible blocking nucleotide is a dideoxynucleotide.