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CN-122012683-A - Gene sequencing method and kit

CN122012683ACN 122012683 ACN122012683 ACN 122012683ACN-122012683-A

Abstract

The application discloses a gene sequencing method and a kit. The method comprises the steps of adopting a sequencing reagent 1 and a sequencing reagent 2 to sequence, wherein the sequencing reagent 1 comprises 4 second nucleotide analogues and 4 first nucleotide analogues, the sequencing reagent 2 comprises 4 first nucleotide analogues, the first nucleotide analogues and the second nucleotide analogues respectively have structures shown in a formula I and a formula II, adopting a scheme I and/or a scheme II to sequence, firstly contacting the sequencing reagent 1 to form a compound, collecting signals, eluting and removing, then contacting the sequencing reagent 2, extending, cutting off a reversible blocking group, circularly completing sequencing, secondly contacting the sequencing reagent 2, extending, then contacting the sequencing reagent 1 to form the compound, collecting signals, eluting and removing, cutting off the reversible blocking group, and circularly completing sequencing. The method adopts the optimized sequencing reagent 1, the sequencing reagent 2 and the sequencing scheme, can perform longer-reading and longer-sequencing, and has higher quality and lower error rate.

Inventors

  • WANG HUI
  • YANG SHUAI
  • YU DI
  • XU JIECHENG
  • CHEN DEXIA
  • LIU ERKAI

Assignees

  • 深圳赛陆医疗科技有限公司

Dates

Publication Date
20260512
Application Date
20260206

Claims (10)

  1. 1. A gene sequencing method is characterized by comprising the steps of sequencing a nucleic acid molecule to be tested by adopting a sequencing reagent 1 and a sequencing reagent 2; The sequencing reagent 1 comprises 4 second nucleotide analogs and 4 first nucleotide analogs, and the sequencing reagent 2 comprises 4 first nucleotide analogs; the first nucleotide analogue has a structure shown in a formula I, and the second nucleotide analogue has a structure shown in a formula II; one (I) ; Two kinds of ; In the formula I and the formula II, 4 second nucleotide analogs and 4 first nucleotide analogs are respectively adenine A, cytosine C, guanine G and thymine T, in the formula I, R 1 is a reversible blocking group, in the formula II, dye is a fluorescent group, linker is a Linker, X is independently selected from any one of CH 2 、NH、S、CF 2 、CBr 2 and Se, and 4 second nucleotide analogs are respectively marked by 4 different fluorescent groups; the step of sequencing the nucleic acid molecules to be detected comprises the step of sequencing at least one of the following schemes after the sequencing primer is hybridized to the nucleic acid molecules to be detected; The method comprises the steps of (1) contacting a sequencing reagent 1, forming a complex by a second nucleotide analogue and a nucleic acid molecule to be tested, a 3 'end of a sequencing primer or a 3' end extended by the sequencing primer under the action of metal ions and polymerase, (2) contacting an imaging buffer to wash away unbound second nucleotide analogue, (3) collecting signals, (4) removing bound second nucleotide analogue after the signal collection is finished, (5) contacting the sequencing reagent 2, polymerizing the first nucleotide analogue to the 3 'end of the sequencing primer or the 3' end extended by the sequencing primer under the action of polymerase, removing a reversible blocking group of the first nucleotide analogue, and repeating the steps (1) to (6) to finish sequencing of the nucleic acid molecule to be tested; the second scheme is that (1) the first nucleotide analogue is contacted with a sequencing reagent 2 and polymerized to the 3' end of a sequencing primer or the 3' end extended by the sequencing primer under the action of polymerase, (2) the second nucleotide analogue is contacted with the sequencing reagent 1 and forms a complex with a nucleic acid molecule to be detected and the 3' end extended by the sequencing primer under the action of metal ions and polymerase, (3) the second nucleotide analogue is contacted with an imaging buffer solution to wash out unbound second nucleotide analogue, (4) the signal acquisition is carried out, (5) the bound second nucleotide analogue is removed after the signal acquisition is finished, (6) the reversible blocking group of the first nucleotide analogue is removed, and the steps (1) to (6) are repeated to finish sequencing of the nucleic acid molecule to be detected.
  2. 2. The method of claim 1, wherein step (1) further comprises contacting the complex with a washing reagent to provide a reaction environment for the complex formed in step (1).
  3. 3. The method of claim 1, wherein step (5) further comprises contacting the first nucleotide analogue with a washing reagent to provide a reaction environment for the polymerization of the first nucleotide analogue in step (5).
  4. 4. The method of claim 1, wherein step (6) further comprises contacting the sample with a washing reagent to wash away unpolymerized first nucleotide analog and provide a reaction environment for removing the reversible blocking group of the first nucleotide analog.
  5. 5. The method of claim 1, wherein step (1) further comprises contacting the first nucleotide analogue with a washing reagent to provide a reaction environment for the polymerization of the first nucleotide analogue in step (1).
  6. 6. The method of claim 1, wherein step (2) further comprises contacting the first nucleic acid with a wash reagent to wash away unpolymerized first nucleic acid analog and provide a reaction environment for the complex formed in step (2).
  7. 7. The method of claim 1, wherein step (6) further comprises contacting the sample with a washing reagent to provide a reaction environment for removing the reversible blocking group of the first nucleotide analog.
  8. 8. A gene sequencing kit is characterized by comprising a sequencing reagent 1 and a sequencing reagent 2; The sequencing reagent 1 comprises 4 second nucleotide analogs and 4 first nucleotide analogs, and the sequencing reagent 2 comprises 4 first nucleotide analogs; the first nucleotide analogue has a structure shown in a formula I, and the second nucleotide analogue has a structure shown in a formula II; one (I) ; Two kinds of ; In the formula I and the formula II, the 'Base' of 4 second nucleotide analogues and 4 first nucleotide analogues are adenine A, cytosine C, guanine G and thymine T respectively, in the formula I, R 1 is a reversible blocking group, in the formula II, dye is a fluorescent group, linker is a Linker, X is independently selected from any one of CH 2 、NH、S、CF 2 、CBr 2 and Se, and 4 second nucleotide analogues are marked by 4 different fluorescent groups respectively.
  9. 9. The gene sequencing kit according to claim 8, further comprising at least one of an imaging buffer, a excision reagent, a washing reagent 1, and a washing reagent 2; The imaging buffer solution comprises 10-100 mM Tris-HCl, 10-50 mM NaCl, 1-2M Betaine, 0.02-0.5% Tween-20, 0.1-1 mM EDTA, 1-70 mM non-catalytic metal ions and 5-70 mM antioxidants; The excision reagent comprises 10-50 mM tris (3-hydroxypropyl) phosphine, 0.2-2M NaCl, 10-100 mM Tris-HCl, 0.02-0.5% Tween-20; the cleaning reagent 1 comprises 20-100 mM NaCl and 25-100 mM C 6 H 5 Na 3 O 7 ·2H 2 O; the cleaning reagent 2 comprises 10-100 mM Tris-HCl, 1-5 mM NaCl and 0.1-1 mM EDTA; optionally, the non-catalytic metal ions comprise at least one of strontium chloride, calcium chloride, barium chloride; Optionally, the antioxidant comprises at least one of sodium ascorbate, trolox, glutathione; Alternatively, the sequencing reagent 1 removes antioxidants on the basis of the imaging buffer, adding a polymerase, 4 second nucleotide analogs and 4 first nucleotide analogs; Optionally, in the sequencing reagent 1, 4 kinds of second nucleotide analogs, namely "Base" are respectively adenine A, cytosine C, guanine G and thymine T, and are respectively labeled as second nucleotide analog A, second nucleotide analog T, second nucleotide analog C and second nucleotide analog G, wherein the concentration of the second nucleotide analog A is 0.02-0.05 mu M, the concentration of the second nucleotide analog T is 0.5-1 mu M, the concentration of the second nucleotide analog C is 0.5-1 mu M, the concentration of the second nucleotide analog G is 0.02-0.05 mu M, and 4 kinds of first nucleotide analogs, namely "Base" are respectively labeled as first nucleotide analog A, first nucleotide analog T, first nucleotide analog C and first nucleotide analog G, wherein the concentration of the first nucleotide analog A is 0.01-0.005 mu M, the concentration of the first nucleotide analog G is 0.01-0.002 mu M, and the concentration of the first nucleotide analog G is 0.01-0.005 mu M; Alternatively, the sequencing reagent 2 comprises 10-100 mM Tris-HCl, 5-50 mM NaCl, 2-20 mM (NH 4 ) 2 SO 4 , 0.01-0.1 mg/mL polymerase, 2-20 mM MgSO 4 , 0.2-1 mM EDTA, and 4 first nucleotide analogs, wherein the first nucleotide analog a, the first nucleotide analog T, the first nucleotide analog C, the first nucleotide analog G are each 0.2-1 μm.
  10. 10. The gene sequencing kit according to claim 8 or 9, wherein the reversible blocking group comprises at least one of methylene azide, allyl, ester, phosphate, hydroxylamine, disulfide bond, photo-cleavable group, 2-nitrobenzyl, azo compound; Optionally, the reversible blocking group comprises at least one of, ; And/or the linker is a cleavable linker or a non-cleavable linker; optionally, the cleavable linker comprises at least one of an electrophilic cleavage linker, a nucleophilic cleavage linker, a photolyzable linker, a cleavage under reducing conditions, a cleavage under oxidizing conditions, a safety pull linker, a cleavage via an elimination mechanism; optionally, the cleavable linker is at least one of alkyl, allyl, azidomethylene, 2-nitrobenzyl, disulfide; Optionally, the non-cleavable linker comprises at least one of a polyethylene glycol chain, a polypeptide chain; and/or the fluorescent group labels of the 4 second nucleotide analogs are not repeated and are selected from AF488、AF532、AF633、AF680、AF660、AF700、AF647、AF594、AF555、AF568、CY3、CY5、CY5.5、CY7、CY7.5、ROX、R6G、ATTO495、ATTO532、ATTO700、ATTO680、ATTO655、ATTO647N、ATTO594、ATTO Rho101、ATTO590、ATTO Thio12、FAM、VIC、TET、JOE、HEX、CAL Fluor Orange560、TAMRA、CAL Fluor Red610、TEXAS RED、CAL Fluor Red635、iFluor488、iFluor514、iFluor532、iFluor546、iFluor555、iFluor568、iFluor590、iFluor610、iFluor633、iFluor647、iFluor680、iFluor700、iFluor710、Quasar705、Quasar670.

Description

Gene sequencing method and kit Technical Field The application relates to the technical field of gene sequencing, in particular to a gene sequencing method and a kit. Background High-throughput sequencing (High-Throughput Sequencing, HTS), also known as Next-generation sequencing (Next-Generation Sequencing, NGS), is a technique that enables rapid sequencing of millions to billions of DNA molecules in parallel. Compared with the traditional Mulberry sequencing, the method has the core advantages of high throughput, low cost and high speed, and thoroughly changes genomics research, so that large-scale sequencing projects, such as a human genome project, are changed from time-consuming yearly engineering to conventional laboratory-finished tasks. The whole flow of high throughput sequencing includes library preparation, cluster generation or DNA Nanosphere (DNB) preparation loading, sequencing reactions. Currently, the mainstream platform technology in the market comprises an illumina, sailu and other companies' synthesis-while-sequencing technology, a Huada intelligent combined probe anchoring polymerization sequencing technology and a Thermo Fisher semiconductor sequencing technology. The sequencing technology of the current mainstream second generation sequencing platform still has short read length, and most error rate is still above 0.1%. Therefore, how to reduce the error rate of high-throughput sequencing and effectively improve the sequencing quality is still a research key point and a difficulty in the technical field of high-throughput sequencing. Disclosure of Invention The application aims to provide an improved gene sequencing method and a kit matched with the sequencing method. In order to achieve the above purpose, the present application adopts the following technical scheme: The application discloses a gene sequencing method, which comprises the steps of sequencing a nucleic acid molecule to be tested by adopting a sequencing reagent 1 and a sequencing reagent 2, wherein the sequencing reagent 1 comprises 4 second nucleotide analogs and 4 first nucleotide analogs, and the sequencing reagent 2 comprises 4 first nucleotide analogs; one (I) ; Two kinds of; In the formula I and the formula II, 4 second nucleotide analogs and 4 first nucleotide analogs are respectively adenine A, cytosine C, guanine G and thymine T, "Base", in the formula I, R 1 is a reversible blocking group, in the formula II, "Dye" is a fluorescent group, "Linker" is a connector, and "X" is independently selected from any one of CH 2、NH、S、CF2、CBr2 and Se, 4 second nucleotide analogs are respectively marked by 4 different fluorescent groups, and sequencing the nucleic acid molecule to be tested comprises sequencing by at least one of the following schemes after hybridization of a sequencing primer to the nucleic acid molecule to be tested: The method comprises the steps of (1) contacting a sequencing reagent 1, forming a complex by a second nucleotide analogue and a nucleic acid molecule to be tested, a 3 'end of a sequencing primer or a 3' end extended by the sequencing primer under the action of metal ions and polymerase, (2) contacting an imaging buffer to wash away unbound second nucleotide analogue, (3) collecting signals, (4) removing bound second nucleotide analogue after the signal collection is finished, (5) contacting the sequencing reagent 2, polymerizing the first nucleotide analogue to the 3 'end of the sequencing primer or the 3' end extended by the sequencing primer under the action of polymerase, removing a reversible blocking group of the first nucleotide analogue, and repeating the steps (1) to (6) to finish sequencing of the nucleic acid molecule to be tested; the second scheme is that (1) the first nucleotide analogue is contacted with a sequencing reagent 2 and polymerized to the 3' end of a sequencing primer or the 3' end extended by the sequencing primer under the action of polymerase, (2) the second nucleotide analogue is contacted with the sequencing reagent 1 and forms a complex with a nucleic acid molecule to be detected and the 3' end extended by the sequencing primer under the action of metal ions and polymerase, (3) the second nucleotide analogue is contacted with an imaging buffer solution to wash out unbound second nucleotide analogue, (4) the signal acquisition is carried out, (5) the bound second nucleotide analogue is removed after the signal acquisition is finished, (6) the reversible blocking group of the first nucleotide analogue is removed, and the steps (1) to (6) are repeated to finish sequencing of the nucleic acid molecule to be detected. In the first and second schemes, the signal acquisition mainly refers to acquiring the signal of the fluorescent group of the second nucleotide analogue in the complex, so that the base type of the second nucleotide analogue can be determined according to the signal of the fluorescent group in the subsequent analysis, and the base type of the correspon