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DNA single molecule sequencing method and sequencing system

A single-molecule sequencing and control system technology, applied in the field of genetic engineering, can solve the problems of high mismatch rate, complex synthesis route, poor recognition speed of enzymes, etc., and achieve the effect of clean reaction

Active Publication Date: 2018-07-06
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Reversible terminators based on disulfide linkage units have been applied in single-molecule sequencing. However, the literature (Nucleic Acids Research, 2008, 36, No. 4e25) reported that reversible terminators based on disulfide bonds were single-color fluorescently labeled four Nucleotides with different bases, in order to ensure that the disulfide bond reversible terminator is used as a single-molecule sequencing reagent to extend only one reversible terminator at a time, a nucleoside monophosphate and diphosphate with a large steric hindrance are connected next to fluorescein. Phosphonic acid, a reversible terminator with such a large steric hindrance, can indeed extend one at a time, but its synthetic route is extremely complicated, and the excessive steric hindrance also makes it difficult for the enzymes involved in DNA chain extension to recognize and slow and misunderstand. High ratio (Michael L. Metzker; Nature Reviews Genetics 2010,11,31.)

Method used

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  • DNA single molecule sequencing method and sequencing system
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  • DNA single molecule sequencing method and sequencing system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1: Synthesis of water-soluble bifunctional linking unit

[0083] 1. Linker 1 (Linker 1), purchased and used directly.

[0084]

[0085] 2. Synthetic route of Linker 2:

[0086]

[0087] The preparation steps of connection unit 2 are as follows:

[0088] 1. Dicyclohexylcarbodiimide (DCC) (80mg; 0.4mmol) and N, N-dimethylaminopyridine (3mg) were added to a solution of ethyl acetate (3mL) dissolved in tribromopropionic acid (compound 1) (43mg, 0.28mmol), at 20 Stir for 5min under the protection of argon at 100°C, then add N-hydroxysuccinimide (compound 2) (72mg; 0.6mmol) and continue to stir for 1h, filter the reaction solution, wash with ethyl acetate, concentrate the filtrate, and purify with a silica gel column to obtain the product (Compound 3) 3-Bromo-2,5-oxo-1-pyrrolidinyl ester.

[0089] 2. Dibromoethanol (compound 4) (187.5 mg, 1 mmol) and KOH (33 mg, 0.6 mg) were added to 25 mL of DMSO solvent and reacted in an ice bath under argon protection...

Embodiment 2

[0111] Embodiment 2: the synthesis of connecting unit and reversible terminator

[0112] connection unit The synthetic route of is as follows:

[0113]

[0114] The specific synthesis steps are as follows:

[0115] 1) Weigh ethylene glycol (6.7ml, 120mmol) and acetic acid (2.4g, 40mmol) into a 100mL single-necked flask and stir, add 0.112mL of concentrated sulfuric acid dropwise to the reaction solution, and stir at 25°C for 24h. Then add 17mL of saturated sodium bicarbonate solution and stir overnight, then add 12mL of water to the reaction mixture, extract with dichloromethane (50mL*8), combine the organic layers, dry with anhydrous sodium sulfate, spin off the solvent, and the residue Use 30:1CH 2 Cl 2 / MeOH was used as the eluent for column chromatography to obtain compound 1 (2.52 g), with a yield of 61%. 1 H NMR (400MHz, CDCl 3): δppm4.20(t, 2H, J=4.8Hz), 3.82(t, 2H, J=4.8Hz), 2.09(s, 3H), 1.93(s, 1H).

[0116] 2) Weigh 2-bromoethanol (9.96g, 80mmol) and sod...

Embodiment 3

[0123] Example 3: Primer P1 is immobilized on the glass surface

[0124] 1. Surface activation (hydroxylation) of glass slides

[0125] The slides (4×4mm) were washed three times with ethanol and water respectively, dried, and placed in hydrogen peroxide (H 2 o 2 , 30%) and concentrated sulfuric acid (H 2 SO 4 , 98% of the mixture (V (H 2 o 2 ):V(H 2 SO 4 )=7:3), heat at 80-90°C for 1 hour, cool down to room temperature naturally, rinse with plenty of water, and blow dry.

[0126] 2. Surface modification of glass slides

[0127] Put the above treated glass slides in absolute ethanol; add APTES (aminopropyltriethoxysilane) to make the mass ratio of APTES to absolute ethanol in the system 1:49, raise the temperature to 60°C, and heat for 2h , and then cooled to room temperature; APTES was connected to the glass surface through a silicon-oxygen-silicon bond, and then washed with ethanol and pure water respectively to obtain a glass slide with amino groups on the surfac...

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Abstract

The invention provides a DNA single molecule sequencing method and a sequencing system, the DNA single molecule sequencing method comprises the following steps: S1 modifying the sursurface of a substrate, connecting a water-soluble bifunctional linking unit to the sursurface of the substrate; and connecting a primer P1 with the water-soluble bifunctional linking unit to obtain the substrate to which the primer is immobilized; S2, placing a mixed solution containing a DNA template to be tested, polymerase, and a four-color fluorescently-labeled reversible terminator on the substrate to which the primer P1 is immobilized, and performing extension to form a fluorescein-containing primer / template complex; S3, imaging the extended primer / template complex to determine nucleotide base species involved in the extension; S4, breaking cleavable linkage units of nucleotides involved in the extension, and performing next extension; and S5, repeating the steps S2 to S4 to obtain the base sequence of the DNA template to be tested. The DNA single molecule sequencing method is capable of perfecting the effect of extending only one reversible terminator in a single sequencing cycle.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular to a DNA single-molecule sequencing method and sequencing system. Background technique [0002] After the completion of the Human Genome Project, DNA sequencing technology has developed rapidly. DNA sequencing (DNAsequencing) refers to the analysis of the base sequence of a specific DNA fragment, that is, the sequence of adenine (A), thymine (T), cytosine (C) and guanine (G). The development of accurate, high-throughput, and low-cost DNA sequencing methods is of great significance for biology and medicine. [0003] DNA synthesis sequencing next-generation sequencing technology has been widely used, but its inherent limitations are also obvious. For example, the sequencing time is long, DNA amplification may introduce a certain error rate, and quantitative measurement is relatively complicated. Therefore, the single-molecule-based three-generation sequencing technology h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C12M1/38C12M1/36C12M1/00C07H19/10C07H19/14C09K11/06
CPCC07H19/10C07H19/14C09K11/06C12Q1/6869C12Q2525/10
Inventor 沈玉梅邵志峰谭连江龚兵李小卫
Owner SHANGHAI JIAO TONG UNIV
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