DNA synthesis sequencing method and system

A technology of synthesis sequencing and control system, applied in the field of genetic engineering, can solve the problems of slow extension reaction, slow speed, accumulation of error information, etc., and achieve the effect of clean reaction

Active Publication Date: 2018-06-22
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] First of all, the synthesis of modified nucleotides protected by 3'-OH is complicated; secondly, in the actual sequencing process, after extension using a reversible terminator protected by 3'-OH, the two reaction sites need to be broken at the same time, that is, the linking unit is 100% broken It is also necessary to deprotect the 3'-OH with 100% efficiency
However, these two sites often cannot be completely broken at the same time under the same conditions, which will inevitably lead to the accumulation of error information, which will eventually affect the read length and efficiency of sequencing
However, DNA sequencing needs to be 100% removed. When the modified nucleotides protected by the 3'-OH participate in DNA chain extension, another huge challenge is that DNA polymerase is not easy to recognize, the extension reaction is slow, and it is easy to mismatch (Ref:Michael L . Metzk

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1: Synthesis of water-soluble bifunctional linking unit:

[0086] Connecting unit 1 is directly purchased as shown in formula I.

[0087]

[0088] The connection unit 2 is shown in formula II,

[0089]

[0090] Its synthetic route is as follows:

[0091]

[0092] Concrete synthetic steps are:

[0093] 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 ℃, then add N-hydroxysuccinimide (compound 2) (72mg; 0.6mmol) and continue stirring for 1h, filter the reaction solution, wash with ethyl acetate, concentrate the filtrate, and purify it with a silica gel column to obtain the product (Compound 3) 3-bromo-2,5-oxo-1-pyrrolidinyl ester;

[0094] 2. Dibromoethanol (compound 4) (187.5 mg, 1 mmol) and KOH (33 mg, 0.6 mg) were added to 25 mL of DMSO solven...

Embodiment 2

[0122] Embodiment 2: the synthesis of connecting unit and reversible terminator

[0123] connection unit The synthetic route of is as follows:

[0124]

[0125] The specific synthesis steps are as follows:

[0126] 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).

[0127] 2) Weigh 2-bromoethanol (9.96g, 80mmol) and sodium...

Embodiment 3

[0135] Example 3: Isothermal Amplification Surface Amplification

[0136] (1) Activate the surface of the slide to make it hydroxylated

[0137] 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, take it out, rinse with plenty of water, and blow dry.

[0138] (2) Surface modification of glass slides

[0139] Place the above treated slides in absolute ethanol; add aminopropyltriethoxysilane (APTES) Make the mass ratio of APTES to absolute ethanol in the system 1:49, raise the temperature to 60°C, heat for 2h, and then cool to room temperature; APTES is connected to the glass surface through a silicon-oxygen-silicon bond, and then rinsed with ethanol and pure water respectively to obtain Glass slides with amino groups on the surface; immerse the glass slides with am...

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Abstract

The invention discloses a DNA synthesis sequencing method and a DNA synthesis sequencing system. The method comprises the following steps: connecting a primer P1 to the surface of a matrix through a water-soluble bifunctional linking unit; adding a solution containing a DNA template to be detected onto the surface of the matrix, and carrying out isothermal surface amplification reaction; adding aprimer Pe to the surface of the matrix, adding an extension reaction solution containing fluorescence labeling reversible terminators of four basic groups onto the surface of the matrix, and carryingout extension reaction; carrying out imaging on DNA double strands after extension, and adding a corresponding chemical reagent, so that the linking units of the fluorescence labeling reversible terminators break; and repeating the steps of DNA chain extension and breakage, and completing a plurality of times of cycle sequencing, thus obtaining the base sequence of nucleotide of the DNA template to be detected. The sequencing method provided by the invention can be used for DNA high-throughput synthesis sequencing, according to the experimental result, under the sequencing condition, the readlength of sequence is at least 73 base groups, if double-end sequencing is adopted, the read length is at least 146, and the accuracy rate is at least 99.98%.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a DNA synthesis sequencing method and a sequencing system. Background technique [0002] With the completion of the Human Genome Sequencing 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] Sequencing By Synthesis (SBS) is one of the second-generation DNA sequencing technologies. Sequencing by synthesis fixes a large number of template DNA fragments to be tested, and hybridizes on the fixed DNA sequencing template to combine with universal DNA primers, and then four base fluorescently labeled nucleotides are sequentially extended and brok...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869C12M1/36C12M1/34
CPCC12Q1/6869C12Q2535/122C12Q2563/107C12Q2533/101
Inventor 沈玉梅谭连江邵志峰龚兵李小卫
Owner SHANGHAI JIAO TONG UNIV
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