Three-color high-throughput sequencing reagent and sequencing method

A high-throughput, sequencing technology, applied in the field of high-throughput sequencing, can solve the problems of reducing signal-to-noise ratio, affecting sequencing read length, sequencing efficiency and sequencing throughput, etc.

Pending Publication Date: 2021-09-03
NANJING SUPERYEARS GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The weakening of the signal will reduce the signal-to-noise ratio and affect the sequencing read length, resulting in a decrease in sequencing efficiency and throughput

Method used

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  • Three-color high-throughput sequencing reagent and sequencing method
  • Three-color high-throughput sequencing reagent and sequencing method
  • Three-color high-throughput sequencing reagent and sequencing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Synthesis of 3'-O-Azuromethyl DATP

[0046] like figure 2 As shown, 410.4 mg (FW 410.4, 1 Hao Moore) is labeled by the base and proton sponge (254 mg, 1.17 Hao Moore) Dry overnight after drying overnight in a vacuum dryer with phosphorus phosphorus. Ester. After adding 134 microlituted fresh distilled trichlorophos (1.17 Hao Mole) at 0 ° C, stirred at 0 ° C for 2 hours. Subsequently 1.817 grams of triaxadium tract (FW 363.33, 5 Hao Moore) and 1.84 ml of tributylamine (185.35, D = 0.778, 7.7 Hao Mocetra) were mixed in 8 ml of no water DMF (dimethylformamide) After mixing, stir after the next time, stirred for 30 minutes. After adding 50 ml of 0.1 M Teab (carbonate) (pH 8.0) at room temperature for 1 hour. After adding 50 ml of concentrated concentrated ammonia at room temperature, stirring overnight, extols off the water and ammonia after dissolving in 15 ml of water. The 3'-O-azide methyl DATP was purified by HPLC (high performance liquid chromatography) using 0.1...

Embodiment 2

[0047] Example 2 Synthesis 3'-O-azide methyl DCTP

[0048] like image 3 As shown, the reactant of 386.4 mg (FW 386.4, 1 Hao Moore) and the proton sponge (254 mg, 1.17 Hao Moore) in P 2 O 5 Drying overnight after drying overnight in a vacuum dryer was dissolved in trimethyl phosphate. Add 134 microlituted fresh distilled POCl at 0 ° C 3 (1.17 Hao Moore) was stirred at 0 ° C for 2 hours. Subsequently 1.817 grams of triaxamate (FW 363.33, 5 Hao Moore) and 1.84 ml of tributylamine (185.35, D = 0.778, 7.7 Hao Mole) were mixed in 8 ml of no water DMF after mixing at room temperature. After stirring for 30 minutes. After stirring 50 ml of TEAb (pH 8.0) at room temperature for 1 hour. After adding 50 ml of concentrated concentrated ammonia at room temperature, stirring overnight, extols off the water and ammonia after dissolving in 15 ml of water. The 3'-O-azide methyl DCTP was purified with EtOAc (EtOAc) EtOAc.

Embodiment 3

[0049] Example 3 Synthesis of 3'-O-Azuromethyl DGTP

[0050] like Figure 4 As shown, 587.6 mg (FW 587.6, 1 Hao Moore) reactants and proton sponges (254 mg, 1.17 Hao Moore) in P 2 O 5 Drying overnight after drying overnight in a vacuum dryer was dissolved in trimethyl phosphate. Add 134 microlituted fresh distilled POCl at 0 ° C 3 (1.17 Hao Moore) was stirred at 0 ° C for 2 hours. Subsequently 1.817 grams of triazine-jam (FW363.33, 5 Hao Moore) and 1.84 ml of tributylamine (185.35, D = 0.778, 7.7 Hao Mole) were mixed in 8 ml of no water DMF after mixing at room temperature. After stirring, stir it for 30 minutes. After stirring 50 ml of TEAb (pH 8.0) at room temperature for 1 hour. After adding 50 ml of concentrated concentrated ammonia at room temperature, stirring overnight, extols off the water and ammonia after dissolving in 15 ml of water. The 3'-O-azide methyl DGTP was purified with EtOAc (EtOAc) / TEAB.

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Abstract

The invention belongs to the field of high-throughput sequencing, and discloses a three-color high-throughput sequencing reagent and a sequencing method. The method comprises the steps that corresponding to-be-labeled basic groups and proton sponge are obtained on the basis of sequencing requirements and stirred for multiple times under different temperatures and environments, and solids obtained through filtering are dissolved in water; a TEAB gradient is used as a negative ion exchange column to purify a solid dissolved in water, and then HPLC (High Performance Liquid Chromatography) is used for purifying to obtain a labeled basic group; the labeled basic group is cleaned and then subjected to fluorescence labeling determination, after determination is completed, a corresponding chemical reagent is used for splitting, sequencing of the current to-be-labeled basic group is completed after cleaning, the processes of reaction, cleaning, basic group reading, splitting and cleaning are circularly conducted on each sequencing process, the used three-color sequencing technology accelerates the sequencing speed like a two-color sequencing technology, base signals are not separately detected, and the sequencing length is not influenced and is increased, so that the sequencing efficiency and the sequencing flux are improved.

Description

Technical field [0001] The present invention relates to the field of high-throughput sequencing techniques, and more particularly to a three-color high-throughput sequencing reagent and sequencing method. Background technique [0002] Currently (80% high-volume DNA sequencing market) high-throughput DNA sequencing technology is 4 color sequencing techniques and 2 color sequencing techniques, and Illumina and Huada gene are used in 4 color sequencing techniques and 2 color sequencing techniques. The 4-color sequencing technique is an ACGT four bases each with a color dye mark to detect the sequencing corresponding base, with a total of four different wavelengths of fluorescent dyes to be sequenced. The 2-color sequencing technique is the first color and the second color to respectively labeled two bases in the four bases, and the third base is labeled with the first color and the second color, respectively, and the fourth base. Base is not marked. The first color and second color ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/96
CPCG01N30/96
Inventor 向国兵吕华
Owner NANJING SUPERYEARS GENE TECH CO LTD
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