Fluorescence-labeled azo-modified nucleotide and application thereof in DNA sequencing

A fluorescent labeling and nucleotide technology, applied in the field of DNA sequencing, can solve the problems of insufficient shearing conditions, low efficiency, short read length, etc.

Active Publication Date: 2015-11-25
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the existing cleavable junction units have many disadvantages, such as the shearing conditions are not mild enough, the efficiency is not high, and the read length is too short when used for sequencing.

Method used

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  • Fluorescence-labeled azo-modified nucleotide and application thereof in DNA sequencing
  • Fluorescence-labeled azo-modified nucleotide and application thereof in DNA sequencing
  • Fluorescence-labeled azo-modified nucleotide and application thereof in DNA sequencing

Examples

Experimental program
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Effect test

Embodiment 1

[0074] Embodiment 1, azo linking unit (a 1 )Synthesis

[0075] The synthesis schematic diagram of the azo linkage unit in this embodiment is as follows image 3 As shown, where m is 2 and n is 1, the specific steps are as follows:

[0076] (1) Synthesis of 1c: 1a (710 mg, 2 mmol) was weighed and dissolved in 5 mL of water, and 0.5 mL of concentrated hydrochloric acid was slowly added in an ice bath. Sodium nitrite (145mg, 2.1mmol) was weighed and dissolved in 10mL of water, slowly added dropwise to the above reaction solution at 0°C, and stirred for 40min. Weigh 1b (694mg, 2mmol) and sodium hydroxide (80mg, 2mmol), add 15mL ethanol and 45mL water into an ice-water bath and stir to dissolve. The generated diazonium salt was slowly added dropwise to the aqueous sodium hydroxide solution of 1b in an ice-water bath, stirred for 5 hours, and a large amount of precipitate was formed. After filtration, the solid was washed three times with 10 mL of water, and dried to obtai...

Embodiment 2

[0078] Embodiment 2, azo linking unit (a 2 )Synthesis

[0079] The synthesis schematic diagram of the azo linkage unit in this embodiment is as follows Figure 4 As shown, where m is 2 and n is 1, the specific steps are as follows:

[0080] (1) Synthesis of 2c: 1a (710 mg, 2 mmol) was weighed and dissolved in 5 mL of water, and 0.5 mL of concentrated hydrochloric acid was slowly added in an ice bath. Sodium nitrite (145mg, 2.1mmol) was weighed and dissolved in 10mL of water, slowly added dropwise to the above reaction solution at 0°C, and stirred for 40min. Weigh 2b (682 mg, 2 mmol) and sodium hydroxide (80 mg, 2 mmol), add 15 mL of ethanol and 45 mL of water into an ice-water bath and stir to dissolve. The generated diazonium salt was slowly added dropwise to the aqueous sodium hydroxide solution of 2b in an ice-water bath, stirred for 4 hours, and a large amount of precipitate was formed. After filtration, the solid was washed three times with 20 mL of water, and d...

Embodiment 3

[0082] Embodiment 3, azo linking unit (a 3 ) and (a 4 )Synthesis

[0083] The synthesis schematic diagram of the azo linkage unit in this embodiment is as follows Figure 5 As shown, where m is 2 and n is 1, the specific steps are as follows:

[0084] (1) Synthesis of 3c: 2a (814 mg, 2 mmol) was weighed and dissolved in 10 mL of water, and 0.5 mL of concentrated hydrochloric acid was slowly added in an ice bath. Sodium nitrite (145mg, 2.1mmol) was weighed and dissolved in 10mL of water, slowly added dropwise to the above reaction solution at 0°C, and stirred for 40min. Weigh 2b (682 mg, 2 mmol) and sodium hydroxide (80 mg, 2 mmol), add 15 mL of ethanol and 45 mL of water into an ice-water bath and stir to dissolve. The generated diazonium salt was slowly added dropwise to the aqueous sodium hydroxide solution of 2b in an ice-water bath, stirred for 4 hours, and a large amount of precipitate was formed. After filtration, the solid was washed three times with 20 m...

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Abstract

The invention discloses a fluorescence-labeled azo-modified nucleotide and application thereof in DNA sequencing. The nucleotide has a structural formula as shown in a formula I, wherein R1, R2, R3, R4, R5 and R6 may not synchronously be H, and m and n are integers of 0 to 10. Compared with the prior art, novel reversible terminators based on azo connection units are synthesized; a denaturing PAGE gel, i.e. a sequencing gel proves that the reversible terminators can only extend a reversible terminator at a time and has extension efficiency of 100% when a template is a plurality of continuous same basic groups; thus, under the action of a reducing agent, highly-efficient, rapid and full shearing can be realized, and great potential and value applied in the DNA sequencing are obtained; meanwhile, raw materials needed by synthesis of the azo-modified nucleotide are simple and easily available, and synthetic process is a conventional chemical reaction and can be used for popularization and application.

Description

technical field [0001] The invention belongs to the technical field of DNA sequencing, and in particular relates to a fluorescently labeled azo-modified nucleotide and its application in DNA sequencing. Background technique [0002] DNA sequencing technology is one of the important means in modern biological research. After the completion of the Human Genome Project, DNA sequencing technology has developed rapidly. DNA sequencing (DNA sequencing) refers to the analysis of the base sequence of a specific DNA fragment, that is, the arrangement 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 to biological and medical sciences. [0003] Sequencing by synthesis (Sequencing By Synthesis, SBS) is one of the next-generation DNA sequencing technologies. The sequencing by synthesis method immobilizes a large number of template DNA fragments to be tested, hybridizes ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H19/10C07H19/20C07H1/00C07C245/08C09K11/06C12Q1/68
Inventor 沈玉梅谭连江邵志峰龚兵杨晴来李小卫
Owner SHANGHAI JIAO TONG UNIV
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