Light-fractured fluorescence-labeling reversible terminal compound and use thereof in DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid) sequencing

A fluorescent labeling and compound technology, applied in the field of medicine, can solve the problems of inability to identify DNA fragments, inability to attack phosphate groups, and reduce the length of sequencing, and achieve the effects of low price, increased yield, and reduced experimental difficulty.

Inactive Publication Date: 2015-10-28
GENEMIND BIOSCIENCES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there are generally two structures of reversible terminators: one is that the 3'-OH group is replaced by other groups, so that the 3'-OH loses the ability to attack the phosphate group and prevents the extension of the next base; the second is Although the structure does not replace the 3'-OH, but an inhibitory molecule is added at the nucleoside position. Due to steric hindrance, the 3'-OH cannot attack the phosphate group
It is worth mentioning that the base substituted by 3’-OH is often not easily recognized by DNA polymerase, thus affecting the sequencing length
Helicos has successfully synthesized terminators with unsubstituted 3’-OH, but the biggest problem is that after the fluorescent label is detached, a long molecule will be left on the base
As carryover molecules accumulate, DNA polymerases will no longer be able to recognize the DNA fragment, reducing sequence length

Method used

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  • Light-fractured fluorescence-labeling reversible terminal compound and use thereof in DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid) sequencing
  • Light-fractured fluorescence-labeling reversible terminal compound and use thereof in DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid) sequencing
  • Light-fractured fluorescence-labeling reversible terminal compound and use thereof in DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid) sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] A method for synthesizing a photocleavable fluorescently labeled reversible terminal compound, comprising any one of the following routes 1-5:

[0083] Synthetic route 1:

[0084]

[0085] Wherein, the reaction conditions of step (i) are: compound A.1 in the presence of tert-butyldimethylsilyl chloride (TBSCl), imidazole (imidazole) and N,N-dimethylformamide (DMF), room temperature, overnight, and then the reaction solution was di-tert-butyl methyl dicarbonate ((Boc) 2 O), in the presence of 4-dimethylaminopyridine (DMAP) and N,N-dimethylformamide (DMF), at room temperature overnight, to obtain compound A.2;

[0086] The reaction condition of step (ii) is: compound A.2 is in Mg(ClO 4 ) 2 , and tetrahydrofuran THF, reacted under the conditions of existence to obtain compound A.3;

[0087] The reaction conditions of step (iii) are: compound A.3 exists in NaH, N,N-dimethylformamide (DMF) and 4-thio-2-nitrobenzyl bromide (4-sulfo-2-nitrobenzyl bromide) Compound A.4 ...

Embodiment 2

[0132] A method for synthesizing a photocleavable fluorescently labeled reversible terminal compound, comprising any one of the following routes 1-5:

[0133] Synthetic route 1:

[0134]

[0135] Wherein, the reaction condition of step (i) is: Compound A.3 is mixed with 4-iodo-2-nitrobenzyl bromide (4-iodo-2-nitrobenzyl bromide), NaH and N,N-dimethylformamide ( DMF) was reacted to obtain compound A.9 under the condition that exists;

[0136] The reaction condition of step (ii) is: compound A.9 is in NaN 3 , CuI, L-proline, NaOH and DMSO were reacted to obtain compound A.10;

[0137] The reaction condition of step (iii) is: compound A.10 is in SiO 2 Compound A.11 is obtained by reacting under the condition of existence;

[0138] The reaction condition of step (iv) is: compound A.11 is in n-tetrabutylammonium fluoride (n-Bu 4 NF), the reaction under the condition that THF exists obtains compound A.12;

[0139] The reaction condition of step (v) is: compound A.12 is in p...

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Abstract

The invention provides a light-fractured fluorescence-labeling reversible terminal compound and use thereof in DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid) sequencing. According to the light-fractured fluorescence-labeling reversible terminal compound and the use thereof in the DNA or RNA sequencing, a light-fractured group is adopted as an inhibition group, and the inhibition group is disconnected from a basic group under the irradiation of certain wavelength; the departure process does not need the assistance of special catalysts or reducers and the like, and side reaction of assistant reagents with DNA polymerase and the basic group cannot occur, so that reversible termination is realized under mild conditions; and in addition, the departure process is free of any interference to a sequencing process, so that sequencing for long-segment single molecules is facilitated.

Description

technical field [0001] The invention relates to the field of medicine, in particular to a photocleavable fluorescent-labeled reversible terminal compound and its application in DNA or RNA sequencing. Background technique [0002] In order to meet the technical requirements of the third generation sequencing, a method of cyclic reversible termination (CRT) is needed to realize the extension of a single base to increase the sequencing speed. That is, when a base with an inhibitory group is added to the DNA chain, it can prevent the extension of the next base. Under mild conditions the inhibitory group can be removed allowing the DNA strand to continue to elongate. For each additional base, real-time sequencing of the DNA chain can be achieved by detecting the fluorescence it carries. Such a base with an inhibitory group is called a terminator. [0003] At present, there are generally two structures of reversible terminators: one is that the 3'-OH group is replaced by other ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H19/16C07H1/00C09K11/06G01N21/64C12Q1/68
CPCC07H1/00C07H19/073C07H19/173C09K11/06C09K2211/1007C09K2211/1029C09K2211/1044C09K2211/1059C09K2211/1074C09K2211/1088C12Q1/6869
Inventor 贺建奎赵陆洋葛良进徐国伟
Owner GENEMIND BIOSCIENCES CO LTD
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