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Photocleavage fluorescent labeling compound and application

A Fluorescent Labeling, Compound Technology

Inactive Publication Date: 2016-12-07
GENEMIND BIOSCIENCES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 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

Method used

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  • Photocleavage fluorescent labeling compound and application
  • Photocleavage fluorescent labeling compound and application
  • Photocleavage fluorescent labeling compound and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] A method for synthesizing a photocleavable fluorescently labeled reversible terminal compound, comprising any one of the following routes 1-5:

[0079] Synthetic route 1:

[0080]

[0081] Wherein, the reaction conditions of step (i) are: compound 10mmol 2.5g A.1 in tert-butyldimethylsilyl chloride (TBSCl) 4.82g 32mmol, imidazole (imidazole) 4.5g 66mmol and N,N-dimethylformazol Under the condition that amide (DMF) 30ml exists, room temperature, overnight, then reaction solution is di-tert-butyl methyl dicarbonate ((Boc) 2 O) 20mmol 4.36g, 4-dimethylaminopyridine (DMAP) 10mmol 1.22g and N,N-dimethylformamide (DMF) 30ml under the condition of existence, room temperature, overnight, after vacuum concentration, the residue was saturated with Ammonium chloride solution washed twice (50ml each time), using CH 2 Cl 2 Extract the combined aqueous layer, dry the combined organic layer with sodium sulfate, concentrate in vacuo, and purify by silica gel column chromatography...

Embodiment 2

[0160] A method for synthesizing a photocleavable fluorescently labeled reversible terminal compound, comprising any one of the following routes 1-5:

[0161] Synthetic route 1:

[0162]

[0163] Wherein, the reaction condition of step (i) is: 2.9g 5mmol compound A.3 in 5.1g 15mmol 4-iodo-2-nitrobenzyl bromide (4-iodo-2-nitrobenzyl bromide), 20mgNaH and 50mlN,N-di The reaction was stirred at room temperature for 4 h in the presence of methylformamide (DMF), dried over sodium sulfate, concentrated in vacuo, and purified by silica gel column chromatography to obtain Compound A. 93.5 g (84%);

[0164] In the above step (i), the 4-iodo-2-nitrobenzyl bromide added can be any value in 10-20 mmol;

[0165] The reaction condition of step (ii) is: the compound A.9 obtained in step (i) is in NaN 3 , CuI, L-proline, NaOH and DMSO were reacted for 2 h, concentrated in vacuo, and purified by silica gel column chromatography to obtain 2.9 g (91%) of compound A.10;

[0166] In the abov...

Embodiment 3

[0234] In order to detect whether the reversible terminal synthesized by the present invention can be applied to DNA / RNA sequencing, this example further tested the A.8, G.8, C.8, T.8, U. 8. Two characteristics of the reversible terminal compounds of A.14, G.14, C.14, T.14, U.14:

[0235] 1) Whether it can be recognized by DNA polymerase and participate in DNA extension reaction as a substrate of DNA polymerase;

[0236] 2) Whether the fluorescent group carried by the reversible terminal can be removed after participating in DNA chain extension, so as to facilitate the next round of extension reaction.

[0237] These two aspects are at the heart of single-molecule high-throughput sequencing-by-synthesis (SMTS). details as follows:

[0238] 1) Preparation of DNA extension reaction system: Mix different reversible terminals in sequence with DNA template (known sequence information and known quality), Klenow (exo-) DNA polymerase, Klenow buffer, and stand at 30°C for 15 minutes...

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Abstract

The invention provides a photocleavage fluorescent labeling reversible terminal compound and application of the compound in DNA or RNA sequencing. Photocleavage groups are adopted as inhibition groups, and the inhibition groups are disconnected from basic groups under irradiation of a certain wavelength. The separation process does not need assistance of special catalysts or reduction agents and the like, side reactions of auxiliary reagents, DNA polymerase and the basic groups do not exist, and accordingly reversible termination can be achieved under mild conditions. Besides, the separation process has no interference with the sequencing process, and long-fragment single-molecule sequencing is benefited.

Description

[0001] This application requires the application number 201510411725.4 submitted to the Chinese Patent Office on July 14, 2015, and its invention title is "a photocleavable fluorescently labeled reversible terminal compound and its use in DNA or RNA sequencing" Chinese patent The priority of the application, the entire content of which is incorporated in this application by reference. technical field [0002] The present invention relates to the field of compounds, and relates to a photocleavable fluorescent labeling compound and its application, in particular to a photocleavable fluorescent labeling reversible terminal compound and its application in DNA or RNA sequencing, especially to a photocleavable fluorescent labeling compound and its application in DNA or RNA sequencing. Labeled compounds and their uses. Background technique [0003] In order to meet the technical requirements of the third generation sequencing, a method of cyclic reversible termination (CRT) is need...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H19/20C07H19/10C07H1/00C07H1/04C09K11/06C12Q1/68
CPCC07H1/00C07H19/073C07H19/173C09K11/06C09K2211/1007C09K2211/1029C09K2211/1044C09K2211/1059C09K2211/1074C09K2211/1088C12Q1/6869
Inventor 葛良进赵陆洋徐国伟
Owner GENEMIND BIOSCIENCES CO LTD
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