Method for analyzing impurities of oligonucleotide sequence based on high-throughput sequencing and application

An oligonucleotide, high-throughput technology, applied in biochemical equipment and methods, microbial measurement/testing, library creation, etc.

Active Publication Date: 2019-03-26
SUZHOU GENESCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved in the present invention is how to quickly, accurately and comprehensively analyze the composition and purity and / or content of each component sequence in the artificially synthesized oligonucleotide sequence

Method used

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  • Method for analyzing impurities of oligonucleotide sequence based on high-throughput sequencing and application
  • Method for analyzing impurities of oligonucleotide sequence based on high-throughput sequencing and application
  • Method for analyzing impurities of oligonucleotide sequence based on high-throughput sequencing and application

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Embodiment 1

[0063] Example 1. A method for analyzing oligonucleotide sequence impurities based on high-throughput sequencing

[0064] 1. Oligonucleotide plus poly tail

[0065] 1. Add a poly A tail to the 3' end of the oligonucleotide sequence, and prepare a tailing reaction system according to the reagents and dosage in Table 1. The oligonucleotide sequence was as follows: 5'-CAGAGCAGCTTGTCTTTTCTTC-3' (SEQ ID NO: 5). Oligonucleotide sequences were synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0066] Table 1 is the tailing reaction system

[0067] Reagent

Amount added (μL)

nuclease free water

13

Oligonucleotides (100 μM)

1

dATP (25μM)

0.5

5×TdT Buffer

4

terminal transferase

1.5

total capacity

20 μL

[0068] 2. React at 37°C for 25 minutes.

[0069] 3. React at 70°C for 10 minutes to inactivate terminal transferase.

[0070] 2. Reverse extension and amplification

[0071] 1. Add the reagents sh...

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Abstract

The invention discloses a method for analyzing impurities of an oligonucleotide sequence based on high-throughput sequencing and application. The method comprises the following steps: constructing a high-throughput sequencing library for analyzing impurities of an oligonucleotide sequence; performing high-throughput sequencing on the high-throughput sequencing library, and analyzing components ofthe oligonucleotide sequence according to the sequencing result, wherein the extension primer sequence used in constructing the high-throughput sequencing library sequentially consist of DNA molecules, N basic groups A or basic group T or basic group C or basic group G as shown in 1-22 positions of a sequence 2; and N is an integer greater than or equal to 6. Experiments prove that the method foranalyzing impurities of an oligonucleotide sequence based on high-throughput sequencing can be used for quickly, accurately and comprehensively analyzing the purity and content of each component in the oligonucleotide sequence.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method and application for analyzing impurities in oligonucleotide sequences based on high-throughput sequencing. Background technique [0002] In recent years, more and more pharmaceutical companies at home and abroad have set foot in gene medicine, investing huge sums of money in the development of new gene medicine to fight against various diseases. Antisense oligodeoxynucleotide technology is a treatment method that uses artificially synthesized or biosynthesized DNA or RNA that can complement RNA to block and inhibit the expression of genes related to the occurrence of diseases. It can be used to treat diseases caused by gene mutations. Tumor or genetic disease. [0003] Antisense technology is a new drug development method. The drugs developed by this technology are called antisense drugs, which involve antisense DNA, antisense RNA and ribozyme. According to the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C40B50/06
CPCC12Q1/6869C40B50/06C12Q2535/122C12Q2525/173C12Q2525/191C12Q1/6806C12Q1/6848C12Q2521/107C12Q2525/161C12N15/1093
Inventor 张佩琢郭良让黄天富
Owner SUZHOU GENESCI CO LTD
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