US 11,597,922 B2 · Method for analyzing impurities of oligonucleotide sequence based on high-throughput sequencing and application

Abstract

The present invention provides a method for analyzing impurities of an oligonucleotide sequence based on high-throughput sequencing. The method of the present invention comprises the following steps: constructing a high-throughput sequencing library for analysis of impurities of an oligonucleotide sequence; subjecting the high-throughput sequencing library to high-throughput sequencing, and analyzing the nucleotide sequence components according to the sequencing results; the sequence of the extension primer used in the construction of the high-throughput sequencing library consisting of the DNA molecule set forth in positions 1-22 of SEQ ID NO: 2 and N bases (A, T, C or G) in sequence; and N being an integer greater than or equal to 6. It is proved by experiments that the method for analyzing impurities of an oligonucleotide sequence based on high-throughput sequencing of the present invention can quickly, accurately, and comprehensively analyze the purity and content of each component in the oligonucleotide sequence.

Claims10

Dependent claims

A method for constructing a high-throughput sequencing library for analysis of impurities of an oligonucleotide sequence, comprising the following steps:

1) Adding a poly tail to the 3′ end of an oligonucleotide sequence to be detected to obtain a poly-tailed product;
2) Performing reverse extension and amplification on the poly-tailed product to obtain a reverse extension and amplification product;
the primers used for the reverse extension and amplification consisting of the oligonucleotide to be detected and an extension primer;
the extension primer sequence consisting of the DNA molecule set forth in positions 1-22 of SEQ ID NO:
2 and N identical bases (A, T, C, or G) in sequence, wherein the N bases are complementary to the bases of the poly tail with N being an integer greater than or equal to 6;
3) Precipitating the reverse extension and amplification product to obtain a precipitated product;
4) Performing end repair, A-tailing reaction, adapter ligation and PCR amplification on the precipitated product sequentially to obtain a high-throughput sequencing library.

4 more elements

Dependencies are inferred from the claim text.

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

Life of this patent

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

Life of this patent

Abstract

Claims10