Method for sequencing unknown flanking sequences flanking known sequences

Abstract

A method for sequencing an unknown flanking sequence at both sides of a known sequence comprises library construction, flanking sequence capturing and sequencing. The method comprises: employing a site directed mutagenesis PCR process, designing an appropriate primer on the known sequence (without mutation sites), taking the constructed library as the template, screening numerous plasmids in the library to obtain a DNA fragment containing the known sequence sites, screening out target sites in the plasmid library by employing the PCR process, and further obtaining an insertion sequence 2 Kb-5 Kb and a plasmid library containing the known sequence, and taking the plasmid library as the target point to obtain the flanking sequence at the both sides of the known sequence. The provided sequencing method helps to realize full view acquisition of genome, helps to solve the technological restriction that researchers cannot describe the full view of a gene and consequently cannot precisely research the functions of the gene when carrying out comprehensive research on the gene, and is beneficial to improve the efficiency of related researches.

Description

technical field [0001] The invention relates to a polynucleotide sequencing method, in particular to a method for sequencing unknown flanking sequences on both sides of a known sequence, which is suitable for screening unknown sequences from genome libraries. Background technique [0002] In recent years, sequencing technology has developed rapidly and has become an important means of biological research. The rise of high-throughput sequencing technology has made it possible to study DNA sequences on a large scale and at low cost. Whole-genome Denovo sequencing is also called genome de novo sequencing, which refers to sequencing the genome of a species without relying on any known genome sequence information, and then applying bioinformatics methods to splice and assemble the sequencing sequences to finally obtain the genome sequence of the species Atlas. However, due to the huge data and complex structure of the whole genome, it has caused certain difficulties in the spli...

AI Technical Summary

Problems solved by technology

This method has cumbersome steps, and is limited by the efficiency of multi-step enzymatic reactions in actual operation, so the scope of application is limited; only the sequence of mRNA can be obtained, and the sequence before and after mRNA cannot be captured and analyzed; it depends on high-quality reverse transcriptase ( It has the ability to read through complex secondary structures, has a high reaction temperature) and high-quality RNA, and has high requirements for samples

[0006] For genome-wide flanking sequence capture and sequencing, researchers often encounter the situation that only a part of the DNA sequence is known when conducting research, and it is impossible to describe the whole picture of the gene when conducting a comprehensive study, and thus cannot accurately study its function

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