Method for determining insertion site of transgenic line by re-sequencing technique

Abstract

The invention discloses a method for determining an insertion site of a transgenic line by a re-sequencing technique. The method comprises the following steps of (1) extracting DNA (deoxyribonucleic acid) of a genome of the transgenic line, wherein the total amount is at least 2g; (2) re-sequencing the genome, wherein the data size is at least 12G; (3) using a T-DNA sequence of a transgenic carrier as a template, and comparing and analyzing the obtained sequencing data, so as to obtain information of the insertion site; (4) according to the information of the insertion site, designing a primer, and performing PCR (polymerase chain reaction) verification, so as to determine the insertion site of each transgenic line. The method for determining the insertion site of the transgenic line has the characteristics of maturity, simplicity and high success rate, and can be widely applied to determine the insertion site of the transgenic line.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering. Specifically, the present invention relates to a new method for quickly determining the insertion site of a transgenic line by using resequencing technology. Background technique [0002] At present, there are many methods for flanking sequence separation, mainly including SON-PCR, TAIL-PCR and linker-PCR, etc. Recently, a method using DNA capture technology to obtain flanking sequences has emerged. SON-PCR and TAIL-PCR are PCR methods invented based on the principle of thermal asymmetry. Due to the randomness of the second strand synthesis and the fact that most transgenic lines are not single-copy, these two methods cause confusion in primer matching, resulting in useful The efficiency of fragments is not high; the adapter-PCR method can greatly improve the acquisition of effective fragments, and three methods for obtaining flanking sequences by adapter-PCR have been announced, but the...

AI Technical Summary

Problems solved by technology

SON-PCR and TAIL-PCR are PCR methods invented based on the principle of thermal asymmetry. Due to the randomness of the second strand synthesis and the fact that most transgenic lines are not single-copy, these two methods cause confusion in primer matching, resulting in useful The efficiency of fragments is not high; the adapter-PCR method can greatly improve the acquisition of effective fragments, and three methods for obtaining flanking sequences by adapter-PCR have been announced, but there are some shortcomings, such as: some methods use blunt-ended junctions, making The connection efficiency is low, which is not conducive to the later PCR amplification; although some methods use sticky end adapters to improve the connection efficiency, the types of enzymes to choose from are limited, the scope of application is not wide, and the versatility is not high; the use of DNA capture The method of obtaining the flanking sequence by technology requires high operation requirements, cumbersome steps, and high technical requirements, resulting in a low success rate

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