A method of acquiring an unknown flanking sequence of a known sequence

Abstract

A method of acquiring an unknown flanking sequence of a known sequence is disclosed. The method includes 1) designing two specific primers according to the known sequence which are labeled as SP1 and SP2, with the SP2 carrying a restriction enzyme recognition site, and designing a pair of reverse PCR primers which are F1 and D1 between the SP2 and a border sequence, 2) amplifying to obtain a PCR product 1 by adopting genome DNA as a template and by using any one degenerate primer and the specific SP1 primer, 3) amplifying to obtain a PCR product 2 by adopting the PCR product 1 as a template and by using the specific SP2 primer and a degenerate primer, with the degenerate primer in the step 3) being same with the degenerate primer in the step 2), but comprising a restriction enzyme recognition site, and 4) subjecting the PCR product 2 to digestion, adding T4 ligase, cyclizing, and amplifying by adopting the cyclized product as a template and by using the F1 primer and the D1 primer to obtain a PCR product 3, thus acquiring the flanking sequence. The method is advantaged by simple and easy operation, a low cost, a high success rate, short time, and the like.

Description

technical field [0001] The invention relates to the field of genome unknown sequence amplification, in particular to a method for obtaining unknown sequences flanking known sequences. Background technique [0002] Flank amplification is a key technique for ascertaining gene sequences. Traditional methods for amplifying unknown sequences can be divided into two types: one is to circularize the sequence and design a reverse primer to amplify the unknown sequence. The enzyme used for digestion is species-specific, and the probability of effective amplification is low, which affects the success. rate; the other is to use specific primers and degenerate primers to amplify unknown sequences. This method has a lot of non-specific amplification, which affects the success rate. The present invention integrates two methods, firstly introduces an amalgamative primer to amplify the flanking sequence, and then amplifies the product after circularizing the product through the sticky end,...

AI Technical Summary

Problems solved by technology

Traditional methods for amplifying unknown sequences can be divided into two types: one is to circularize the sequence and design a reverse primer to amplify the unknown sequence. The enzyme used for digestion is species-specific, and the probability of effective amplification is low, which affects the success. The other is to use specific primers and degenerate primers to amplify unknown sequences. This method has a lot of non-specific amplification, which affects the success rate.

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