Method for quickly building differential expression gene library

Abstract

The invention discloses a method for rapidly constructing a differentially expressed gene library, comprising the following steps: first extracting total mRNA of goose bursa infected with goose parvovirus and non-infected goose parvovirus; using the obtained mRNA to synthesize double-stranded cDNA for enzyme digestion Digestion, and then divided into 2 tubes, adding different adapters for connection; first perform forward subtractive hybridization on the double-stranded cDNA with adapters, and then perform reverse subtractive hybridization on the forward subtractive hybridization products, and then reverse subtractive hybridization Perform the first PCR amplification, dilute the amplified product, and use the diluted solution as a template to perform the second PCR amplification; finally, the product of the second PCR amplification is subjected to denaturing gradient gel electrophoresis, and stained after electrophoresis. The differentially expressed gene library of goose parvovirus stressing the bursa of Fabricius is obtained; the gene library constructed by the method of the invention has no false positive, and has the characteristics of fast speed, high sensitivity, strong specificity, low cost, convenient operation and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a method for rapidly constructing a differentially expressed gene library. Background technique [0002] Suppression subtractive hybridization (suppression subtractive hybridization, SSH) technology is designed by DiatchenkoL in 1996, based on suppression PCR and subtractive hybridization technology to find differentially expressed genes. It has the characteristics of stability and sensitivity, and is currently the most One of the most promising research tools, it has shown important value in the isolation and identification of new genes and has been widely used. However, this technique has the following defects: ① There are still about 10-40% false positives when the enriched cDNA fragments are cloned (Gurskaya NG, Diatchenko L, Chenchik A., et al. Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction : cloning of Jurkat cell transcripts induced by ph...

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Problems solved by technology

However, this technology has the following defects: ① There are still about 10-40% false positives when the enriched cDNA fragments are cloned (Gurskaya NG, Diatchenko L, Chenchik A., et al. Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction : cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate. Anal Biochem. 1996; 1: 90-97); ②Although this method enriched differentially expressed genes, some genes were repeatedly cloned, ③This method cannot predict how much library capacity can contain all the differential genes, so that the detected expressed genes are relatively small; ④This method has many steps and high cost, and ordinary laboratories are unable to carry out

However, due to the fact that DGGE technology "can detect differences but not enrich differences, and does not need differences", it cannot detect some genes with an abundance of less than 1%, and these genes with an abundance of less than 1% are often key functional genes. Unable to be "enriched" and not detected, so that it cannot be applied in the construction of differentially expressed gene libraries

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