Method for quickly building differential expression gene library

Abstract

The invention discloses a method for quickly building a differential expression gene library. The method comprises the following steps of: firstly extracting total mRNA of infected goose parvovirus and uninfected goose parvovirus goose bursas of fabricius; synthesizing double strands cDNA for enzyme digestion by utilizing the obtained mRNA, and then separating into 2 pipes, respectively adding different adaptors for connecting; carrying out forward subtractive hybridization on the double strands cDNA with the adaptors, then carrying out reverse subtractive hybridization on a forward subtractive hybridization product, carrying out the primary PCR (polymerase chain reaction)) amplification after reverse subtractive hybridization, and carrying out the secondary PCR amplification by taking diluent as a template after diluting an amplification product; and finally carrying out denaturing gradient gel electrophoresis on a product of the second PCR amplification, and dying after electrophoresis to obtain the differential expression gene library of the goose parvovirus stress bursa of fabricius. The gene library built by the method does not have false positive. The method has the characteristics of high speed, high sensitivity, strong specificity, low cost, convenience in operation, and the like.

Description

[0001] technical field [0002] The invention belongs to the field of biotechnology and relates to a method for rapidly constructing a differentially expressed gene library. Background technique [0003] 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 indu...

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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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