PCR (polymerase chain reaction) method for eliminating genes

Abstract

The invention discloses a PCR (polymerase chain reaction) method for eliminating genes, which can eliminate non-target genes in a mixed gene population. The key points of the technology disclosed by the invention are as follows: making or artificially synthesizing the non-target genes into short-sequence gene eliminating primers, performing enzyme cutting on the mixed gene population containing target genes, adding a restriction enzyme cutting site with a base pair mismatch and a joint with Poly (dA), purifying, using the mixed gene population as a template, restoring the correct enzyme cutting site through the gene eliminating primers and annealing extension of the template in the PCR, and then using the heat-resistant restriction enzyme cutting site to eliminate the joint of the non-target genes so as to prevent amplification of the non-target genes. The reaction is cycled by utilizing a PCR instrument: 30s at the temperature of 94 DEG C, 40s at the temperature of 60 DEG C, 8min at the temperature of 75 DEG C, 12-18 cycles are performed for enriching the target genes, and then the conventional PCR is adopted for further amplifying the target genes. The method disclosed by the invention has the advantages of high efficiency, high speed, simplicity and convenience in operation, low cost, high repeatability, good separation effect and low false positive rate.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a method for gene elimination PCR, in particular to a method for eliminating non-target genes in a gene population by PCR to achieve the purpose of separating target genes, and is mainly used in biology, medicine, Agronomy, animal husbandry and veterinary medicine, environmental science, food science and other technical fields of gene elimination and gene separation related to molecular biology. Background technique [0002] PCR, polymerase chain reaction (Polymerase Chain Reaction, PCR) is a technology invented by Kary Banks Mulis, a scientist of PE-Cetus Company in the United States in 1985, which can rapidly amplify a specific gene or DNA sequence in vitro. This new technology is based on the characteristics of rapid replication of DNA sequences in organisms, to achieve rapid amplification of certain specific DNA sequences in vitro, and to obtain millions of copies of specific D...

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Problems solved by technology

But at the same time, it also has unavoidable shortcomings such as high false positives: the excess of the driver-cDNA in the second subtraction hybridization can cover up the cDNA with a difference in the abundance of the tester-cDNA; in the SSH principle, it is considered that after the second hybridization, the Intra-strand annealing of fragments with the same adapter is better than inter-strand annealing, which makes the inverted repeat sequences at both ends of the fragments form a structure similar to "pan handle" and cannot be amplified. However, it has been confirmed in our experiments that "pan handle" can also efficiently amplify increase

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