PCR efficient amplification method of long-sequence gene without base mutation

Abstract

The invention discloses a PCR efficient amplification method of a long-sequence gene without base mutation. The method is characterized in that an amplification system comprises 5 ul of 10*Buffer forKOD-Plus-Ver.2, 5 ul of 2mM dNTPs, 3 ul of 25mM MgSO4, 1.5 ul of Primer-F(10uM), 1.5 ul of Primer-R(10 uM), 1 ul of Template DNA, 1 ul of KOD-Plus-Neo:1 and the balance of ddH2O, wherein the total amount is 50 ul. The PCR process comprises the following steps: performing denaturation at 94 DEG C for 2 minutes; performing denaturation at 98 DEG C for 10 seconds; annealing at 60 DEG C for 30 seconds; extending at 68 DEG C for 1 minute; performing 4 circulations; extending at 68 DEG C for 10 minutes; and performing heat preservation at 4 DEG C. By the method, an accurate gene segment can be improved and guarantee is provided for the subsequent gene function.

Description

technical field [0001] The invention relates to the technical field of genetic engineering molecular cloning, in particular to a method for efficiently obtaining long-sequence genes without base mutations by PCR amplification and TA cloning. Background technique [0002] The type and sequence of bases are the carriers of biological genetic information. In organisms, the process of proofreading during DNA replication is very strict, and there are various mechanisms in the body to avoid and repair mismatches. [0003] With the development of biotechnology, in vitro gene amplification technology is becoming more and more perfect. In vitro gene amplification is based on DNA polymerase chain reaction. However, in the process of in vitro amplification, base mismatches are often caused by primer concentration, annealing temperature, template purity, cycle number and lack of 3'-5' exonuclease activity of DNA polymerase. [0004] As the amplified target fragment is longer, the DNA ...

AI Technical Summary

Problems solved by technology

Although the codon has degeneracy, base mutations in in vitro amplification are often missense mutations, which cause changes in amino acids and affect protein functions. Therefore, in the study of gene cloning and gene function analysis, avoid base mutations. Genetic mutation is the primary technical issue

[0005] However, in vitro PCR lacks these processes, so the mismatch rate of DNA replication in organisms is much lower than that of PCR. In the process of amplification using PCR technology in vitro, the accurate base pairing mainly depends on the DNA polymerase used. , a typical DNA polymerase has a mismatch rate of about 1×10 -5 / bp

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