Isothermal PCR nucleic acid augmentative method based on dI modified primer and endonuclease V

Abstract

The invention relates to an isothermal PCR nucleic acid amplification method based on dI modified primer and endonucleaseV. The amplified target nucleic acid primer is completely matched with the template; 1 to 5 dI nucleic acids are on the 10 to 30 site at the 5' end; 10 to 30 nucleic acids are at the downstream of the dI nucleic acids. The reaction components of isothermal PCR comprise dI modified forward primer and reverse primer, target nucleic acid template, four kinds of deoxyribonucleoside triphosphates dATP, dTTP, dCTP and dGTP, DNA polymerase, endonucleaseV, single DNA chain binding protein and helicase. After the initial thermal denaturation and annealing hybridization, the mixture of isothermal PCR carries out the PCR reaction. During the amplification process, the circulation and regeneration of the primer-template compound is realized by endonucleaseV, which induces the DNA synthesis reaction; the single DNA chain binding protein and helicase can improve the efficiency of isothermal PCR. The amplification of the target nucleic acid of the invention does not rely on the thermal cycler; the invention has easy and convenient operation, which can be used for a large scale and high yield amplification of the target nucleic acid.

Description

technical field [0001] The invention relates to a new nucleic acid amplification method, in particular to an isothermal PCR nucleic acid amplification method based on dI modified primers and endonuclease V, which can be used for isothermal amplification of target nucleic acids and belongs to the technical field of genetic engineering. Background technique [0002] Polymerase Chain Reaction (PCR) is a routine molecular biology technique with numerous applications. Conventional PCR is mainly composed of three steps: 1) thermal denaturation of the target nucleic acid template molecule, 2) hybrid annealing between the free primer and the denatured target nucleic acid template molecule, 3) thermostable DNA polymerase catalyzed binding to the target nucleic acid template molecule The primers on the DNA synthesis reaction. The three-step reaction of conventional PCR is repeated in cycles to achieve exponential multiple amplification of the target nucleic acid. Routine PCR require...

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Problems solved by technology

[0004] The defects of the existing isothermal PCR technology mainly include: 1) the amplified product is a double-stranded DNA mixture of different lengths, which needs to be digested with restriction endonucleases to form DNA fragments of uniform length; 2) phi 29 DNA polymerase and Isothermal PCR catalyzed by T7 DNA polymerase can only amplify relatively short circular target nucleic acid template molecules, and often produces non-specific amplification bands; 3) Although isothermal PCR based on loop-structured primers can amplify highly complex line 4) Isothermal PCR based on loop-structured primers requires two pairs of primers, the operation of the amplification reaction is complicated, and the DNA synthesis reaction triggered by any pair of primers fails. Both will cause the failure of target nucleic acid amplification