A pcr method to obtain high-fidelity and "a" products at the 3' end

Abstract

The invention discloses a PCR method for obtaining high-fidelity products with "A" added at the 3' end. The method is as follows: firstly, each reaction component and high-fidelity Taq DNA polymerase are added to the PCR reaction system, and then the following reaction procedures are performed. PCR amplification: (1) 95°C pre-denaturation for 2min; (2) 95°C, 15sec; annealing temperature, 30sec; 72°C, extension time; 28 cycles; (3) Directly add ordinary Taq DNA polymerization to the reaction PCR tube Enzyme and mix; (4) 95°C, 15sec; annealing temperature, 30sec; 72°C, extension time; 3 cycles; (5) 72°C, 10min. In the present invention, ordinary Taq DNA polymerase is added in the last 3 cycles of PCR reaction using high-fidelity Taq DNA polymerase, and the PCR product with "A" added at the 3' end with high-fidelity can be obtained after the PCR is completed, which overcomes the current high-fidelity The problem that "A" cannot be directly added to the 3' end of the product during PCR eliminates the need to purify the high-fidelity PCR product before adding "A", which simplifies the processing steps before TA cloning of the high-fidelity PCR product.

Description

technical field [0001] The invention relates to a PCR method for obtaining high-fidelity products with an "A" added to the 3' end, belonging to the technical field of PCR methods. Background technique [0002] PCR technology is the most classic and commonly used experimental method in the field of molecular biology. It is mainly used for gene detection and cloning. One of the most popular methods in the field of biology. In terms of gene acquisition, although there are many methods to choose from, such as chemical synthesis, PCR technology has irreplaceable advantages and is still the most important method. [0003] The basic process of PCR technology for gene cloning is to first obtain the DNA template or cDNA template of the target gene, then design and synthesize the upstream primer and downstream primer of the target gene, and select the appropriate Taq DNA polymerase and PCR program to obtain the PCR product through efficient amplification , the target gene DNA, the p...

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Problems solved by technology

[0005] In summary, although the target DNA fragment amplified with ordinary Taq DNA polymerase has a protruding "A" at the 3' end, it often contains a mutation site inside; the PCR product amplified with high-fidelity Taq DNA polymerase has a 3' The ends are blunt ends, and TA cloning cannot be performed directly. It is necessary to use a special "A" kit to add "A" to the 3' end of the PCR product before TA cloning; while ordinary Taq DNA polymerase and high-fidelity Taq DNA Although the direct mixed use of polymerases (such as Taq Platinum DNA Polymerase of a biological company) partially solves the above problems, the high fidelity of PCR products and the efficiency of adding "A" at the 3' end of the product are significantly reduced

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