Method for amplifying DNA by using thermophilic primase

Abstract

The invention relates to a method for amplifying DNA by using thermophilic primase, and belongs to the technical field of molecular biology. According to the method, thermophilic primase A is introduced into a reaction system, is TtDnaG2, and has an amino acid sequence represented by SEQ ID NO.1; the method comprises: denaturing double-stranded DNA molecule at a high temperature, cooling to synthesize RNA primers, heating, cooling to a temperature of 0 DEG C, standing, adding DNA polymerase, and extending at a certain temperature to achieve the amplification of the long DNA fragment; and the method is especially suitable for the amplification of whole genome DNA, has characteristics of simple operation and low cost, is suitable for amplifying large fragments or irregular DNA molecules so as to be used for whole genome sequencing, and has good application prospect.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for amplifying DNA by using a thermophilic primase. Background technique [0002] DNA amplification technology is widely used in current biological research. PCR technology uses thermophilic DNA polymerase, by adding artificially synthesized DNA primers, annealing at high temperature, denaturing at low temperature, and extending at an appropriate temperature. Amplification of specific DNA fragments. PCR technology has been greatly developed at present, but due to the limitations of the current technology, to amplify a specific DNA fragment, to obtain a DNA fragment of a specific length and sequence, the amplification product can only be a fragment of 20kbp at most. Amplify. For the amplification of some longer DNA fragments (usually whole genome DNA), methods such as MDA, MALBAC, and DOP-PCR are commonly used at present. site, to achieve overall amplification of longe...

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Problems solved by technology

Due to the defects of the random primer itself, the traditional amplification method itself also has a certain amplification preference, and with the increase of the number of amplifications, this preference will accumulate, and finally the amplification product will be uneven. DNA fragments are amplified many times, some may be less

Since a variety of DNA polymerases can also use RNA as primers to amplify DNA, it has been reported that primases are used to synthesize RNA, and DNA polymerases use RNA as primers to replace random primers for whole-genome amplification. Method (pWGA): T7pWGA utilizes the gp4 protein of T7 bacteriophage to simultaneously complete the two processes of unwinding and priming at room temperature, and completes the amplification of DNA molecules at room temperature by adding DNA polymerase and single-strand binding protein, but the The method is complicated to operate, and various proteins such as gp4, single-strand binding protein gp2.5, and T7 DNA polymerase need to be added, and gp4 is only in a specific DNA sequence (3'-CTGG(G / T)-5' or 3'-CTGTG -5') to synthesize RNA primers, so the practicability is relatively low, and the sequence specificity is high; T4pWGA is another method for DNA amplification using primase, which is purified in vitro by constructing a T4 phage replicon All 8 proteins of the T4 phage replicon were used to replicate DNA, but this method needs to use too many proteins, so it is only of theoretical significance; it has also been reported recently that a DNA can be used as a substrate. The method of amplifying the primer enzyme TthPrimPol with low sequence specificity has also obtained better results, but the primer enzyme used in this method still has certain sequence specificity.

[0004] If the specificity of the amplification priming sequence can be reduced, the application space of the priming enzyme in the field of DNA amplification can be greatly expanded, but there is no report on using the priming enzyme to amplify DNA non-specifically and efficiently

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