Artificial synthesis method for long chain DNA

Abstract

The invention provides a method for synthesizing long chain DNA. The main point of the technology of the invention lies in that: the characteristics of the DNA secondary structure are introduced to primer design; combining a new step by step temperature reducing method (at the same time, joinase chain-reaction or T4 joinase chain-reaction are used), each primer positioned in same chain is matched with single-chain oligonucleotide primer of bridge-type and connected with each other sequentially; after step by step temperature reducing reaction (or 40 joinase chain-reaction or T4 joinase chain-reaction), a large amount of single chain DNA module is obtained, and then after PCR reaction, the required DNA double chain is amplified. The invention has low cost, simple steps, fast speed, low mutation rate and large application value.

Description

Technical field: [0001] The invention relates to a method for synthesizing nucleotides, in particular to a method for artificially synthesizing long-chain DNA. Background technique: [0002] Nucleotides have a wide range of application value in biotechnology. At present, the methods and principles of short-chain DNA synthesis mainly include the following methods: [0003] ①Artificially synthesize all short oligonucleotide primers (30-40mer) required for splicing into the entire double-stranded DNA. The design of oligonucleotide primers is based on the Tm value and the application of current computer software, and then the oligonucleotide Primers were phosphorylated, purified, and spliced. [0004] ② Use two-step PCR for synthesis reaction or another synthesis method to perform a ligation reaction with ligase before this step, and then further PCR amplification, and finally obtain the whole gene sequence. [0005] However, the above two synthetic methods have certain limita...

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Problems solved by technology

[0005] However, the above two synthetic methods have certain limitations:

[0006] ① There are a lot of oligonucleotide primers synthesized, and each length is less than 40 nucleotides. For example, a DNA double strand of 700 bps needs to synthesize at least 37 primers. In addition, the design of the primers must be within a certain Tm value range, otherwise the upper and lower The complementary regions of the two chains are difficult to pair, resulting in non-specific binding and synthesis failure

[0007] ②The cost of synthesis is higher

[0008] ③Complicated synthesis steps and high purification requirements

[0009] ④Two-step PCR will inevitably cause a higher mutation rate

[0010] ⑤The success rate is limited based on the secondary structure and total number of primers

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