Nucleic acid synthesis method based on bidirectional isothermal extension

Abstract

The present invention relates to a nucleic acid splicing method based on bidirectional isothermal extension. The method is as follows: an extension system is set up, the extension system comprises a starting double strand, a group of oligonucleotides which are mutually different and orderly spliced, mixed enzyme with connection, polymerization and restriction endonuclease activity, and a reaction buffer matching with the mixed enzyme; in the cooperation of multiple kinds of enzymes, and starting from the starting double strand, the oligonucleotides can be isothermally spliced for synthesis of a target DNA long chain. The nucleic acid splicing method has the characteristics of high success rate, simple design and simple operation, high automation, and the like, so that the nucleic acid splicing method has a potential low cost advantage, and has potential values of application in promotion of gene synthesis, and development of biological engineering, biomedical and bioinformatics fields.

Description

technical field [0001] The invention relates to a method in the field of nucleic acid synthesis, in particular to a nucleic acid splicing method for bidirectional isothermal extension. Background technique [0002] With the development of biotechnology and biomedicine, more and more attention has been paid to the design and modification of nucleic acid, especially DNA sequence. Traditional gene amplification, cloning, recombination and mutation methods can only obtain DNA sequences that exist in nature or are close to those in nature, while DNA chemical synthesis technology can synthesize user-specified oligonucleotide sequences, but due to the efficiency of chemical reactions And the problem of error rate, these oligonucleotide sequences are usually less than 120-200 bases in length. To obtain DNA sequences with a length of hundreds or even millions of bases, these oligonucleotides need to be spliced ​​together. The common splicing methods are: 1) ligase-based method (here...

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Problems solved by technology

[0006] All of the above methods require the use of overlapping oligonucleotides, so there is an endogenous problem of mishybridization, especially when there are repeated sequences or inverted repeats in the sequence, which can cause misligation or misligation. Mis-extension, or even complete splicing, both ligase and polymerase methods use only one enzyme (ligase or polymerase) during splicing, but more steps are required before transforming cells (ie, PCR amplification, gel-tapping purification, restriction endonuclease digestion and ligation), so it is difficult to use for high-throughput synthesis

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