Nucleic acid synthesis method based on bidirectional isothermal extension

Nucleic acid synthesis method based on bidirectional isothermal extension

  • CN 104,212,791 A
  • Filed: 06/03/2013
  • Published: 12/17/2014
  • Est. Priority Date: 06/03/2013
  • Status: Active Application
First Claim
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1. , based on the nucleic acid synthesis methods that two-way isothermal extends, comprise the following steps:

  • Set up an extension system, extend system by an initial double-strand, one group of mutually different and oligonucleotide that can splice in order, one containing connecting, the mixed enzyme of polymerization and restriction enzyme enzymic activity, and supporting reaction buffer forms with mixed enzyme;

    Under the cooperation of multiple enzyme, these oligonucleotide are initially carry out isothermal splicing with start-of-chain, synthesis target dna long-chain;

    Each oligonucleotide in described oligonucleotide group is the hairpin structure with 2-10 base overhang, and hairpin structure inside is containing an endonuclease recognition sequence, and the 5'"'"' end of oligonucleotide does not have phosphorylation;

    Described splicing is as follows;

    Initial double-strand is connected with certain hairpin structure existed in system by ligase enzyme, and polymerase extension makes hairpin structure open, and restriction endonuclease sites becomes double-strand;

    Restriction enzyme cuts the site becoming double-strand;

    Then extend and undertaken by 3 step cycle below, until synthesis target target dna long-chain;

    (1) end that cutting produces is connected with certain hairpin structure existed in system by ligase enzyme;

    (2) polymerase extension makes hairpin structure open, and restriction endonuclease sites becomes double-strand;

    (3) restriction enzyme cuts the site becoming double-strand.

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