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Nucleic acid synthesis method based on bidirectional isothermal extension
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A synthetic method and isothermal technology, applied in DNA preparation, recombinant DNA technology, etc., can solve problems such as difficult high-throughput synthesis, wrong hybridization, and inability to splice
Active Publication Date: 2014-12-17
WUXI QINGLAN BIOLOGICAL SCI & TECH
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[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 endonucleasedigestion and ligation), so it is difficult to use for high-throughput synthesis
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[0122] The starting double strand is a pJW vector digested by BglI, the pJW vector is a modified pUC19 vector, the BtsI and BglI sites on it have been removed through mutation treatment, and two BglI sites have been introduced into the multi-cloning site, After digestion with BglI, the two ends of this linear vector are the 3' hanging sticky ends of 'AGG' and 'GGT' respectively.
[0123] The designed oligonucleotides are listed in Table 4.
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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 DNAlong 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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