A primer-free gene synthesis method based on plasmid library

Abstract

The invention provides a primer-free gene synthesis method based on a plasmid library, and the method is as follows: constructing a plasmid library containing 16384 plasmids (any combination sequence of four basic groups of 47 and 7bp), starting from a constructed plasmid containing 7 bp, dividing a selected target gene according to each 82bp, successively reconstructing four resistance genes Kana, cam, gem and spc by enzyme digestion and enzyme connection operation to obtain a plurality of 82bp plasmids, and assembling the 82bp plasmids into long fragment genes. According to the method, after construction of the plasmid library containing the 16384 plasmids (any combination sequence of four basic groups of 47 and 7bp), various target genes can be synthesized without any primer. The synthesis method has the advantages of simple operation, extremely low mutation rate, no need of sequencing, low cost and less amount of screening, is conductive to constructing a mutant library, can realize solution operation, and is easy to realize automation.

Description

Technical field [0001] The invention relates to a gene synthesis method, which is a primerless gene synthesis method based on a plasmid library, and belongs to the technical field of genetic engineering. Background technique [0002] In recent years, the research progress of synthetic biology is very fast, especially gene synthesis technology. Whole gene synthesis is based on the gene sequence of a certain protein, designing and synthesizing overlapping single-stranded oligonucleotides, and then splicing the full length by overlapping extension PCR method. Gene synthesis does not require a template, and is one of the means to obtain genes. The molecular modification and artificial organization of genes through total gene synthesis is becoming a routine laboratory method. Therefore, it is very important to establish a method that can accurately and efficiently design and synthesize long-segment genes in a relatively inexpensive and short time. [0003] As the research of synthetic...

AI Technical Summary

Problems solved by technology

The non-PCR-mediated synthesis method is mainly a kind of DNA solid-phase synthesis technology, such as Sloning technology. Although the error rate of this technology is very low, because the primer needs to be specially modified during synthesis, the primer The cost of synthesis is relatively high, and considering the cost, only 3 bases can be added each time, so the cycle of synthesizing large genes will be relatively long, so it is not practical

[0004] Gene synthesis technology will be applied in more and more fields and play a huge role, but the high error rate and high cost of bases in synthetic genes limit the application of whole gene synthesis methods, and it is necessary to find new Gene synthesis method to solve the problems existing in the existing technology, so that gene synthesis technology can be more widely used

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