Primer-free gene synthesis method

Abstract

The invention discloses a primer-free gene synthesis method and belongs to the field of artificial synthesis of genes. The invention discloses a pNew carrier plasmid, and the pNew carrier plasmid has a nucleotide sequence shown in the formula of SEQ ID NO.33. The invention also discloses a carrier bank which is constructed from pNew carrier plasmids and contains 16384(47) plasmids. The invention further discloses a primer-free gene synthesis method. The primer-free gene synthesis method comprises the following steps of 1, carrying out grouping on targeted gene DNA sequences to be synthesized according to a ratio of 82bp per fragment, 2, finding plasmids corresponding to the grouped fragments in the constructed plasmid bank, 3, carrying out enzyme digestion on the corresponding plasmids, carrying out screening by antibiotic and carrying out reconstruction to obtain a plasmid containing 82bp of the targeted gene sequences, and 4, splicing the gene fragments by a golden-gate cloning reaction to obtain a complete targeted gene. The primer-free gene synthesis method is free of primers, has a low synthesis cost, a very low mutation rate and high accuracy, can synthesize special gene sequences such as a highly repetitive sequence and Poly A, has simple processes and can realize automatic operation.

Description

technical field [0001] The invention relates to a gene synthesis method, in particular to a library-based primer-free gene synthesis method, which belongs to the technical field of gene synthesis. Background technique [0002] In recent years, research on synthetic biology, especially gene synthesis technology, has progressed very rapidly. At present, the whole gene synthesis is based on the nucleotide sequence of a certain protein, firstly designing and synthesizing overlapping single-stranded oligonucleotides, and then splicing the full-length gene sequence by overlapping extension PCR method. Template is currently one of the effective means to obtain genes. [0003] So far, the gene synthesis methods that have been reported all rely on chemically synthesized oligonucleotide primers, mainly including the following methods: (1) chemical synthesis, because the principle of chemical synthesis itself has defects to a certain extent, These defects determine that the DNA fragm...

AI Technical Summary

Problems solved by technology

This solid-phase synthesis technology greatly reduces the error rate of synthesis, but due to the need to use specially modified primers in the synthesis process, this will lead to an increase in the cost of primers, which will lead to an increase in the cost of subsequent gene synthesis

At the same time, solid-phase synthesis technology relies on special instruments and equipment, which cannot meet the needs of ordinary laboratories and scientific research units

[0004] With the progress of synthetic biology research, especially the continuous deepening of the research on gene synthesis technology, it is urgent to develop a new gene synthesis method to establish a relatively low price, accurate and efficient synthesis in a short period of time. The method of long-segment genes to solve the problems of high error rate and high synthesis cost in the existing gene synthesis technology

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