PRIMER-FREE GENE SYNTHESIS METHOD

摘要

The present invention relates to the field of artificial synthesis of genes. Disclosed is a primer-free gene synthesis method. First, disclosed is a pNew carrier plasmid, wherein the pNew carrier plasmid has a nucleotide sequence shown in SEQ ID NO.33. Also disclosed is a carrier bank that is constructed by using pNew carrier plasmids and contains 16384(47) plasmids. Further disclosed is a primer-free gene synthesis method. The method comprises the following steps: (1) grouping DNA sequences of target genes to be synthesized according to a length 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 using an antibiotic and carrying out reconstruction to obtain a plasmid containing a target gene sequence of 82bp; and (4) splicing the gene fragments by means of a golden-gate cloning reaction to obtain a complete target gene. The gene synthesis method of the present invention is free of primers, has low synthesis costs, very low mutation rate and high accuracy, can synthesize special gene sequences such as a highly repetitive sequence and Poly A, has simple operations and can realize automatic operations.