Primer-free gene synthesis method

Gene synthesis is an efficient and cost-effective alternative to molecular cloning for custom gene production, where the DNA is manufactured by assembling strings of oligos together.Gene synthesis is the process of chemically synthesizing double-stranded DNA molecules in vitro. The main concept of gene synthesis is to assemble custom oligos into long DNA molecules. Email :marketing@medicilon.com.cn web: www.medicilon.com

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.

The present invention relates to a method of gene synthesis, particularly to a primer based on free gene synthesis method of library synthesis gene belongs to the technical field. Background technique

In recent years, synthetic biology, in particular studies on gene synthesis technology very quickly. At present, the whole gene synthesis in accordance with the nucleotide sequence of a protein, the first designed and synthesized overlapping single-stranded oligonucleotides, and then spliced ​​by overlap extension PCR method of the full-length gene sequence, without this gene synthesis process template, is one of the effective means to obtain genes.

So far, it has been reported that the gene synthesis methods are dependent on the chemical synthesis of the oligonucleotide primers, including the following methods: (1) chemical synthesis, chemical synthesis because the principle itself, there is a degree of defects, these defects determines the chemical synthesis of DNA fragments can not be too long, a chemical synthesis method is mainly used for synthesis of oligonucleotides; (2) synthesis of PCR mediated methods, this method can synthesize large DNA fragments, and the cycle It is relatively short, but the error rate of synthesis of PCR-mediated relatively high, for the synthesis of DNA greater than 2kb above its higher error rates, so in order to get the correct cloning large DNA fragments, may be necessary to repeat the cloning and sequencing of work this extra increase in the cost of gene synthesis, but also because it is mediated PCR method requires a high temperature DNA polymerase and dNTP and other reagents, it will also increase the cost of gene synthesis; (: :) 3 non-PCR-mediated method So far the reported non-PCR-based DNA synthesis method is mainly solid-phase synthesis of DNA technology. Such solid phase synthesis technique, which greatly reduces the error rate of the synthesis, but due to the synthesis process requires the use of specially modified primers, which would lead to increased cost of the primer, resulting in a subsequent increase in cost of gene synthesis. At the same time, solid phase synthesis techniques rely on special equipment, can not meet the needs of ordinary laboratories and research units.

Advances in synthetic biology as, in particular, the deepening of gene synthesis technology research methods, is urgently needed to develop a new method of gene synthesis, to be able to establish a relatively low price, accurate and efficient synthesis in a short time the method for long gene, gene synthesis to solve the prior art that the error rate, synthesis and high cost. SUMMARY

The main object of the present invention is to provide a primer-free gene synthesis method;

To achieve the above object, the present invention provides a first pNew vector plasmid used for gene synthesis; its nucleotide sequence is shown in SEQ ID N0.33;

The present invention further provides a method of constructing the vector plasmid pNew, the method comprising:

(1) Construction of vector backbone to obtain a linear pNew nucleotide sequences shown in SEQ ID NO.21;

(2) Amplification of the nucleotide sequence of SEQ ID 5SGCK resistance gene fragment and the nucleotide sequence shown in SEQ ID N0.27 N0.32 shown in 3KCGS;

(3) the linear vector backbone pNew, 5SGCK resistance gene and resistance gene fragment was cloned into a complete 3KCGS carrier, that was the pNew vector plasmid.

The present invention is a plasmid as a template to pet23a to ori-PF / ori-PR as primers amplified by PCR linear vector backbone pNew (1.9kb), the nucleotide sequence as shown in SEQ ID N0.21; during PCR by primer ori-PR BseRI introduce restriction enzyme recognition sites.