Industrialized 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 discloses a method of gene synthesis industrialization, comprising the steps of: (1) to be synthetic DNA sequence analysis and optimization, to obtain the target DNA sequence; (2) according to the target DNA sequence, the oligonucleotide is designed to extend nucleotide chain; (3) the synthesis and purification of oligonucleotide; (4) splice oligonucleotides to form DNA fragments; (5) the DNA fragment was cloned into the vector and the recombinant plasmid; (6) to recombinant plasmids were sequenced; (7) the correct sequencing recombinant plasmid quality verification. The present invention has a high success rate, high throughput, high speed, low cost, and simple in design, wide range of applications, operating standardization can promote an industrial scale, help reduce costs, shorten cycle time and increase the synthesis of synthetic quality.

As one of the basic techniques of molecular biological manipulation, molecular cloning has been successfully used more than twenty years, is a necessary means scientists have conducted gene cloning, detection, recombinant expression and functional studies, the development of bio-engineering has played a revolution sexual role. In the era of rapid development of today's gene, conventional molecular cloning techniques can not meet the requirements for efficient and accurate genetic manipulation researchers need, the emergence of gene synthesis technologies is to fill the vacancy, an important technical means to obtain the target gene. Obtained by gene synthesis the gene of interest, can be obtained not only does not exist in nature or very low levels of gene template sequence, and can improve the expression level · to optimize or modified to achieve the purpose of gene function by sequence.

The gene synthesis technology for human-modified organisms has opened up a new direction, in any area associated with genes are required for the synthesis of artificial genes, and utilization of plasmid or viral vector delivery to target cells or tissues, in order to achieve a variety of purposes. With the development of proteomics, bio-pharmaceutical and other industries deepening of the study, the whole gene synthesis demand is also increasing. In the foreseeable future, gene synthesis will play a significant role in the field of life sciences and new energy, new materials, artificial life, DNA vaccines, biological medicine and the like.

The gene synthesis, there are two ways, one synthetic gene synthesis company's business, and second, their own local laboratory synthesis genes. Lab own synthetic genes generally require reference academic articles, synthesized according to the method references, but most are just synthesis of individual genes, and the cost is high, the success rate is low, and therefore have limited reference value. The synthetic gene synthesis company's business is the professional and technical personnel to operate, and a synthetic short period of restriction sites may or gene sequence of the gene modification, codon optimization can also be designed in accordance with their wishes researchers give nature is difficult to obtain even non-existent advantage genes, so I decided to synthetic gene synthesis company's business become the main channel for gene synthesis.

The present gene synthesis consists of three steps: First, the chemical synthesis of oligonucleotide fragments, usually synthetic oligonucleotide fragments 150bp~200bp; the second is enzymatically chemically synthesized oligonucleotides manual stitching, get a longer sequence; third is a long sequence, based on further DNA sequences spliced ​​get longer, until it reaches the required length. Artificial gene synthesis, splicing oligonucleotide is a major rate-limiting step of the synthetic gene. Commonly used gene-splicing methods are the following two kinds:

The first method is to connect enzyme, an oligonucleotide is first activated, necessary to bring the 5'-phosphate group, and then annealed with respective complementary oligonucleotide fragments with sticky ends formed double-stranded oligonucleotide fragments, then ligase (T4DNAS Ligase, Taq Ligase, etc.) to connect them to each other to form a complete gene or a large fragment of the gene (Khorana HG.Science.V203, 614-625.1979). Since all ligases require 5'-phosphorylated and 3'-hydroxyl terminal connection, so before the reaction, the primers used for intermediate connection needs to be 5'-phosphate treatment, which makes the cost of gene synthesis greatly increase. Meanwhile, in order to ensure the efficiency of the synthesis, was synthesized ligase single longer sequence length is restricted, usually not more than 500bp.

The second method is PCR method, the two long oligonucleotides complementary nucleotide 3'-terminal fragments anneal to each other, the resulting single-stranded DNA as a template in the Klenow fragment of E. coli DNA polymerase action, the corresponding complementary strand synthesis, double-stranded DNA fragment is formed, may be treated to insert an appropriate carrier. PCR method also commonly used in the synthesis of longer sequences, such as Smith et al (Hamilton 0.Smith, ClydeA.Hutchison III, Cynthia Pfannkoch et al.PNAS.V100, 15440-15445.2003) Tag Ligase After connecting a pair of primers, and then the connection was synthesized PCR products were obtained ΦΧ174 bacteriophage genome of the full length of 5386bp. PCR method does not require any modification of the oligonucleotide chain, and therefore costs than connecting enzymatic synthesis is greatly reduced, but due to a number of parallel oligonucleotide chain reaction, the longer the sequence to be synthesized, then extending the possibility of errors occur becomes. Isothermal unidirectional growth gene synthesis method (Patent No. ZL 200610028886.6) is a modified PCR method, the DNA oligonucleotides designed to join the hairpin and restriction sites, adding a number in the same reaction system enzymes include polymerase and exonuclease restriction enzyme, so that multiple DNA oligonucleotides way grown under the same temperature conditions, after the complementary role of hybridization in the three enzymes, and then the next round aggregation extension. The method uses a plurality of DNA oligonucleotide chain reaction simplifies operation under the same conditions, reduce the cost, but there are many shortcomings, such as oligonucleotide design complexity, a variety of enzymes at the same time multi-step reaction leading to splice low efficiency, long DNA sequence of the synthetic gene synthesis success rate is low and the production of small, can not be directly used for downstream operations, such as cloning, ligation PCR amplification and so on and so again, it can not be used for longer genes, whole genes synthetic or genome, harder to promote in actual production.