Gene synthesis method

Abstract

The invention provides a gene synthesis method. The method comprises the steps that 1, a to-be-synthesized gene sequence is divided into a first continuous fragment with the length being 55-100 bp, the first continuous fragment is designed to be a forward primer, a gene sequence staggered with the first continuous fragment at the portion of the length of 15-20 bp serves as a second continuous fragment, and continuous reverse primers are designed on a reverse complementary sequence of the second continuous fragment; 2, the forward primer is mixed with the reverse primers; 3, the primer mixture is boiled, annealing is conducted, T4DNA ligase is added, and the primers are connected into a double-stranded DNA gene with the two ends provided with the long sticky tail ends; 4, the synthesized double-stranded DNA gene is connected to a pUC57-GFP-Amp carrier, the sequence of the pUC57-GFP-Amp carrier is shown as a sequence table 1, escherichia coli cells are converted, a non-fluorescing bacterial colony is reversely screened, and sequencing verification is conducted. Accordingly, the method does not reply on PCR amplification, and the defects that the experimental procedure in PCR amplification is tedious, and the PCR technical error rate is high are overcome.

Description

technical field [0001] The invention relates to the field of gene synthesis, in particular to a gene synthesis method. Background technique [0002] At present, gene synthesis technology generally uses DNA polymerase to simulate DNA replication in vitro. Primers with homologous sequences can extend each other under the action of DNA polymerase to obtain target DNA fragments. Finally, the obtained DNA fragments are separated and purified and cloned into Sequencing verification in the specified vector, we call this technology PCR-based gene synthesis technology. The principle of this technology is simple and easy to use, but the operation process is very complicated. The whole process needs to go through tedious experimental steps such as PCR system preparation, on-board amplification, electrophoresis detection and recovery, enzyme digestion and connection, transformation, etc. At the same time, the PCR process will The amplification of the mutation itself leads to synthesis ...

AI Technical Summary

Problems solved by technology

The principle of this technology is simple and easy to use, but the operation process is very complicated. The whole process needs to go through tedious experimental steps such as PCR system preparation, on-board amplification, electrophoresis detection and recovery, enzyme digestion and connection, transformation, etc. At the same time, the PCR process will The amplification of the mutation itself leads to synthesis errors; and high-level structures such as highly repetitive sequences, high GC, high AT, inverted repeats, and palindromes fail to be synthesized due to the failure of PCR to amplify normally, which has become the first choice of PCR-based gene synthesis technology A major difficulty and bottleneck

Corresponding to it is a large amount of manpower, material resources and time consumption, thus greatly limiting the synthetic throughput and per capita production capacity

With the continuous deepening of gene research, currently "reading genes" (gene sequencing) has a fully automatic high-throughput sequencer, but "writing genes" (gene synthesis) all rely on artificial synthesis, so cumbersome experimental steps, current technical means The inherent short board has become the speed-limiting step for writing genes

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