Method for synthesizing DNA fragments and assembling synthetic genes in Escherichia coli through one-step method

Abstract

The invention provides a method for synthesizing DNA fragments and assembling synthetic genes in Escherichia coli through a one-step method. The method mainly comprises the following steps: constructing LIC vector plasmid LIC-A; preparing a LIC-A linear vector; constructing a receptor vector puc-Kana for assembling the DNA fragments; selecting target genes, and dividing small fragments of about 300bp for synthesizing; and assembling multiple small-fragment DNA into large-fragment genes by utilizing a Jinmen cloning reaction. The method is simple in operation, the operations of cloning and sequencing are not needed to be repeatedly performed, direct annealing is realized, the cloning efficiency is high, luminous report genes such as sfgfp and gfp are used, the screening method is intuitive, the error rate of the synthesis method is extremely low, the period of the synthetic genes is short, the cost is greatly saved, and the time is saved. Moreover, the method is suitable for synthesizing various genes and is particularly effective for large-gene synthesis.

Description

Technical field [0001] The invention relates to the study of a gene synthesis method, which is a one-step method for synthesizing DNA fragments and assembling synthetic genes in Escherichia coli, belonging to the technical field of genetic engineering. Background technique [0002] Synthetic biology is a branch of life science that has just emerged in the 21st century. In recent years, the research of synthetic biology has made rapid progress, especially gene synthesis technology. Whole gene synthesis is based on the gene sequence of a certain protein, designing and synthesizing overlapping single-stranded oligonucleotides, and then splicing the full length by overlapping extension PCR method. Gene synthesis does not require a template, and is one of the means to obtain genes. The molecular modification and artificial organization of genes through total gene synthesis is becoming a routine laboratory method. Therefore, it is very important to establish a method that can accurate...

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Problems solved by technology

[0003] As the research of synthetic biology becomes more and more popular, people's exploration of life is no longer limited to macro organisms, but starts from the simpler basic unit gene, and since the 1970s, molecular biology and genetic engineering Rapid development, so far the methods of DNA synthesis mainly focus on three methods: (1) chemical synthesis method, because the principle of chemical synthesis determines that the DNA fragments synthesized by chemical method cannot be too long, so it is mainly used for oligonucleotide synthesis Synthesis; (2) PCR-mediated synthesis method, this method can synthesize large DNA fragments, and the cycle is relatively short, but so far, the error rate of PCR-mediated synthesis method is relatively high, especially for the synthesis of more than 2kb Due to the high error rate of the above DNA, in order to obtain correct cloning, it is necessary to do repeated work of cloning and sequencing, which will be very costly; and because it is a PCR-mediated method, reagents such as DNA high-temperature polymerase and DNTP are required , so the cost will be relatively high; (3) non-PCR-mediated method, the non-PCR-based DNA synthesis method reported so far is mainly the solid-phase synthesis technology of DNA

This solid-phase synthesis technology has indeed greatly reduced the error rate in the synthesis, but because the primers need to be specially modified during the synthesis, and the method needs to build a library, the cost of the primers will be relatively high; This method can only add 3 bases each time, so the synthesis speed will be relatively slow, especially for the synthesis of large DNA fragments, the cycle is particularly long, although it can be automated, it is not very practical for general laboratory research

[0004] In view of the high error rate and high synthesis cost in the current gene synthesis method, it is necessary to find a new gene synthesis method to solve the problems existing in the existing technology

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