A high-throughput DNA multi-site precise base mutation method

Abstract

The invention discloses a high-throughput DNA multi-site precise base mutation method. The method provided by the present invention is a method for efficiently introducing mutations at multiple sites of target gene fragments based on overlap extension PCR high-throughput; the primers used are not degenerate primers, but mutation primers that accurately design the target mutation sequence; the length of the mutation site is not Limited to a single amino acid, it can be designed as 1-5 consecutive amino acids; primers are not synthesized separately, but are synthesized in batches by an oligonucleotide synthesizer on an integrated chip, and the number of primers can reach 10 4 -10 5 Type / batch; the mutation method of the present invention can control the mutation frequency to a certain extent by adjusting the reaction conditions. The mutation library construction method of the present invention can obtain a relatively high proportion of effective mutants, and the positive rate of mutants (non-synonymous amino acid mutation) can reach 80%; the mutant library constructed by the present invention undergoes subsequent high-throughput screening It becomes a powerful tool for obtaining ideal protein and nucleic acid transformation bodies.

Description

technical field [0001] The invention relates to the technical field of gene manipulation, in particular to a high-throughput DNA multi-site precise base mutation method, in particular to a high-throughput synthetic mutation primer based on overlap extension PCR using precisely designed oligonucleotide chips Library, a method for simultaneously introducing mutations into multiple sites of a target gene fragment. Background technique [0002] In 1967, Spiegelman et al. proposed the idea of ​​directed evolution of biomolecules at the molecular level in vitro. Directed evolution technology is a process of using laboratory methods to repeatedly modify genetic diversity, combined with high-throughput library screening to obtain organisms with ideal traits. It has become one of the most widely used and effective techniques in the field of protein engineering. Directed evolution belongs to the category of irrational protein design. Compared with the rational mutation method, the co...

AI Technical Summary

Problems solved by technology

The disadvantages are: (1) Error-prone PCR generally only introduces a small part of sequence changes (mainly point mutations), so it is generally suitable for smaller gene fragments (<2kb)

(2) A large number of redundant mutations such as synonymous mutations, stop codon mutations, and reading frame disruptions caused by the randomness of the introduction of mutations make the mutation library constructed by error-prone PCR less efficient, and the effective mutant ratio accounts for about 0.1 -0.2%, increasing the cost of the experiment, causing difficulties in downstream screening experiments, making it difficult to obtain ideal mutants

The disadvantages are: (1) when amplifying the upstream and downstream fragments respectively, only high-fidelity or mixed DNA polymerases without tailing performance can be used, but DNA polymerases with no template tailing performance cannot be used; (2) ) When the target site is close to the end of the target gene, the segmented amplified fragments are too small to be recovered, which will easily cause the terminal mutation frequency to drop; (3) the number of degenerate primers required for multi-point saturation mutation is large, and the cost High, and it is difficult to ensure that each point saturation mutant can be obtained within a limited number of experiments

The disadvantages are: (1) Due to the need to amplify the full length of the plasmid, the effective amplification efficiency of PCR is low, time-consuming, and it is easy to introduce random mutations at non-target positions; (2) PCR amplification and mutagenesis efficiency are easily affected by plasmid vectors. The effect of size is that larger plasmid vectors are not easy to amplify and have low mutagenesis efficiency; (3) Although the method based on homologous recombination in E. coli to circularize PCR products in vivo is simple, it requires subsequent DpnI enzyme digestion or expression The special E. coli strain of DpnI is used to eliminate the template plasmid, and it is necessary to optimize the corresponding electroporation conditions according to actual experiments to obtain higher mutagenesis efficiency; (4) due to the low overall mutagenesis efficiency, this method requires iterative mutagenesis Multiple experiments are required to obtain multi-point mutagenesis, which is time-consuming and costly

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