Gene saturation mutation library and its construction method and application

Abstract

The invention relates to the technical field of biology, in particular to a gene saturation mutagenesis library as well as a construction method and application thereof. The construction method of the continuous multipoint saturation mutagenesis library in long-sequence DNA is suitable for high-throughout protein functional screening; annular double-helix DNA plasmid containing a gene of expressing a target protein is taken as a template; a pair of positive and negative mutation primers with sequences completely complementary reversely are adopted; and saturation mutagenesis of a plurality of basic groups which are continuously arranged in the long-sequence DNA can be realized by one-step reaction through polymerase chain type reaction; and the gene saturation mutagenesis library has the characteristics of being simple, quick and high in mutagenesis success rate, and can be used to realize quick screening positioning and efficient mutagenesis modifying on protein functional sites on amino acid level.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a gene saturation mutation library and its construction method and application. Background technique [0002] Protein modification and screening technologies are widely used in heterologous expression of natural genes, optimization of protein drugs such as vaccines and antibodies, artificial synthesis of cell factories and even artificial life forms, and are of great significance to human health and social development. In the research and engineering of protein function, the key amino acid sites that affect protein function must be determined first, and the engineered protein with desired function and activity can be obtained through multiple rounds of mutation and screening of these sites. The usual strategy is to use multiple rounds of error-prone PCR to randomly mutate the target gene, and initially screen out some suspicious sites. Subsequently, further exploration around the su...

AI Technical Summary

Problems solved by technology

[0003] However, although error-prone PCR can randomly introduce multiple base mutations into the target gene at one time, its randomness also limits the mutation efficiency of this method, for example, some sites are repeated in multiple rounds of mutation However, the sequence in some regions has not been mutated, and the number of mutated bases in each round is also different, which makes it almost impossible to achieve a comprehensive exploration of the target gene sequence in a limited number of experiments

On the other hand, the various site-directed mutagenesis methods currently used can only achieve mutations of 1-3 adjacent bases in a single round of experiments, while multi-point mutations require that the interval between each mutation site be tens to hundreds More than one base pair, and usually requires multiple PCR reactions in series to achieve the ultimate mutation purpose

These methods are inefficient and cumbersome for mutations of multiple bases arranged consecutively in the specified sequence, and have poor compatibility with long-sequence DNA

Images