Novel method for building ultra-high capacity gene library based on combination principle and PCR

Abstract

Directed evolution of enzyme is an effective way to change enzyme properties and improve utility value thereof. However, the existing methods for directed evolution all rely on existing genes, and only perform reconstruction on the basis of the well-discovered genes. Therefore, the existing methods can do nothing for absent or newly-minted screening targets in nature. The invention uses random segments with shared joints, connects, combines and reunites the random segments by a PCR method to obtain a huge gene library which is randomly combined. In theory, the library consists of random gene sequences, and screens at random target traits so as to create novel genes and break through the limitation of the previous direction evolution method which can only perform reconstruction on the existing genes. The invention (building an ultra-high capacity gene library) enables the directed evolution and medicine screening not to be limited by gene library diversity, provides a huge resource library for screening, and plays an active role in some targets which are difficult to screen the existing gene library such as screening of target receptor, discovering of unnatural condition enzyme, detection of new target substances and the like.

Description

technical field [0001] The invention relates to a method for establishing a gene library in molecular evolution, using random fragments with shared joints, and performing complementary, overlapping extension and reunion on the random fragments by means of PCR to obtain a randomly combined huge gene library. Background technique [0002] As biocatalysts, enzymes have the advantages of high catalytic efficiency, strong specificity, mild reaction conditions, and environmental friendliness. The U.S. Department of Energy, the Department of Commerce and other departments predict that biocatalysts will become a necessary tool for the sustainable development of the chemical industry in the 21st century. It is the core of industrial biotechnology, but the properties of natural enzymes are difficult to adapt to various industrial production conditions. Directed evolution of enzymes (Directed Evolution, DE) is an effective way to change the structure and properties of enzymes to meet ...

AI Technical Summary

Problems solved by technology

Error-prone PCR has certain limitations in practical application: first, the mutation speed is slow; second, only point mutations are introduced; third, the gene fragments involved are too short (less than 800bp)

[0006] The existing methods of establishing mutant libraries have greatly promoted the evolution of enzymes, especially the production of biocatalysts suitable for pharmaceutical and industrial production, but these methods can only rely on existing genes for modification and screening, and are difficult to develop In the new sequence space (especially when the function changes greatly), for screening targets that do not exist in nature, such as the prevention of emerging viruses or the screening of unnatural traits, it is difficult to use the original gene transformation to achieve

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