Method for Directed DNA Evolution using Combinatorial DNA Libraries

摘要

The present invention provides a method of rapid directed DNA evolution based on single-stranded combinatorial DNA mutant library. The single- and double-stranded mutant library are constructed either separately or simultaneously using editing primers that contain mutated nucleotides and are targeted to different regions of the parent DNA sequence. The mutant library is then inserted into expression vectors and mutants with desired property are obtained by high throughput screening. Evolution of promoters, enzymes and metabolic pathways were successfully achieved using this method and mutants with excellent properties were obtained. The method of the present invention is simple, rapid, and efficient. It can be used for directed evolution of regulatory sequence such as promoters and ribosome binding sites, and is especially suitable for introducing diverse mutations into protein encoding genes, leading to rapid directed evolution of gene of interest.

主权项

1. A method of rapid directed DNA evolution using a combinatorial DNA mutant library, comprising the steps of:
a) preparing a single-stranded DNA mutant library, wherein editing primers with mutated nucleotide(s) that are targeted to different regions of the same strand of a parent DNA are used as internal primers in a PCR amplification to introduce combinatorial mutations into said parent DNA, and wherein single-stranded DNAs with said combinatorial mutations constitute said single-stranded DNA mutant library; b) preparing a double-stranded DNA mutant library, wherein said single-stranded DNA mutant library is used as a template to generate said double-stranded DNA mutant library; and c) assembling said double-stranded DNA mutant library into expression vectors and screening for mutants with desired properties.