Method for constructing mutagenesis libraries in situ

摘要

Method and kit for constructing a random mutagenesis library. The method comprises providing a first expression vector comprising a target polynucleotide fragment, providing a pair of Vector-primers that are complementary to the vector portion of the first expression vector, wherein the Vector-primers comprise a second selection marker gene, and wherein the Vector-primers allow PCR amplification of the target polynucleotide; and performing a PCR reaction using the first expression vector as the template with the Vector-primers under error-prone PCR conditions in the presence of a thermostable DNA ligase, generating a second expression vector which comprises the second selection marker gene and a mutated target polynucleotide.

主权项

1. A method of constructing a random mutagenesis library, comprising:
i) providing a first expression vector comprising a target polynucleotide fragment, wherein the first expression vector comprises a vector portion and a target portion, wherein the vector portion comprises a first selection marker gene, and a first region and a second region flanking the first selection marker gene, wherein the target portion comprises a target polynucleotide capable of being expressed; ii) providing a pair of Vector-primers that are complementary to the vector portion of the first expression vector in the first and second regions respectively, wherein the Vector-primers comprise a second selection marker gene, and wherein the Vector-primers allow PCR amplification of the target polynucleotide; and iii) performing a PCR reaction using the first expression vector as the template with the Vector-primers under error-prone PCR conditions in the presence of a thermostable DNA ligase, generating a second expression vector which is circular and comprises the second selection marker gene and a mutated target polynucleotide wherein the second expression vector does not comprise the first selection marker.