Using Recombineering to Generate Point Mutations: galK-Based Positive-Negative Selection Method

摘要

Recombineering is a recombination-based highly efficient method of genetic engineering. It can be used to manipulate the bacterial chromosomal DNA as well as any episomal DNA. Recombineering can be used to insert selectable or non-selectable DNA fragments, subclone DNA fragments without the use of restriction enzymes and also to make precise alterations including single nucleotide changes in the DNA. Here we describe a galK-based two-step method to generate point mutations in the bacterial artificial chromosome (BAC) insert using the recombineering technology. It takes advantage of the ability to select and also counter-select for the presence of galK.