Direct Cloning of Mutant Alleles from the Bacterial Chromosome into Plasmid Vectors In Vivo

摘要

Genetic analysis can be performed by mutagenesis of a gene in single copy on the chromosome or mutagenesis of a cloned gene in plasmid vectors. There are several advantages of the singlecopy approach. Overexpression of a gene product from a plasmid vector may obscure the phenotype of potentially interesting mutants with weak effects on the gene. Also, overexpression of certain gene products from plasmid vectors can be toxic or perturb physiologically important interactions with other gene products, resulting in the isolation of secondary mutations, which can confuse the analysis of the mutations of interest. However, the direct isolation of mutations in the chromosomal copy of a gene also has a major disadvantage: the mutant alleles often need to becloned for further analysis. In vitro cloning of a large number of mutant alleles is very timeconsuming, requiring a number of labor-intensive steps such as isolation of chromosomal DNA, screening for rare clones, etc. Alternatively, polymerase chain reaction (PCR)-based cloning methods can be used. However, cloning PCR fragments frequently results in secondary mutations, and PCR amplification of specific large DNA fragments is often inefficient.