Efficient cloning of single-copy genes using specialized cosmid vectors: isolation of mutant dihydrofolate reductase genes.
A method for the efficient cloning of single-copy genes from restriction digests of mammalian DNA is described. The method is illustrated by the cloning of several mutant genes as well as the wild-type gene for Chinese hamster dihydrofolate reductase (DHFR; 7,8-dihydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). This gene is isolated within a 41-kilobase Bgl I fragment by using cosmid (plasmids containing a cohesive-end site) vectors that have been constructed especially for this purpose. Two cosmids are used: one contains a short region from the 5' flanking region of the dhfr gene, and the other contains a short region from the 3' flanking region. These two regions contain the Bgl I sites that bound the dhfr gene. Bgl I leaves staggered ends that are different depending on the DNA sequence within the enzyme binding site. When these cosmids are cut with Bgl I and hybridized with total Bgl I-cut genomic DNA, they preferentially associate with the fragment bearing the dhfr gene, since it has the same Bgl I ends. An approximately 500-fold enrichment for the dhfr gene in cosmid libraries from Chinese hamster ovary cells was achieved by using this method coupled with a single-step size fractionation. As a result, only several hundred cosmid colonies need to be screened in order to clone a dhfr gene from a particular mutant Chinese hamster ovary cell. This method should facilitate the repetitive cloning of any gene or gene fragment.
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005822:Genetic Vectors; D009154:Mutation; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
D013762:Tetrahydrofolate Dehydrogenase