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. 1985 Feb;82(4):1189–1193. doi: 10.1073/pnas.82.4.1189

Efficient cloning of single-copy genes using specialized cosmid vectors: isolation of mutant dihydrofolate reductase genes.

G Urlaub, A M Carothers, L A Chasin
PMCID: PMC397220  PMID: 3883353

Abstract

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.

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Selected References

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