A strategy employing PCR technology to facilitate the amplification of DNA segments inserted in plasmid vectors is described. Nine oligonucleotide primers specific for vector sequences bracketing cloning sites in seven commonly used vectors were designed. We used these primers for the amplification of 25 different inserts ranging in size from 0.4-4.8 kb. Vector PCR-generated products used as radiolabeled DNA probes in Southern hybridization compared favorably with conventionally prepared probes. This strategy was successfully applied to single colonies of bacteria containing recombinant plasmids for direct amplification of the plasmid insert from the bacterial lysate. Vector PCR enabled the production of microgram quantitites of DNA from limited amounts of starting material without the time-consuming steps required for bacterial culture and purification of plasmid DNA. The amplification reaction is independent of the DNA segment to be amplified, rendering the method universally applicable.
|Original language||English (US)|
|Number of pages||7|
|State||Published - Nov 6 1991|
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)