Cloning of prokaryotic genes by a universal degenerate primer PCR

Authors


  • Editor: David Studholme

Correspondence: Liyan Ping, Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Hans-Knoell-Street 8, Jena 07745, Germany. Tel.: +49 3641 57 1214; fax: +49 3641 57 1202; e-mail: lping@ice.mpg.de

Abstract

A PCR approach was developed using a hexameric degenerate primer, which reflects the Shine–Dalgarno sequence of prokaryotic transcripts, hitherto named SD-PCR. In standard PCR reactions, the sizes and melting temperatures of the two primers are usually designed to be as equal as possible, while SD-PCR uses a single long gene-specific primer pairing with a much-shorter universal degenerate primer. This approach can be used in PCR walking to clone either the upstream or the downstream region of a known sequence. We have successfully applied the method to template DNAs of different GC contents as well as complex mixtures composed of highly contaminating DNA(s).

Ancillary