Cloning of prokaryotic genes by a universal degenerate primer PCR.
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).
Ping
Liyan
L
Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Jena, Germany. lping@ice.mpg.de
Vogel
Heiko
H
Boland
Wilhelm
W
D001419:Bacteria / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
D001426:Bacterial Proteins; D017931:DNA Primers