Open AccessCloning of prokaryotic genes by a universal degenerate primer PCR
Author(s) -
Ping Liyan,
Vogel Heiko,
Boland Wilhelm
Publication year - 2008
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2008.01311.x
Subject(s) - primer (cosmetics) , in silico pcr , biology , primer dimer , gene , cloning (programming) , genetics , polymerase chain reaction , computational biology , dna , clone (java method) , microbiology and biotechnology , chemistry , multiplex polymerase chain reaction , computer science , organic chemistry , programming language
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).