Cloning of prokaryotic genes by a universal degenerate primer PCR

FEMS Microbiology Letters
Liyan PingWilhelm Boland

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).

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Citations

Nov 1, 2011·Plant Molecular Biology·Xiaoshu TangLiyan Ping
Aug 28, 2012·Journal of Bacteriology·Wenjing WangLiyan Ping
Aug 19, 2011·The FEBS Journal·Claudia LeoniLuigi R Ceci
Feb 24, 2012·FEMS Microbiology Ecology·Xiaoshu TangLiyan Ping

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