Recombinant Circle PCR
dkim at nmsu.edu
Fri Mar 10 14:07:59 EST 1995
I was looking into directionally cloning PCR products into a plasmid, and
stumbled on an article in PCR Methods and Applications 3:S141-S148 (1994)
"PCR Mutagenesis and Recombination in Vivo". This article describes a
method for combining PCR products in vivo by transfecting linear DNA
fragments with homologous ends into E coli.
Previous investigators have shown that DNA ends containing short regions
of homology undergo intramolecular recombination in vivo in Escherichia
coli, including RecA-minus E. coli strains used routinely for cloning, and
that E coli can mediate intermolecular reconbination betweeen a short,
single-stranded oligonucleotide and a restriction endonuclease-digested
plasmid. Recombination PCR is a method for making DNA joints in vivo by
the recombination of PCR-generated homologous DNA ends in E coli.
The article refers to two BioTechniques papers as well:
12(4):528-535? and 10(1):62-66 (1991)
I don't hear about this process in any other papers that I've run into,
and I expect to go through quite a bit of trouble to subclone PCR
-mutagenized products in-frame into an expression vector using more
conventional methods (adapters, restriction site fill-in, etc). This
method seems to be much faster and more flexible.
Before I start designing primers, I'd like to know if anyone has
experience with this process. Is it easier than more conventional
methods? Does it really work as advertised in these papers?
dkim at verdi.nmsu.edu
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