vmiao at oregon.uoregon.edu
Tue Sep 28 22:38:26 EST 1993
In article <9309281323.AA05870 at net.bio.net>, FOF at FDACFSAN.BITNET wrote:
> THis may be a stupid question, but what exactly is ligation-mediated PCR?
See Pfeifer et al., 1989. Science 246: 810-813. Genomic sequencing and
methylation analysis by ligation mediated PCR.
LM-PCR is a method of sequencing directly from genomic DNA if you have a
primer for the region of interest. The DNA is chemically cleaved as if for
Maxam Gilbert chemistry, producing a whole bunch of fragments that end in
G, A, T or C. You denature the DNA and let the primer anneal to the
fragments that are related the region you are interested in. By primer
extension off that primer, you make only those fragments double stranded
and blunt ended; these molecules are able to be ligated to a linker (the
linker is staggered at one end, and blunt on the other, so the ligation is
directional). Now you have a bunch of fragments that have two known ends
(your primer, and the linker) and you can use PCR to amplify the various
fragments, run them on a gel and determine the sequence. The reason you
might want to go to all this trouble would be to examine conditions of DNA
that are lost upon cloning (e.g. methylation), or to examine footprints of
DNA binding proteins in vivo.
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