sequence polymorphisms

Bruce Roe BROE at AARDVARK.UCS.UOKNOR.EDU
Fri Nov 4 07:16:17 EST 1994


Hi,
	The major problems we've had are to obtain pcr products
of sufficient quality to yield high signal/noise.  This basicly
seems to depend on how the product is purified after the pcr rxn.
Even a protocol that works for one student does not work for another.
The bottom line for me is to clone the product. What we do is:

	1. a quick fillin with klenow and the 4 dNTP's to blunt end
	2. clone into smaI/CIP treated pUC (from Pharmacia)

Since we've got the ds template isolation to routinely work quite well, we
then sequence 6-12 separate isolates of the cloned pcr product. In the past
2 years of using this "blunt end cloning approach" we have not noticed any pcr
misincorporation artifacts.  That's over several hundred clones sequenced.  

It's my belief that all the talk of Taq polymerase causing or rather allowing
misincorporation is:
	1. an artifact of dNTP and Mg concentration in the original
	 pcr reaction.  As I recall, a few years ago there was such a
	 study published that pointed this out. (Sorry for not having
	 the reference handy) 
	2. alot of hype by companies that are trying to get around the
	 PE/Cetus patent.

In the case of long pcr's (ala W.Barnes), we've not investigated the quality
of the product from anything longer than 1-2kb for cloning/sequencing because
of fears that strange things may happen.

Finally, as for sequencing pcr products, it's my thought that the original
poster of the message was not observing misincorporation, but rather had low
quality template and thus was observing sequencing artifacts due to a low
signal to noise ratio or slightly nicked template.  Remember, UV light can
cause nicking of DNA in the presence of ethidium bromide and even a single band
on a gel can get nicked upon prolonged exposure to UV during extraction and
purification from the gel.  If you're curious about this, try the expt where
you run some DNA fragment out on a gel (with EtBr), expose to UV for various
times and after eluting it, re-run it on the gel.  It's an "eye opening"
experience.

Cheers......bruce
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 \  Bruce A. Roe               Professor of Chemistry and Biochemistry /
 /  University of Oklahoma     INTERNET: BROE at uoknor.edu               \
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