melting temps of oligos with mismatches etc

Bruce Roe broe at aardvark.ucs.uoknor.edu
Sat Apr 25 08:38:00 EST 1992


In article <1992Apr24.053538.1 at lure.latrobe.edu.au>, micprf at lure.latrobe.edu.au writes...
>Hello again molbio freaks,
>	First of all thanks to the several people who answered my
>query about EcoP1. Now another. Does anyone know how to calculate
>melting temperatures for a pcr primer when there are mismatches
>and gaps when the primer is aligned with the template? We have an
>unexpected PCR product that we think we can explain on the basis
>of partial base pairing of one primer but there are several mismatches
>and a gap of 3 nucleotides out of 34. Please send replies to
>fisher at mpib-martinsried.mpg.dbp.de. Many thanks for your pearls of
>wisdom. Paul Fisher.

Paul,
	Under "usual" PCR conditions, you will get some primers binding
with a match of as little as 14 or 15 bases.  If this match occurs such
that the 3' end is base paired, then there is a likelyhood of primer
extension and a background band being observed.  We, and others have
observed this quite often.  All that is needed is for a few mis-matched
primings to occur and create a new template which will have an EXACT
match for your primer.
	Although Tm calculations can give one a idea of what's going
on, it may be better to search your target sequence using a program
which will find mis-matches in your primer sequence and not to use
primers which give a match over say 14-15 nucleotides (or in some
cases even less as we once had a 9-mer sequence which primed just
fine and their Tm was calculated to be 19 degrees under the salt
concentrations we were using).
	Good luck,
	--bruce
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 \  Bruce A. Roe                     Dept. Chemistry and Biochemistry /
 /  BROE at aardvark.ucs.uoknor.edu     University of Oklahoma           \
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