Why PCR reamp = smears.

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Sun Aug 28 15:38:37 EST 1994


Hello,

I've been reading this thread about PCR products producing smears on 
reamp, and trying to think of a reason for it.

For example, Mruiel Liberto writes: "I have diluted the original
1:1000.  Could purity be causing a problem, even after a thousand fold 
dilution (into water) ???"   Good point.

The consensus response seems to be to dilute it more severely.  I
wonder if the explanation isn't something like this.  If you dilute
the original 1:1000, that's only 2^10.  So in ca. 10 cycles you should be
back where you started.  Now if you're going 20 or 30 cycles, you're
forcing the polymerase to work under conditions where reannealing of
the template is inhibiting further extension.  So you're prone to
aberrations like blocked partial extension and maybe even strand
switching.  This might make your smear.

This is pure conjecture.  Take it with a (diluted) grain of salt.

Steve Hardies, Dept. of Biochemistry, Univ. of Texas HSC at San
Antonio
Hardies at thorin.uthscsa.edu




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