Why PCR reamp = smears. (fwd)

szcooley at chip.ucdavis.edu szcooley at chip.ucdavis.edu
Tue Aug 30 11:22:55 EST 1994


Robert Preston (rapr at MED.PITT.EDU) wrote:

: This replies to the following:
: Forwarded message:
: > From: HARDIES at thorin.uthscsa.edu
: > Subject: Re: Why PCR reamp = smears.
: >[edits] 
: > 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.


: I think Steve Hardies and others who say to go for the high dilutions are
: on the mark, due to the excessive taarget input without high dilution.
: It seems to be difficult for people to relate to the incredible capacity
: of the PCR exponential process, so amounts of target can be way too 
: excessive for the "standard" number of cycles used for PCR (20-30).
: It helps to think of copy number in the input DNA, NOT nanograms.  In a
: routine human genomic 30cycle rxn, we use 50 ng DNA. That's 15,000 copies
: of (single-copy/haploid genome) target, and it gives good gel-visible yield
: (i.e., 100's of ng of product) after 30 cycles, and 30 cycles is probably
: overkill.  BUT, if the target is, say, 0.01 ng of some 200 bp PCR product
: that you want to hyperamplify, that's already 50,000,000 copies in the input,
: and 30 cycles is way way way way way way way way tooooo many.  You'd get
: all sorts of smear-mischief going on in the late cycles.  If I INSISTED
: on using 0.01 ng of 200 bp input target (way more than enough for 30 cycles)
: then the reasonable thing to do would be to reduce the number of cycles
: to give 50,000,000/15,000 = 3,300-fold less amplification.  That would be
: 12 cycles less, since 2(exp 12)=3300, so I'd run 18 cycles and be smear-free.
: I could also go home earlier.  You can test this yourself by setting up
: multiple duplicate PCRs in one of your smear-experiments, pulling out
: one tube after 10 cycles, one after 12, one after 14, etc., and I'll bet
: you'll see that a real nice band appears in the lower cycles and then
: degenerates into smear after too many cycles.

: Rob Preston
: rapr at med.pitt.edu


Hey! Somebody try this! The result would be interesting!


#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%
%                                                        #
#   Dr. Michael Cooley         Practice Random Kindness  %
%   mbcooley at ucdavis.edu    and Senseless Acts of Beauty #
#                                                        %
%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#




More information about the Methods mailing list