Contamination in Nested PCR
leach at mbcrrc.bu.edu
Wed Oct 26 09:05:51 EST 1994
In article <38ljej$145a at msuinfo.cl.msu.edu>, mcmanusp at student.msu.edu
(Patty McManus) wrote:
> I'm having problems with carryover contamination in my nested PCRs.
> I've heard about using dUTP followed by uracil DNA glycosylase, but I
> do not understand the logic of this method!
> Here's how I see it:
> 1) dUTP replaces dTTP in first round of PCR
> 2) an aliquot of first round product is added to tube with all components
> for the nested PCR plus UDG enzyme
> 3) UDG will cleave DNA with dUTP incorporated but leaves "natural" DNA
> My question is: Doesn't this defeat the purpose of nested PCR? I mean,
> if you destroy the amplified product (has dUTP incorp'ed) from the first
> round, then why do two rounds? The amount of template (bona fide, natural
> DNA with T) in the second round will be even less than in the first!
> I know folks have been using this method to eliminate crossover contamination
> for years, but danged if I can figger it out. Please help! Thanks.
UV everything for 30 minutes tips/tubes/pipettes racks....etc..
BTW the dUTP method above only works if the contaminant contains dUTP!!
If the source of contamination is from DNA made/synthesised etc.. prior to
use of the dUTP method...then you're screwed!!
..... Martin Leach Email:leach at mbcrrc.bu.edu
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