Contamination in Nested PCR

[Martin Leach]

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
> intact
> 
> 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

-- 

.....          Martin Leach                Email:leach at mbcrrc.bu.edu 
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