Whole genome amplification?

Brett Beitzel bbeitzel at jeeves.ucsd.edu
Mon Mar 17 12:37:06 EST 1997


I was trying basically the same thing a couple of years ago with
microdissected DNA.  We had some luck using a primer first described by
Telenius (sometime in the '80s, I think,)  which is a semi-degenerate
primer (6 deg bases out of 24.)  The main problem, which I was never really
able to get around, was that I had to use low annealing temp for the first
rounds of PCR, which allowed for lots of primer dimer formation (which
overwhelmed the reaction.)  You can reduce this primer dimer somewhat by
using very low primer concentration initially (somewhere in the nM to pM
range, look for references on "booster PCR" for more detailed
descriptions,) but it is still extremely difficult to get a good, clean PCR
reaction.  One method that I didn't try extensively, but other people I
have talked to claim works well for them, is linker-adapter PCR.

Hope this helps some,
Brett Beitzel
bbeitzel at jeeves.ucsd.edu



In article <332D1DAF.46EF at sun-recomgen.univ-rennes1.fr>, Yvon CAVALOC
<cavaloc at sun-recomgen.univ-rennes1.fr> wrote:

> I am currently trying to amplify the whole human genome starting with
> very low amounts of DNA (< 5pg) using a degenerate PCR strategy. Since I
> cannot amplify more than 200 fold the genome, I would like to find
> people experienced in this topic. Has anybody been able to obtain a
> strong general amplification with a totally degenrated primer (N15, N10,
> N6?) or is it better to use degenerated primer having fixed 5' sequence?
> Thank you for help
> 
> Yvon CAVALOC
> UPR 41 CNRS
> Faculte de Medecine
> 2, Av du Pr L Bernard
> 35043 RENNES cedex - France
> 
> cavaloc at sun-recomgen.univ-rennes1.fr



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