mysterious PCR question

Dr. Duncan Clark news at hgmp.mrc.ac.uk
Thu Jul 19 04:07:58 EST 2001


In article <3.0.6.32.20010718135113.008aaad0 at mail.montclair.edu>, the
eminent Dr. James J. Campanella at BIOSCI/MRC Human Genome Mapping
Project Resource Centre wrote
>I was amazed that I even got any amplification at all at the
>1:1000 dilution.

Why? A standard say 500bp PCR with 1ug yield will generate 1.8x10E12
copies of DNA. A thousand fold dilution is basically no where near
enough. A 1000 fold dilution is only equivalent to 10 cycles of
amplification so if you diluted 1000fold and run more than 10 cycles you
are looking for trouble i.e. smear.

What is the smear? Thermus and archael pols have been shown to indulge
in de novo synthesis  i.e. DNA synthesis without either starting
template or primers. Two papers, at least, published in NAR in the last
three or four years. Alternatively it could be long oligomers with
sequence homology to your primers. 

Duncan  
-- 
It might look like I'm doing nothing, but at the cellular level I'm really
quite busy.

Duncan Clark
GeneSys Ltd.
Tel. +44 (0) 1252376288
FAX +44 (0) 8701640382




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