PCR lore

nvahsen at hot.hgmp.mrc.ac.uk nvahsen at hot.hgmp.mrc.ac.uk
Wed Mar 29 16:14:58 EST 2000


Dear Susanne, joke or not, I think the discussion should still be
open.

Is there really much about software and science about PCR primer
design? Or is the success of primer software only showing how robust
PCR is towards primer choice? Has anyone seriously compared different
primer design software? Are balanced GC an Tm and avoiding to bad
hairpins/(cross-)dimers enough to make a good primer?
 
I almost think so although for long PCR false priming may be a problem
which could be overcome by software searching for priming sites.
Much PCR primer "art" is based on Rychlik's work. However, in his
paper Rychlik, W. et al., Optimization of the annealing temperature
for DNA amplification in vitro [published erratum appears in Nucleic
Acids Res 1991 Feb 11;19(3):698]. Nucleic Acids Res. 18
(21):6409-6412, 1990 are many errors in the formulas. Also the
thermodynamic data he uses for oligo stability calculation is outdated
(see Biopolymers 44:217,1997), so is "Oligo". Maybe Oligo 5 no more..
In Rychlik, "Priming efficiency in PCR" Biotechniques 18 (1):84-90,
1995 we read about good and bad primers regarding 3' and 5' stability.
Same problem. Recalculating his data with modern thermodynamic data
sets makes me doubt his conclusion. Also I calculated several 3' and
5' stabilities from successful and unsuccessful primer pairs I use and
found nothing conclusive.
For these calculations I use recent thermodynamic data (SantaLucia,
PNAS 95:1460, 1998) implemented in a spreadsheet software (Schutz and
von Ahsen. Spreadsheet software for thermodynamic melting point
prediction of oligonucleotide hybridization with and without
mismatches. Biotechniques 27 (6):1218-1224, 1999). or go to
http://server1.medikc.med.uni-goettingen.de/meltcalc.htm to get the
software.

But many people say Oligo works fine. I use Primer3 most times, very
exact Tm predictions, good model and free...
Or do we in the end get our best PCRs from sacrifices in the dark
room?

Nicolas





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