GC melt

nvahsen at hot.hgmp.mrc.ac.uk nvahsen at hot.hgmp.mrc.ac.uk
Sat Nov 6 04:28:47 EST 1999


Well, this began with a GC-melt question and ended with a discussion
on primer design.

For GC-melt I pretty much suspect (and T. Zhang already gave the
refenrences for this (promega)) that most of these reagents are
containing DMSO and betaine. I want to suggest reading of N. Baskaran,
et al., Uniform amplification of a mixture of deoxyribonucleic acids
with varying GC content. Genome Res. 6 (7):633-638, 1996. Basically,
they recommend 7%DMSO and 1M betaine to use for the tough templates.

For primer design I think that Rychlik satisfies us, because his
arguments "make sense to (us)" (Tom Knight). However, I think that
Rychlik's theory is largely unproven, at least in the references I
list below. The problem is that he calculated stabilities with the
nearest neighbor model based on outdated thermodynamic data (namely K.
J. Breslauer et al., Predicting DNA duplex stability from the base
sequence. Proc Natl Acad Sci U S A 83:3746-3750, 1986, discussed in R.
Owczarzy et al., Predicting sequence-dependent melting stability of
short duplex DNA oligomers. Biopolymers 44 (3):217-239, 1997.
.). Breslauer's pioneering work had its time, but today one should use
better thermodynamic data like those of J. SantaLucia Jr's group. If I
recalculate some of Rychlik's data with these parameters there is no
consistent finding about 5' and 3' stability and primer "quality".
Furthermore if I calculate these data for several good and not so good
performing primer sets that I use, it is simply impossible to make a
prediction. 
The software we use for these stability predictions was written by a
colleague of mine and me (E. Schütz and N. von Ahsen) and is supposed
to be published in the December'99 issue of BioTechniques.

Any comments on this are greatly appreciated.
Nicolas von Ahsen



W. Rychlik and R. E. Rhoads. A computer program for choosing optimal
oligonucleotides for filter hybridization, sequencing and in vitro
amplification of DNA. Nucleic Acids Res. 17 (21):8543-8551, 1989.
W. Rychlik, W. J. Spencer, and R. E. Rhoads. 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.
W. Rychlik. Priming efficiency in PCR. Biotechniques 18 (1):84-90,
1995.
W. Rychlik. Selection of primers for polymerase chain reaction.
Mol.Biotechnol. 3 (2):129-134, 1995.





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