>Re: GC-rich sequencing
Mr. T. Weissensteiner
tweissen at hgmp.mrc.ac.uk
Fri Aug 1 10:02:28 EST 1997
Re: GC-rich Sequencing
In article < Xref: hgmp.mrc.ac.uk bionet.molbio.methds-reagnts:58009>
Patrick Grealish (eris at injersey.com ) wrote :
> I have tried successfully to use GC-melt(a CLONTECH product) with the
> standard ABI PRISM seq kit and it works at 1M just fine.
> [ ]
> I have great suspisions that GC-melt is really only TMAC
> (tetramethylammonium chloride).
> [ ]
> I have not tried TMAC in sequencing, but side-by-side in a PCR rxn with
> GC-melt it worked equally as well, while DMSO and no additives gave no
> bands at all.
I doubt that CLONTECH's wondrous "GC-melt" is TMACl which, to my knowledge,
has never been used successfully at more than 120 mM in any published PCR
The likely reasons are that while the TMA+ ion may under certain conditions reduce the difference in the Tm of A/T and G/C base pairs,
it is also an electrolyte. It therefore stabilizes dsDNA (it was first
recommended as a substitute for NaCl in Southern hybridizations !),
as well as destabilizes protein/DNA interactions at high concentrations.
To me, it seems much more like "GC-melt" could be betaine. This compound
shares the A/T, G/C base pair isostabilizing property of TMA+, but without
the detrimental properties of electrolytes mentioned above.
For example, Mytelka et al. (see references below) found that 2 M betaine abolished pausing of T7-, Klenow- and Taq-polymerase when sequencing or
amplifying GC-rich regions. In my own PCR experience the optimal amount of
betaine is between 0.8 - 1.5 M, depending on template, primers, and final
salt concentration in the reaction.
Betaine solutions can have a maximal concentration of 5.6 M. In case this
Is similar to the "GC-melt" stock solution this would be a clue.
P.N. Hengen. 1997. Methods and reagents - Optimizing multiplex
and LA-PCR with betaine
Trends in Biochemical Sciences 22, pp.225-226
D.S. Mytelka and M.J. Chamberlin. 1996. ANALYSIS AND SUPPRESSION OF
DNA-POLYMERASE PAUSES ASSOCIATED WITH A TRINUCLEOTIDE CONSENSUS
Nucleic Acids Research 24, pp. 2774-2781
T. Weissensteiner and J.S. Lanchbury. 1995. How to control
preferential amplification in PCR typing reactions: lessons from
an assay for alleles of the HLA-B locus. European Journal of
Immunogenetics 22, p. 100
T. Weissensteiner and J.S. Lanchbury. 1996. Strategy for
controlling preferential amplification and avoiding false
negatives in PCR typing reactions. Biotechniques 21, pp 1102-8
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