>Re: GC-rich sequencing

[Mr. T. Weissensteiner]

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
method.
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.


Review

    P.N. Hengen. 1997. Methods and reagents - Optimizing multiplex 
    and LA-PCR with betaine
    Trends in Biochemical Sciences 22, pp.225-226

References

    
    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


Thomas Weissensteiner
Autoimmunity Group
Jenner Institute for Vaccine Research
Compton / Newbury
Berkshire RG20 7NN

T    :  0044 1635 577920
FAX  :  0044 1625 577901

email : tweissen at RETURNMEhgmp.mrc.ac.uk
(remove capitalized letters when using this address !)

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