betaine in LA PCR

Paul N Hengen pnh at ncifcrf.gov
Fri Apr 18 15:53:38 EST 1997


Wayne M. Barnes (wayne at BARNES1.WUSTL.EDU) wrote:

> The reference below is to using betaine in PCR.  We find it
> enhances LA PCR up to 7 kb.  We worry about a negative
> effect on fidelity, but we don't have data on this yet.  We
> leave out the DMSO from the reference below, except for 1%
> sometimes.
> 
> Add 1.3 M final betaine, from a 4 M or 5 M stock.  
> 
> Take one or two degrees off the melting temp of your PCR cycles.
> 
> We find that even ordinary 5 kb PCR is enhanced, not just the high 
> GC targets targeted by the reference below.
> 
> 
> [2] Baskaran, N., Kandpal, R.P., Bhargava, A.K., Glynn, M.W., Bale, A., &
> Weissman, S.M. (1996) Uniform amplification of a mixture of
> deoxyyribonucleic acids with varying GC content, Genome Research 6:633-638.

What do you think the betaine (N,N,N-trimethylglycine) is doing to help with
the processivity of Taq? In another reference listed below (Mytelka), a study
was done with adding 2M betaine to reactions with T7 DNA polymerase (Sequenase)
when used on dsDNA. They have some data (not shown) that the fidelity of the
enzyme is not altered, only the ability to bypass secondary structures that
cause polymerase pausing, ie.  BALFs seen in sequencing gels.  The betaine is
thought to help in a way different from simply stabilizing the enzyme.  So,
what is it doing? They think it is increasing the hydration of GC pairs from
the minor groove (?), although not stated that clearly in the paper.

BTW, they also tried Taq polymerase with 1-1.5 M betaine added and say it
helped in yields of some longer PCR products. I'd be interested in seeing a
comparison of 1-2 M betaine added to various buffers with and without K+
(Woodford1995) and with and without DMSO (Filichkin1992) for Taq processivity
and fidelity.  They also found that TMANO (trimethylamine N-oxide) also worked.
However, I checked and found that TMANO is about 10-fold more expensive from
Sigma than betaine.

@article{Mytelka.Chamberlin1996,
author = "D. S. Mytelka
     and M. J. Chamberlin",
title = "Analysis and suppression of {DNA} polymerase
pauses associated with a trinucleotide consensus",
journal = "Nucleic Acids Res.",
volume = "24",
number = "14",
pages = "2774-2781",
comment = "2M betaine for PCR cycle sequencing",
year = "1996"}

@article{Woodford1995,
author = "K. Woodford
     and M. N. Weitzmann
     and K. Usdin",
title = "The use of {K}+--free buffers eliminates a common
cause of premature chain termination in {PCR} and {PCR} sequencing",
journal = "Nucleic Acids Res.",
volume = "23",
number = "3",
pages = "539",
comment = "potassium free buffer for PCR;
cycle sequencing through high GC content",
year = "1995"}

@article{Filichkin1992,
author = "S. A. Filichkin
     and S. B. Gelvin",
title = "Effect of dimethyl sulfoxide concentration
on specificity of primer matching in {PCR}",
journal = "BioTechniques",
volume = "12",
number = "6",
pages = "828-830",
comment = "use of DMSO in PCR; cycle sequencing through high
GC content; gelvin at bilbo.bio.purdue.edu",
year = "1992"}

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