Nucleic Acids Research, Vol 26, Issue 14 3340-3347, Copyright © 1998 by Oxford University Press
DG McDowell, NA Burns and HC Parkes
The polymerase chain reaction is an immensely powerful technique for
identification and detection purposes. Increasingly, competitive PCR is
being used as the basis for quantification. However, sequence length,
melting temperature and primary sequence have all been shown to influence
the efficiency of amplification in PCR systems and may therefore compromise
the required equivalent co-amplification of target and mimic in competitive
PCR. The work discussed here not only illustrates the need to balance
length and melting temperature when designing a competitive PCR assay, but
also emphasises the importance of careful examination of sequences for
GC-rich domains and other sequences giving rise to stable secondary
structures which could reduce the efficiency of amplification by serving as
pause or termination sites. We present data confirming that under
particular circumstances such localised sequence, high melting temperature
regions can act as permanent termination sites, and offer an explanation
for the severity of this effect which results in prevention of
amplification of a DNA mimic in competitive PCR. It is also demonstrated
that when Taq DNA polymerase is used in the presence of betaine or a proof
reading enzyme, the effect may be reduced or eliminated.
ARTICLES
Localised sequence regions possessing high melting temperatures prevent the amplification of a DNA mimic in competitive PCR
Department of Analytical Molecular Biology, Laboratory of the Government Chemist, Queens Road, Teddington, Middlesex TW11 0LY, UK. dgm@lgc.co.uk
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