Nucleic Acids Research, Vol 26, Issue 19 4401-4408, Copyright © 1998 by Oxford University Press
J Serth, F Panitz, H Herrmann and J Alves
Competitive PCR is a frequently used technique for quantitation of DNA and
mRNA. However, the application of the most favourable homologous mutated
competitors is impeded by the formation of heteroduplex molecules which
complicates the data evaluation and may lead to quantitation errors.
Moreover, in most cases a single quantitation of an unknown sample requires
multiple competitive reactions for identification of the equivalence point.
In the present study, a highly efficient and reliable method as well as the
underlying theoretical model is described. The mathematical solutions of
this model provide the basis for single-tube quantitation using a
homologous mutated competitor. For quantitation of Human Papilloma Virus
16-DNA, it is shown that single tube quantitations using simple PAGE
separation and video evaluation for signal analysis permit linear detection
within more than two orders of magnitude. In addition, repeated single-tube
competitive PCRs exhibited good precision (average standard deviation 5%),
even if carried out as nested high cycle PCR for quantitation of low
abundant sequences (intraassay sensitivity <2 x 10(2) copies). This
evaluation method can be applied to any DNA separation and detection method
which is capable of resolving the heteroduplex fraction from both
homoduplex molecules.
ARTICLES
Single-tube nested competitive PCR with homologous competitor for quantitation of DNA target sequences: theoretical description of heteroduplex formation, evaluation of sensitivity, precision and linear range of the method
Klinik fur Urologie/Forschung OE6247, Abteilung Biometrie, D-37073 Gottingen, Germany. serth.juergen@mh-hannover.de
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. E. Clark and T. L. Karr Distribution of Wolbachia Within Drosophila Reproductive Tissue: Implications for the Expression of Cytoplasmic Incompatibility Integr. Comp. Biol., April 1, 2002; 42(2): 332 - 339. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Watzinger, E. Horth, and T. Lion Quantification of mRNA expression by competitive PCR using non-homologous competitors containing a shifted restriction site Nucleic Acids Res., June 1, 2001; 29(11): e52 - e52. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Serth, M. A. Kuczyk, U. Paeslack, R. Lichtinghagen, and U. Jonas Quantitation of DNA Extracted after Micropreparation of Cells from Frozen and Formalin-Fixed Tissue Sections Am. J. Pathol., April 1, 2000; 156(4): 1189 - 1196. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G.J.S. Jenkins, H.S. Suzen, R.A. Sueiro, and J.M. Parry The restriction site mutation assay: a review of the methodology development and the current status of the technique Mutagenesis, September 1, 1999; 14(5): 439 - 448. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Serth, F. Panitz, U. Paeslack, M. A. Kuczyk, and U. Jonas Increased Levels of Human Papillomavirus Type 16 DNA in a Subset of Prostate Cancers Cancer Res., February 1, 1999; 59(4): 823 - 825. [Abstract] [Full Text] [PDF]
|
|