Mathematical considerations of competitive polymerase chain

Connolly, AR, Cleland, LG and Kirkham, BW (1995) Mathematical considerations of competitive polymerase chain. Journal of Immunological Methods, 187 2: 201-211. doi:10.1016/0022-1759(95)00185-2


Author Connolly, AR
Cleland, LG
Kirkham, BW
Title Mathematical considerations of competitive polymerase chain
Journal name Journal of Immunological Methods   Check publisher's open access policy
ISSN 0022-1759
1872-7905
Publication date 1995-12-01
Sub-type Article (original research)
DOI 10.1016/0022-1759(95)00185-2
Volume 187
Issue 2
Start page 201
End page 211
Total pages 11
Place of publication Amsterdam, Netherlands
Publisher Elsevier BV
Language eng
Abstract Reverse transcriptase polymerase chain reaction (PCR) is used frequently to monitor gene expression. It is generally regarded as a qualitative technique, although refinements have been made to improve quantification. The object of this study was to develop competitive PCRs to allow reliable quantification of the rat T cell cytokines interferon-γ (IFN-γ), interleukin-2 (IL-2) and interleukin-4 (IL-4). Truncated constructs of cDNA for these cytokines were prepared using appropriate pairs of standard and specially constructed primers designed to allow subsequent co-amplification of the purified competitor construct and the target cDNA. A high resolution capillary electrophoresis (CE) system was used for PCR product detection. The performance of the system was compared with a mathematical model that describes and predicts the exponential nature of the PCR reaction. Co-amplification of the competitor and target were achieved. A high level of resolution and accuracy was achieved using CE to detect and quantify the PCR products. The rates of generation of the respective products conformed closely but not exactly to the predictions of the mathematical model. The competitive PCRs estimated initial numbers of target cDNA within 1.1–5.0-fold relative to the amount of starting material as assessed by conventional spectrophotometric absorbance prior to dilution and amplification. A convenient and flexible competitive PCR strategy has been developed with accurate resolution of products and reliable quantification. Assay variability was far less than biological variability likely to be encountered in experiments investigating immunological responses in rats or other animals. Copyright © 2011 Elsevier B.V. All rights reserved.
Keyword Quantitative polymerase chain reaction
Rat cytokine
Capillary electrophoresis
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collection: Australian Institute for Bioengineering and Nanotechnology Publications
 
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Created: Tue, 16 Nov 2010, 10:04:00 EST by Dr Ashley Connolly on behalf of Aust Institute for Bioengineering & Nanotechnology