Target and reporter molecule signal quantification: Final report (The validity of competitive PCR as a quantitative method)

Date: 30/04/2000	Ref No: LGC/VAM/2000/026	Type: Report
Author(s): Birch L, Calder A
Organisation/Publisher: LGC

Competitive PCR is now widely used to quantify the amount of an unknown sample in a reaction.  However, there is still controversy over the validity of this method as a quantitative technique.  Therefore this project aimed to assess the accuracy of quantification by competitive PCR.  To achieve this, a model system was developed with four competitors differing in size from 4-16bp.  Each of the competitors was designed to have a similar length, G-C content and to amplify from a single primer pair.  One of the competitors was used as a pseudo-target for quantification purposes.  A multiple tube and single tube assay was performed and three different methodologies were used to assess the accuracy of the competitive PCR model system.  Each method was also assessed for reproducibility, user friendliness, cost and level of technical competency required.  The work presented here demonstrates that, competitive PCR can be used to quantify an unknown target and has highlighted some important points to consider when designing competitive PCR assays.