Construction of Polycompetitors for Competitive PCR

Abstract

Many different protocols are now available for competitive polymerase chain reaction (PCR) and most rely on the use of a mimic or competitor that serves as a reference for quantitation (1–4). The success (or failure) of all these protocols is critically dependent on the design, construction, and utilization of these constructs. This protocol provides detailed instructions for developing individual mimics, or competitors, for use in competitive PCR reactions. Individual competitors can be joined together in logical order in one plasmid, producing a single reagent, or polycompetitor, with multiple specificity. Although the protocol has been used successfully in producing cytokine polycompetitors, for both human and mouse (5), it should work well for almost any molecule of interest, provided sequence information is available. If a polycompetitor is to be synthesized, careful planning is especially required for a trouble-free outcome. Detailed restriction-endonuclease maps of the cloning vectors and PCR products to be cloned must be used in the design of primers and to plan appropriate strategies for incorporation of individual competitor constructs. Although many different cloning vectors may be used, in order not to be too general, this protocol provides detailed information using a commercially available vector, pGEM 11Z, and steps used in the construction of a specific polycompetitor, the human polycompetitor for T-cell cytokines, pDC10. The general principles, however, are applicable to the construction of polycom-petitors for any genes, using many different commercially available vectors.