Quantification of polymerase chain reaction products by affinity-based collection

To quantify the amount of specific polymerase chain reaction (PCR) product, it is usually not sufficient to measure the total amount of DNA synthesized in the reaction. Depending on the conditions and experimental design, the PCR can create various amounts of product that do not correspond to the desired template. Such erroneous products may arise by the amplification of sequences by essentially random priming or by the creation of primer–dimer artifacts. Once a polymerization product has been formed, it becomes a true PCR template and amplified in subsequent cycles. Consequently, a qualitative step, such as hybridization with a probe internal to the PCR primers, should be included in the analysis. This chapter discusses the application of the affinity-capture technology for the quantification of mixtures of closely related sequences .In this modification of the procedure, the relative concentration of two sequences differing from each other, for example, by a point mutation is accurately determined. Both sequences behave identically in the amplification and one of them serves as an internal standard for the quantification of the other sequence.