The power of polymerase chain reaction (PCR) is greatly enhanced by techniques that accurately, sensitively and reproducibly quantify DNA samples. These real-time or quantitative PCR (qPCR) techniques rely on the ability to detect the PCR product at each cycle during the exponential phase. The PlexorTM technique requires only two primers for sensitive and specific quantification. PlexorTM technology takes advantage of the highly specific interaction between two modified nucleotides for qPCR analysis. These two novel bases, iso-dG and iso-dC, form a unique bonding interaction when incorporated in double-stranded DNA and pair only with each other. In PlexorTM reactions, one PCR primer is synthesized with an iso-dC residue and a fluorescent label at the 5' end. The second PCR primer is unlabeled. Iso-dGTP nucleotides, which are conjugated to the quencher Dabcyl, are included into the nucleotide mix containing the four naturally occurring nucleotides. During the amplification reaction dabcyl-iso-dGTP is incorporated at the position complementary to the iso-dC residue. Incorporation of the dabcyl-iso-dGTP in close proximity to the fluorescent label effectively quenches the fluorescent signal. A web-based primer design program for use with the PlexorTM systems is available to design primers for single and multiplex qPCR reactions. For more information, please visit www.promega.com/techserv/apps/qpcr/.
|