Promega's (www.promega.com) new Plexor technology for gene-expression analysis and SNP profiling allows users to multiplex real-time quantitative PCR (RTQPCR). The method requires only two novel primers for sensitive and specific quantitation, according to the company.
In addition, Plexor runs on most commercial RTQPCR instruments, including those made by Applied Biosystems, Roche, Bio-Rad, Cepheid, and Stratagene. Whereas other RTQPCR systems produce an increase in fluorescence, Plexor reportedly quenches fluorescence.
The Plexor chemistry can be used to identify and quantitate specific DNA sequences in genomic DNA, mitochondrial DNA, cDNA, or viral DNA samples. In multiplex reactions, one primer for each target must carry a different fluorescent label.
The types and number of fluorescent labels that can be used depend on the detection capabilities of the real-time instrument. While developing Plexor, Promega experts worked with scientists at the University of Wisconsin, who were skeptical that multiplexing was possible. The current dogma is that quantitation of high-copy number transcripts interferes with quantitation of low copy-number transcripts.
The Promega team proved that for up to four primer pairs, Plexor's primer chemistry detects low copy-number genes in the presence of high copy-number genes.
Because many bench scientists share real-time instruments, they are often limited to running one or two plates a week. Plexor's multiplexing ability allows them to move control reactions into target reactions as internal controls, which "opens up half a plate to run more experiments and increases productivity," said Kyle Hooper, Ph.D., genomics product manager at Promega.
Alpha testers gave Promega the first targets tested in Plexor. To further validate the new platform for SNP genotyping, "we need help from researchers with thousands of samples," said Dr. Hooper.
Promega plans to release Plexor by the end of 2005. Customers will design their assay primers at a special website, then select a collaborating oligonucleotide manufacturer to construct the primers with the novel primer chemistry. A Plexor kit contains all the reagents needed to run the reactions, notes the company.
The Plexor software imports raw data that is exported from current real-time PCR instruments and converts it to analyze fluorescent quenching.