The gold standard for gene-expression analysis, qPCR, has been extensively studied. Standard protocols have been published and multiple instruments for cycling with real-time detection are commercially available. So, how can there be anything new to talk about?
Well, some laboratories have designed new methods for signal generation that offer additional benefits, while the focus on infectious disease has forced other researchers to concern themselves with PCR primer design in the development of validated assays.
All these scientists, whose work will be presented at CHI’s upcoming “Quantitative PCR—Getting the Basics Right” conference in San Diego, have novel tips, tricks, and techniques to offer that can help solve specific issues in qPCR applications.
At Johnson & Johnson, Elisa Mokany, Ph.D., a scientist in nucleic acid analytic technology, and her colleagues developed a novel detection methodology based on multicomponent nucleic acid enzymes (MNAzymes). MNAzymes, which form in response to accumulating target amplicon, use a nonprotein-based enzymatic reaction to cleave a generic reporter probe, thereby separating the fluorophore from the quencher moiety.
The ability to accomplish this is found in the elegant design of the MNAzyme. They are composed of two oligonucleotide components, referred to as partzymes A and B. Each partzyme consists of a target-specific sensor arm, a partial catalytic-core sequence, and a generic probe-specific reporter arm.
The sensor arms of the two partzymes are designed to bind to adjacent sequences in the target amplicon (DNA or RNA). This allows the catalytic core to form and provides a binding site for the generic reporter probe on the reporter arms of the MNAzyme. Self assembly of the MNAzyme is facilitated by binding to a specific target sequence in the amplicon. Subsequent cleavage of the bound generic reporter probe results in fluorescent signal generation.
The ability of MNAzymes to discriminate between closely related sequence variants is based on the design of the target-specific sensor arms. With strategic design of the sensor arms, MNAzymes have been shown to discriminate between single base polymorphisms without bias.
“MNAzymes provide an extremely flexible detection method that is compatible with most pre-existing PCR primer sets under qPCR cycling conditions,” says Dr. Mokany. “We have demonstrated their effectiveness in seven different real-time PCR machines. The fluorescent signal is only generated in the presence of the specific target amplicon. This signal is linear over a broad range without background.”
The most important contribution of MNAzymes, however, is in their application for multiplex analysis. The catalytic core and reporter arms are generic. A typical multiplex analysis is set up with a series of generic reporter probes with different fluorophores that bind to the respective reporter arms. In closed-tube reactions, Dr. Mokany reports that she has been able to demonstrate detection of different signals in a multiplexed reaction for simultaneous quantification of up to five target transcripts without cross talk.
Daniel Adlerstein, Ph.D., head, molecular diagnostics, at Diasorin, and his colleagues developed an efficient signal-generation alternative by incorporating a quenched fluorophore on the 5´ tail of the PCR primer. The key to FLAG (fluorescent-amplicon generation) is the use of a thermostable endonuclease PspGI that is stable throughout the cycling process. The signal evolves when PspGI cleaves off the quencher moiety using the restriction site that separates the quencher and fluorophore on the accumulating amplicon.
The story doesn’t stop there, however. The group at Diasorin also uses PspGI to selectively amplify mutated codon 12 KRAS sequences in the presence of the wild-type sequence. Preferential amplification of mutant KRAS sequences (codon 12) is enabled, because the recognition site for PspGI overlaps the wild-type sequence in codon 12 of the KRAS oncogene. Cleavage at this site prevents the accumulation of wild-type amplicons, resulting in preferential amplification of the low-abundance mutant variants.