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Apr 1, 2008 (Vol. 28, No. 7)

New Solutions Make qPCR a Rapidly Advancing Field

Novel Approaches to Generating Signals and Designing Primers Improve Applications

  • This version of REMS PCR (restriction enzyme-mediated selective PCR) is preferred over traditional REMS PCR due to the use of the thermostable PspGI endonuclease, according to Dr. Adlerstein. In traditional REMS PCR, the restriction enzyme used loses activity over a few cycles allowing wild-type amplicons to start accumulating and overwhelming the low-abundance mutant signal.

  • Click Image To Enlarge +
    PCR Systems and Thermocyclers

    Before FLAG, the operator would have to open up the reaction tube and periodically add additional enzyme or perform a sequential two-step amplification/degradation process to knock down the wild-type amplicons. This is clearly not optimal for a high-volume diagnostic assay and could also lead to carry-over contamination of other reaction tubes. The thermal stability of PspGI allows the reaction to continue in a closed tube.

  • “Signal generation for FLAG is universal,” says Dr. Adlerstein, “but real-time REMS PCR is restricted to KRAS sequences.” To further extend the utility of this methodology as a diagnostic, Dr. Adlerstein and his team have developed a method for KRAS mutation genotyping in the closed-tube reactions. Genotyping is facilitated by the use of mutant-specific PNA probes added to the master mix in separate, parallel reactions.

    It is well known from the literature that PNA probes have a higher affinity for DNA than DNA probes. Based on this observation, PNA clamping enables genotyping, because a perfect PNA/DNA hybrid formed on the target sequence can not be displaced, whereas a single-base mismatch will allow displacement by the polymerase and amplification of the target. When run in parallel, amplification will be seen in all reactions that have a mismatch between the PNA probes and the target but not in the reaction with the perfect match.

    Primer Optimization

    qPCR has emerged as a powerful virologic technique for measuring viral replication and viral loads in humans and animal models. The animal model used by Shane Crotty, Ph.D., assistant professor of vaccine discovery, and his team at the La Jolla Institute for Allergy & Immunology is lymphocytic choriomenigitis virus (LCMV) infection in mice. LCMV is the best understood mouse model for chronic viral infection with parallels to HCV and HIV in humans. As reported in the literature, LCMV presents a state of constant replication in tissues with release of viral particles into the serum. Dr. Crotty’s laboratory has designed a qPCR assay to monitor persistence of infection and viral load by tracking level changes in tissues and serum over time.

    “The key to designing a robust, functional assay is to design PCR primer pairs that have specificity in all tissues that will be used in the diagnostic assay,” says Dr. Crotty. “For LCMV, tissue samples are processed from liver, kidney, brain, and serum. In each of these tissue samples, the complexity of the cellular RNA populations is significantly different and in all cases, present at levels significantly above that of the viral RNA.”

    To avoid the risk of false negatives or false positives, the viral RNA primer pairs must have high specificity in all tissues. An optimal primer set that selectively amplified viral RNA in all tissues for this assay was empirically defined.

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