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

Novel Approaches to Generating Signals and Designing Primers Improve Applications

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.

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.

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.

PCR Systems and Thermocyclers
“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.

With the standardization of the sample-prep steps and the conventional qPCR reactions using SYBR® Green detection methodology, Dr. Crotty and his team developed a sensitive, robust assay for the detection of viral infection in vivo. The assay is more than 1,000-fold more sensitive than standard plaque assays for tracking LCMV infection in mice, he reports.

In the molecular diagnostics reference laboratory at Idexx Reference Laboratories, the requirement that real-time PCR provides analytical and diagnostic specificity and sensitivity is key to molecular diagnostics for infectious agents. On a daily basis, Christian Leutenegger, Ph.D., regional head of molecular diagnostics, and his laboratory are faced with two challenges: to maintain a portfolio of fully validated qPCR assays and to maintain the quality control for each assay.

As for the first challenge, PCR primer-sequence design is the solution. The specificity of the reaction is based on the balance between false-positive and false-negative signals. The former comes from cross-reactive signals from related but not etiological agents. False-negative signals come from sequence variation based on selection pressure and normal drift in the genetic isolates.

Regardless of the type of nucleic acid in the infectious agent including viral, parasites, fungi, or bacteria, all infectious agents demonstrate sequence variation. For optimal sequence design, it is important to query all the public databases for identification of both highly conserved and highly variable regions within infectious agents. This is a numbers game, so when the number of published sequence variants is high, the investigator can have confidence in the validity of the defined conserved and variable regions of the genomes.

Dr. Leutenegger and his colleagues routinely resequence positive samples to look for new sequence variants and then add the new sequence information to their working database. Being vigilant to do the bioinformatics workup for all qPCR assays is fundamental to proper assay design and validation. They apply this process in cycles, initially designing and validating the assay, then resequencing and redesigning, and finally revalidating the diagnostic assay.

PCR primers designed to bind within highly conserved regions risk high levels of cross reactivity to related strains that are not the etiological agent. PCR primers designed to bind within highly variable regions yield high specificity but risk high incidence of false-negative results. The answer is to design in a balance between these extremes.

Quality control in the laboratory is the other challenge, having the confidence in every negative result and every positive result. To maintain quality control in the laboratory process, controls from sample prep through amplification and finally resequencing are strictly enforced. The direction of the workflow is designed to minimize the chance for contamination.

“We accept the fact that no PCR laboratory is without contamination,” states Dr. Leutenegger, “so we employ a further level of control by incorporating the AmpErase system in all PCR assays.” This means that all amplification assays are subject to enzymatic digestion of any contaminating amplicons based on the incorporation of uracils into the product.

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