August 1, 2013 (Vol. 33, No. 14)

Kate Marusina Ph.D.

The conceptual and practical simplicity of quantitative real-time PCR (qPCR) has made it a choice tool for many molecular analysis applications. Because of its speed, sensitivity, and specificity, qPCR has been displacing conventional PCR in the vast majority of its applications. Some believe that it is only a matter of time before qPCR becomes a major player in diagnostics.

A large swath of publications reporting reverse transcriptase qPCR (RT-qPCR) data underscores a great interest in this technique. Yet there still exists a relative lack of consensus in how best to perform the RT-qPCR experiments.

“We can’t simply assume that RT-qPCR always accurately quantitates gene expression levels,” says Chaminda Salgado, head of CMC bioassay and genomics at NDA Analytics. “Each step of the procedure has technical challenges. Optimization and validation of each step is key.”

He adds that the “advancement of qPCR into the diagnostic realm will depend on standardization of at least four key technical components: Sample handling and assessment; RT strategy—enzyme selection and RT priming; normalization during analysis; and increase in hardware speed—for point-of-care applications.”

To date, standardization has been hampered by lack of sufficient experimental details in scientific reports. Frequently, qPCR publications omit critical analysis parameters and justification for reference gene selection, hindering critical evaluation of the quality of the results. To enable inter-laboratory comparisons, qPCR-based clinical diagnostic assays will require more stringent standardization.

Salgado emphasizes that primer design, reverse transcriptase selection, and choice of reference genes are vital underpinnings for the progression of RT-qPCR from a research technique to a market-ready in vitro diagnostic tool.

The first step toward this goal may be implementation of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. These call for reporting of minimal essential and desired information to ensure qPCR relevance, accuracy, correct interpretation, and repeatability. Properly implemented, the MIQE guidelines could help promote consistency among laboratories and potentially zero in on the methods that show potential utility for diagnostic applications.

“QPCR is a sensitive method, but many clinically important analyses require detection of minority events below qPCR current limits of sensitivity. Quantitation of rare mutations on the background of wild-type DNA is of prime importance in several fields of medicine,” says Mike Makrigiorgos, Ph.D., from the Dana-Farber Cancer Institute. “Specifically, subclonal mutations in cancer cells may determine if a particular tumor is resistant to chemotherapy. We envision that monitoring the quantity of these mutations during cancer treatment may inform management decisions for the remainder of the therapy or subsequent rounds of therapy.”

Dr. Makrigiorgos’ team focuses on development of techniques for identification of rare events and translating them into diagnostic applications. A few years ago, they developed COLD-PCR (co-amplification at lower denaturation temperature) to enrich samples with mutations irrespective of where they occur in DNA sequence. The technology takes advantage of a small but detectable difference in melting temperatures of mismatched sequences. Denaturing and re-annealing genomic DNA creates mismatched DNA duplexesFurther denaturation of these duplexes at critical denaturation temperatures preferentially releases the mismatched strands, which then can be amplified by PCR. Two consecutive rounds of COLD-PCR result in a 100-fold enrichment of mutations.

“Further enrichment with COLD-PCR was challenging due to polymerase errors and mis-priming events,” Dr. Makrigiorgos says. “We used the same scientific principle to develop an alternative enrichment method that does not involve enzymatic amplification.”

DISSECT (differential strand separation at critical temperature) also uses differential denaturation of DNA heteroduplexes. However, it avoids enzymatic steps being entirely based on repeated cycles of denaturation and hybridization on magnetic beads coated with wild-type target gene sequences. Wild-type DNA remains attached to the beads, while the mutant DNA is released and collected. Because the sequences are not altered during DISSECT, the method is compatible with downstream applications including qPCR and sequencing.

“The method is exceedingly simple, and is easy to automate, multiplex, and scale up,” explains Dr. Makrigiorgos. “DISSECT can simultaneously enrich diverse targets for multiple mutations in the same tube using a single denaturation temperature.” In a proof-of-principle study, three to four rounds of DISSECT produced up to 100- to 400-fold enrichment of mutations in three selected oncogenes.

Researchers at the Dana-Farber Cancer Institute have developed an alternative enrichment method that does not involve enzymatic amplification. DISSECT avoids enzymatic steps as it is entirely based on repeated cycles of denaturation and hybridization on magnetic beads coated with wild-type target gene sequences.

Finding Multiple Mutations

“Many important diagnostic applications require discrimination of multiple sequence variants present in the same sample,” says Kenneth Pierce, Ph.D., senior research scientist at Brandeis University. “Under these conditions, technical challenges for detection based on the classical probe-target hybridization are significant. We developed a novel PCR-based approach that overcomes these challenges and opens doors for a variety of diagnostic and species identification applications.”

This approach, linear-after-the-exponential (LATE)-PCR is an elegant adaptation of the asymmetric PCR method. Because of the unique primer design, LATE-PCR efficiently generates single-stranded DNA after the period of exponential double-stranded amplification. Single-stranded DNA is a superior target for product detection using complementary DNA probes.

“Single-stranded DNA offers an opportunity to use low annealing temperatures for detection,” Dr. Pierce says. “This means that a single universal probe can be used to detect sequences of high diversity. Conversely, we can use the temperature gradient to obtain information about specific mutations in a given sequence.”

The team used this novel approach to study antibiotic resistance of gram negative bacteria. Mutations in beta lactamase enzymes give rise to an extended spectrum of antibiotic resistance. Rapid detection of a specific mutation may help with selection of the appropriate medical treatment.

One detection approach combines LATE-PCR with Lights-On/Lights Off probes. Rather than detecting each mutation with its own color probe, as many current PCR-based applications do, Lights-On/Lights-Off uses the same color to detect many mutations at once in the same tube. Each Lights-On probe consists of a quencher and a fluorescent moiety, and Lights-Off probe has only a quencher moiety. Each Lights-Off probe quenches the Lights-On probe when both are bound to the target in a close proximity. The signals from all contiguous Lights-On probes create a composite fluorescent contour, which is mathematically converted into a sequence-specific fluorescent signature. The fluorescent probes will bind to mismatched sequence at a lower temperature and will produce a distinct fluorescent contour.

“We found that each mutation in lactamase’s hot spots produced its own specific signature,” Dr. Pierce says. Brandeis University owns LATE-PCR and its allied technologies and is prepared to license them for commercialization.

“Current diagnostic tests rarely provide immediately actionable information,” asserts David Dolinger, Ph.D., evp, Seegene. “Medicine, and in particular diagnostic medicine, needs to evolve from art to science where diagnostic assays are based on signs and symptoms of disease.”

Seegene believes two of its technical achievements will transform diagnostics.

Dual Priming Oligonucleotides (DPO™) eliminate typical “noise” problems in multiplex PCR. DPO primers consist of two distinct annealing regions separated by a unique polydeoxyinosine linker. The larger portion, called the “stabilizer,” binds to target DNA resulting in stable annealing. However, extension will occur only if the shorter arm selectively binds to the target sequence. Built-in thermodynamic constraints create an internal control so that specific extension only occurs when both arms anneal.

“The DPO technology opened doors for literally unlimited multiplexing,” Dr. Dolinger affirms. “The second piece of the puzzle required solving the detection of the targets in a multiplex reaction. Current fluorescent detection technologies can only deliver three to five answers per sample. We wanted to achieve at least an order of magnitude more.”

Seegene’s approach involves detection and measurement of the “catcher,” an artificial single-stranded template labeled with a quencher and a fluorescent moiety. In its free form, the catcher does not fluoresce.

And then there is the “pitcher,” a single-stranded nucleotide composed of a sequence that binds to a target sequence that also has a tagging portion. As the PCR reaction initiates, the DPO primers and the pitcher hybridize to the target gene. Once the DNA extension reaches the pitcher, Taq polymerase with 5′ nuclease activity cleaves the tagging portion. The tagging portion uniquely binds to the catcher and, if cleaved at the correct base, could be extended to form the duplex catcher. The duplex causes the physical separation of the quencher and the fluorophore, producing a fluorescent signal.

Fluorescence signatures generated from the combination of four On Probes and four Off Probes distinguish a penicillin-resistant variant (green) of the bacterial TEM gene from two variants (blue, red) with extended-spectrum antibiotic resistance. [Brandeis University]