Since the origin of PCR in 1983, there have been many enhancements and adaptations. Current trends, recently cited in a report by HTStec (qPCR Assay Trends, 2008), indicate a focus on faster reagents, shorter run times, and new applications such as expression profiling and multimarker diagnostics.
However, there are still some limitations including high instrument and consumable costs as well as data analysis. Automation of qPCR is expected to improve assay quality and throughput. The recent “Developments in Real-Time PCR Research and Molecular Diagnostics” conference shed some light on advances that will address current challenges.
Researchers at Qiagen-DxS developed a novel, real-time Taq extension assay to assess the strength of the amplification refractory mutation system (ARMS) switch. ARMS is allele-specific PCR, where the primer acts like a switch for PCR; when it matches, PCR occurs. It is used to detect gene mutations and SNPs. Jane Theaker, R&D team leader, noted that existing Taq polymerase activity assays involve using radioactivity, are laborious, and are not in real-time.
“We didn’t have an easy way of assessing the strength of the ARMS switch.” She added that it’s difficult to dissect out ARMS extension from other whole PCR effects such as total amplification efficiency, presence of primer-dimers, and side products. The new approach also provides the ability to vary parameters not practical in PCR. It also saves time and costs by using specific primers, she said.
Traditional PCR utilizes primers specific to a sequence; in ARMS, primers are modified at the 3´ end. The modification is designed to make them allele specific. The ARMS switch acts like a switch for PCR by making that last base specific for the allele under investigation.
The assay principle only generates extension products; no PCR cycling occurs. There are 4,096 separate data points (64 primer combinations x 64 template combinations). Any combinations where all three bases are mismatched because they would be highly unlikely to misprime are excluded.
Theaker claimed that the assay provides an actual measurement via mathematical modeling. “We can put a finger on how the Taq extension works, rather than basing it on a threshold cycle you might get with real-time PCR. This is useful in one PCR application but may not be applicable across all PCR situations where you have different templates. We’re just looking at the efficiency of the 3´ end of the primer and that’s what gives us specificity and will be applicable across many different templates.”
She added that they haven’t proved that this is a better way of assessing what will occur in PCR, but that “some of the initial data leads us to believe that we can use extension data to predict what’s going on in PCR.”
Applications for the new assay include when allele-specific PCR is required—such as for genotyping, designing companion diagnostics, and for developing new diagnostic assays. However, Theaker said it won’t be launched as a product, but rather the information will be publicly available after it’s published.
A micro real-time PCR platform consisting of a microscope cover slip on top of a micromachined silicon chip integrated with a heater and temperature sensor has been developed by a team at KIST-Europe. Measuring about 5 x 10 x 4 cm, Pavel Neuzil, Ph.D., team leader, clinical diagnostics, and co-developer, said the impetus behind making the system was to develop something to detect H5N1 virus in undeveloped countries.
“This allows the detection of any gene in about 15 minutes versus 30 minutes to 2 hours in a normal PCR platform,” he remarked, adding that the hardware is about 10 to 20 times cheaper, and the consumables almost 100 times cheaper. He predicts that manufacturing costs will be close to $150 versus $5,000 to up to $20,000.
The DNA sample is placed on the microscope cover slip and encapsulated with mineral oil, creating a virtual reaction chamber. Dr. Neuzil said the platform can be used for any sample, depending on the dimers added.
“Basically, you can use any sample and look for HIV, paternity testing, and such.” The microscope cover slip is disposed of once the reaction is completed, avoiding sample cross-contamination, and a new one may be added for the next run. The labeling system used is SYBR Green I. The optical system is derived from those used in DVD players.
Although the system can’t compete with big machines used in labs or hospitals, Dr. Neuzil noted this micro platform’s potential applications include point-of-care, homeland security (Anthrax), education, and field applications. He is hoping it will soon be manufactured.