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Oct 15, 2009 (Vol. 29, No. 18)

qPCR Faces Growing Pains Head-On

Establishment of Standards and Evolution of Methodologies Help the Field Mature

  • Molecular Beacons

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    Scientists at New Jersey Medical School are using a molecular beacon approach to expedite qPCR research.

    New qPCR technologies involving molecular beacons were described by Fred Kramer, Ph.D., professor at New Jersey Medical School. Like the faithful lighthouses that send out signals to ships at sea, molecular beacons provide biological signals useful in applications that range from rapid pathogen identification to genetic screening.

    “We have been developing ways to rapidly identify potentially lethal sepsis-causing bacteria. Identification in a clinical setting often takes several days, during which time a patient is treated with a broad-spectrum antibiotic. This strategy occasionally fails and contributes to the development of antibiotic-resistant strains. With a molecular-beacons approach in a qPCR setting, we can identify pathogens in one reaction in only one or two hours.”

    Molecular beacons are single-stranded oligonucleotide hybridization probes with a unique architecture. They form a stem-and-loop structure in which the loop contains a probe sequence complementary to the target. One terminus of the stem contains a fluorophore and the other a quencher. Like a molecular switch, molecular beacons light up only when the probe hybridizes to its target.

    According to Dr. Kramer, molecular beacons can be designed to be either “finicky” or “sloppy”. “Finicky probes are shorter (~18–26 nucleotides) and form hybrids with perfect complementarity. These are useful for genetic screening, detection of SNPs, and pharmacogenetic applications. Sloppy probes, however, are longer (~40 nucleotides) and hybridize to a broad range of species we want to identify.”

    This feature helps identify pathogens in a clinical sample. “We use a sloppy probe set with four differently colored fluorophores in a single gene amplification reaction,” he said. “The molecular beacons hybridize at a relatively low temperature. As the temperature is slowly raised, each of the four probe-target hybrids melts apart and signal is lost. The resulting set of four melting temperatures creates a specific signature that identifies which of potentially hundreds of species is present in the sample.”

    Dr. Kramer has licensed the molecular-beacons technology for an assortment of clinical tests that range from HIV-1 identification to tuberculosis screening. While his current research is still at the development stage, he is optimistic that advanced molecular-beacon designs will enable new clinical diagnostic applications.

  • FFPE Samples

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    Detection of differential expression of two microRNAs in laser-dissected specimens from tissue FFPE sections (Exiqon)

    Another emerging technology that uses modified primers was discussed by Adam Baker, Ph.D., director of diagnostic product development at Exiqon. “It is critical for us to be able to analyze microRNAs accurately and robustly in blood-based and precious clinical FFPE samples. Analyzing microRNAs from these types of specimens can be exceedingly difficult due to low RNA yields and insufficient quality.”

    Exiqon’s new qPCR system allows high-quality microRNA profiling to be carried out in samples with low RNA yields. According to Dr. Baker, the new qPCR platform, called miRCURY LNA™ Universal RT microRNA PCR, features two important advancements.

    “First, because the system is based on a universal reverse-transcription reaction, the same first-strand cDNA pool can be used as template in all microRNA PCR amplification assays. This means that up to 740 different microRNAs can be profiled from just 40 nanograms of total RNA. Second, the qPCR assays use two microRNA-specific primers containing locked nucleic acids (LNA™). LNA nucleosides are “locked” by a methylene bridge connecting the 2´-O atom and the 4´-O  atom enabling both higher affinity base pairing and enhanced mismatch discrimination.”

    Dr. Baker noted that this has allowed the development of exceptionally sensitive and specific assays that enable accurate quantification of low microRNA levels and discrimination between highly related microRNA sequences. “Using this system we have been able to carry out sensitive and robust screening in clinical cancer samples. As a result, we are developing diagnostic tests based on these results. We are gearing up to launch the new assay system this year.”


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