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May 1, 2011 (Vol. 31, No. 9)

qPCR Evolving to Meet Emerging Needs

Method Now Utilized in Epigenetics Research and Characterization of Circulating Tumor Cells

  • Predesigned Assays

    Click Image To Enlarge +
    According to Integrated DNA Technologies, PrimeTime assays are more sensitive than competitors’ assays. A comparison of a competitor’s WDR3 assay and the equivalent IDT PrimeTime predesigned qPCR Assay is shown for five 10-fold dilutions of the target.

    Designing a good qPCR assay requires careful consideration of a number of parameters that can spell the difference between success and failure, according to Mark Behlke, M.D., Ph.D., CSO, Integrated DNA Technologies (IDT). “The design of the assay must carefully determine parameters such as primer placement, specificity, avoidance of single nucleotide polymorphisms, oligonucleotide interactions, and accurate melting-temperature calculations.”

    One answer is the use of predesigned assays that shift the burden of these choices to the manufacturer. But not all assays are created equal, according to Dr. Behlke. “Unfortunately, databases are dynamic, not static. Some commercial assays were designed using now-outdated databases. In other cases, a scientist might be using a primer design format that was done with older, less accurate melting-temperature algorithms. Even worse, a researcher may not be able to find out from the company the sequence information that could be verified or even a correct quote for a research publication.”

    To overcome this problem, IDT offers its PrimeTime® predesigned qPCR assays. Scott D. Rose, Ph.D., director, molecular genetics, reports, “We’ve developed these assays for all genes in the human, mouse, and rat transcriptomes. The assays are very specific and eliminate concerns such as cross-reactivity within each genome and primer interactions. Importantly, we also freely provide all the information a researcher will need to know, including full sequence disclosure of both primers and probes as recommended by the MIQE guidelines.”

    The IDT technology employs its ZEN double-quenched probes. Dr. Rose indicated that “these assays provide sensitivity down to 10 copies or less. We were able to reduce background fluorescence by decreasing the length between the reporter dye and the internal quencher to only nine base pairs. Traditionally probes have 20–30 bases between the dye and quencher.

    “Thus, the ZEN quencher is always within close proximity of the reporter, resulting in a much lower initial background fluorescence, Lower background results in greater sensitivity for the assay. With the ZEN system, even long probes—up to 50 base pairs—are effectively quenched because the distance between the quencher and the reporter dye is fixed and is independent of probe length.”

  • Circulating Nucleic Acids

    Analysis of nucleic acid fragments present in plasma, serum, and other body fluids such as urine can enable specific detection of tumor types from a simple blood sample, said Martin Horlitz, Ph.D., senior scientist, Qiagen. “These circulating DNA and RNA fragments can originate from malignant tumors, a developing fetus, and also from viral or bacterial infections. Assessing the presence of these nucleic acids via qPCR could help diagnose cancer at an early stage or assess the genetic makeup of a developing fetus.”

    Circulating cell-free nucleic acid fragments have distinctive properties. “They are present in blood mostly as shorter fragments of less than 500 base pairs or as nucleotides at a concentration of about 5–100 ng/mL. Because of their low concentration and small fragment sizes, they present a challenge to isolate. Qiagen offers the QIAamp Circulating Nucleic Acid Kit for the quick and efficient purification of these circulating nucleic acids from samples up to 5 milliliters.”

    The kit is based upon the selective binding of nucleic acids to a silica-based membrane that provides for improved recovery of fragmented nucleic acids. Dr. Horlitz explained, “The purification is highly efficient with reproducible yields and can be easily done on a vacuum manifold. There is no organic extraction or ethanol precipitation needed. Thus, there is complete removal of contaminants and inhibitors.”

    However, even with nucleic acids efficiently isolated, running a qPCR presents another challenge. “Concentration and size distribution of isolated nucleic acids can vary between samples and even in the same individual when samples are collected at different times. Hence, the use of internal controls is recommended to independently track the success of both nucleic acid extraction and PCR quantification.”

    Qiagen offers the QuantiFast Kits for singleplex and multiplex real-time PCR and RT-PCR, both of which are SYBR Green and probe-based. “The QuantiFast Kits enable fast and reliable quantification of fragmented circulating nucleic acids of differing sizes and low concentration levels.”

    According to Dr. Horlitz, “qPCR of circulating nucleic acids will likely have an impact on molecular testing. While it is already used for certain molecular diagnostic applications, it is still in its infancy. But it will have many applications that range from genotyping to routinely checking for the presence of tumors.”

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