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

RT-PCR: Ready for Prime Time?

  • Finding Multiple Mutations

    Click Image To Enlarge +
    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]

    “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.

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