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Feb 15, 2012 (Vol. 32, No. 4)

Aiming to Optimize qPCR Steps

  • Advantages of Digital qPCR

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    Engineers test a new PCR instrument developed at Lawrence Livermore National Laboratory that can reportedly process biological samples in less than three minutes. [Jacqueline McBride/LLNL]

    Reginald Beer, a principal investigator and associate program leader at Lawrence Livermore National Laboratory, will discuss the advantages of digital qPCR at the meeting, as well as the potential for ultra-fast qPCR. Beer was reportedly the first to demonstrate real-time, droplet-based digital qPCR on a chip in 2007, and he also has developed an instrument capable of detecting both DNA and RNA targets in picoliter droplets.

    Beer notes that digital qPCR involves partitioning targets in such a way that there is either one copy or no copies of the target in each PCR well. This offers significant advantages in absolute quantification as a result of the high accuracy in knowing the starting concentration of template due to Poisson statistics. The technology is complex compared to bulk PCR, however, and it is not appropriate for all qPCR applications, he reports.

    Digital qPCR is ideal for low-copy number detection in a sample with a high concentration of interfering templates, such as applications involving rare mutation detection, absolute quantitation, copy-number variation, and haplotyping. Advances that Beer sees coming in digital qPCR include instrument and process simplification and higher throughput.

    Emphasizing his belief that “speed will serve the broad scientific community,” Beer also touts his team’s recent work developing an ultra-fast PCR instrument that can complete 30 PCR cycles in under three minutes. Such an instrument would have numerous benefits, including higher throughput, faster results for clinical applications, and widespread utility in forensics and food safety.

    The device reportedly achieves extremely fast thermal cycling, making possible heating and cooling rates of 45°C per second. “We were really encouraged by the fact that off-the-shelf polymerases worked at these speeds, and there are a lot of parameters that can be adjusted to potentially go even faster.” Dr. Beer and his team are currently working to develop an optical detector for an ultra-fast qPCR instrument.

  • LATE-PCR

    J. Aquiles Sanchez, a senior research scientist and an expert in cancer genomics at Brandeis University, has used LATE-PCR with Thermalight™ Lights-On/Lights-Off probes for the simultaneous qPCR mutation scanning of all four of the key EGFR exons (18–21) related to tyrosine kinase inhibitor (TKI) sensitivity/resistance in non-small-cell lung cancer (NSCLC). Current technologies for such mutation detection utilize DNA sequencing or high-resolution melting analysis, but are only able to interrogate one exon at a time, he explains.

    With LATE-PCR, mixtures of different genotypes can be reliably distinguished down to the 10% level, even when the total number of starting copies is very small. The approach is “remarkably robust” and does not require quantification, Dr. Sanchez notes, adding that the clinicians he is collaborating with are ecstatic over the possible availability of this tool, which would permit rapid and cost-effective screening of small biopsy samples.

  • Nondestructive Isolation of mRNA

    Karl Hasenstein, a plant physiologist and professor in the biology department at the University of Louisiana-Lafayette, is working to develop a technique for the nondestructive sampling of mRNA from cells. The goal is to allow for quantitating mRNA in different regions of cells and at different times without harming the cells.

    Hasenstein says that his group uses a steel needle coated with oligo dT that can be inserted into cells to bind to the oligo dA tail of mRNA, and thus retrieve the mRNA without damaging the cell. The needles are actually acupuncture needles with a tip diameter of less than 15 microns.

    To establish proof of principle, Hasenstein has used this coated needle approach to successfully collect mRNA and assess the distribution of two critical genes (BICOID and NANOS) in fruit fly egg cells. This coated needle-based mRNA sampling “can be completed in 60 seconds without damaging the specimen,” he says.

    When the needles are coated reliably at the same density with oligo dT, the approach reportedly allows for absolute quantification without the need for reference genes. In addition, the success of this coated needle approach could “ultimately eliminate the need for biopsies that can have many negative consequences.”

  • A Discrete Dynamical System

    Steven Smith, professor of molecular science, urology, and urologic oncology at the Beckman Research Institute and Medical Center at the City of Hope, is working on an algorithm that would address certain diagnostic anomalies in qPCR reactions. His algorithm treats qPCR as a discrete dynamical system and models qPCR using Michaelis-Menten kinetics over a 50 cycle experiment.

    Use of this algorithm “may permit improved cycle optimization and better quantitation,” Dr. Smith suggests. He emphasizes that while most approaches to empirical or semi-empirical description of PCR utilize continuous functions, the data is actually discontinuous and a discrete approach to the analysis of this dynamical system has certain conceptual and practical advantages. The algorithm yields average values for dwell time, the maximal velocity, and the Michaelis constant of the DNA polymerase over the course of the reaction.


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