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Jan 15, 2009 (Vol. 29, No. 2)

Enhancing PCR for Diagnostic Applications

Usage Ranges from the Functional Analysis of Genes to the Diagnosis of Disease

  • qPCR

    Stephen Bustin, Ph.D., professor of molecular science at Barts and the London School of Medicine’s Institute of Cell and Molecular Science, will present at “qPCR2009.” Dr. Bustin is currently working on a real-time quantitative (qPCR) assay for the detection of Clostridium difficile, the bacterium responsible for a huge number of hospital-acquired infections. He expects that the assay will be more sensitive, more informative, and much less costly than current PCR-based assays. 

    Such assays, he notes, cost in the $20–$30 range per assay, and dedicated instrumentation is required. His goal is to develop a set of affordable tools that allow not just detection, but also a more detailed molecular characterization of C. difficile. Currently, he notes, there are many problems in the field of qPCR, and the problems are proving to be durable. 

    Dr. Bustin authored a paper citing such limitations in 2000, “but we haven’t progressed a lot since then,” he observes. In fact, “the field is littered with papers that are meaningless,” with the problems beginning with sample preparation, and extending to “how you normalize, analyze, and report results.”

    “It is clear that a high percentage of publications utilizing qPCR technology, and especially those aiming to profile cellular RNA levels, report poorly designed, executed, and interpreted experiments and results,” Dr. Bustin states.

    “Considerations of mRNA transcription, in vivo stability, regulation by miRNAs, tissue specificity of splice variants, allele-specific difference in expression, the lack of concordance between most mRNAs and their specified proteins, and the critical importance of post-translational modifications and questions of tissue heterogeneity all describe serious issues that are not being addressed in an adequate manner,” he concludes. “It will require a significant amount of courage, and a sea change in attitude from the research community to deal with this quagmire.”

    On a positive note, there are several developments that Dr. Bustin finds encouraging. Among these he cites the introduction of less expensive, optimized reagents that make reaction assembly simpler and more consistent; the development of more intuitive analysis software to help with assay setup and project management; the introduction of advanced algorithms that allow more accurate quantitation; and the extension of the technology into novel areas such as high-throughput, nanoliter qPCR—specifically, microfluidic digital PCR, which is “an exciting new development that extends the scope of qPCR technology.”

  • Digital Applications

    Click Image To Enlarge +
    According to Fluidigm, microfluidics-based digital PCR makes the technique more sensitive and cost effective.

    Ken Livak, Ph.D., senior scientific fellow at Fluidigm, explains the value of his company’s microfluidic chips for digital PCR and the system’s capability to analyze gene expression at the single cell level. Applications include cancer and stem cell research. 

    Noting that digital PCR has been around since the 1990s, but that it remains tedious and expensive, Dr. Livak says that microfluidics-based digital PCR makes the technique more sensitive and cost effective.  The BioMark digital array functions as “the integrated circuit for biology,” Dr. Livak explains. “Reaction chambers within the chip are the size of a pencil dot, which contributes directly to the system’s stingy use of sample and reagents.”

    The Fluidigm system has been used to identify and quantify ABL tyrosine kinase domain point mutations in samples from 28 patients. The digital array with its nanoscale channels, valves, and pumps partitioned samples into 12 panels, each panel containing 765 chambers. There is an improvement in detection of rare mutations as a consequence of partitioning before PCR, Dr. Livak observes.

    He cites the example of a mixture containing a molecule of T3151 ABL in 100,000 molecules of unmutated ABL. If partitioned into 1,000 separate chambers, the chamber containing the single mutant molecule would only contain about 100 molecules of the unmutated ABL. This provides a 1,000-fold increase in relative concentration and allows for a theoretical 1,000-fold improvement in the detection sensitivity of PCR reactions.

    Such sensitive detection of the T3151 mutation and other mutations may allow investigators to study the biology of clonal selection and evolution in the context of tyrosine kinase inhibitor therapy, resistance, and progression in chronic myelogenous leukemia, Dr. Livak concludes. 

    In addition, he notes, the BioMark system “lets you do higher- and higher- throughput analysis without a lot of robotics, making it more affordable so it can be used in more labs by more scientists.”

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