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Jul 1, 2010 (Vol. 30, No. 13)

Real-Time PCR Advances Show Promise

Emerging Enhancements Address Limitations of This Essential Laboratory Technique

  • Rapid Quantification of DNA Libraries

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    Agilent Technologies’ Brilliant III UltraFast qPCR MasterMix features a newly engineered Taq that reduces cycling time and improves sensitivity, the company reports. The firm says the Brilliant III reagents’ faster extension rate along with hot-start technology minimize nonspecific amplification.

    In order to address some of the current challenges of qPCR, Agilent Technologies has launched a mutant Taq DNA polymerase. “There is a big drive for high throughput,” explained Raza Ahmed, Ph.D., product specialist. “The two ways this is done is by increasing the number of reactions per plate and by decreasing the assay run time. Unfortunately, as you shorten the run time, you compensate for a loss of sensitivity and performance.”

    The Brilliant III UltraFast qPCR MasterMix contains a mutant Taq polymerase that can accommodate these challenges and tolerate any inhibitors in the sample (it has been tested in whole blood and various cells). “This provides a 60 percent time savings, and we now have the fastest enzyme on the market, enabling a qPCR reaction in 30 minutes.”

    Rather than using an antibody-based enzyme, Dr. Ahmed explained that Agilent modified a chemical reagent to minimize background noise from nonspecific amplification. This is because the antibody has to be specific to activate Taq and there is potential for it to fall off. “This chemical reagent is much faster and you can go as short as three minutes on preamplification with 90 percent polymerase activity.”

    The overall advantages include ability to perform fast qPCR without losing sensitivity, amplifying targets even in the presence of PCR inhibitors, and minimizing nonspecific amplification via a modified, chemical hot start.

    Current applications include pathogen detection, gene expression, and genotyping. A new application for single-cell analysis will be launched soon, and Dr. Ahmed said they are considering splice variants and methylation.

    The company also launched a library quantification kit for next-generation sequencing that includes the Brilliant III enzyme. Primers specifically designed for the Illumina genome sequence analyzer are key. “This is important because the current issue with sequencing is that researchers are unable to obtain high-quality data from the DNA sequences because there are issues having the correct amount of DNA to put on the sequencers. This is the big bottleneck at the moment.” The Brilliant III reportedly enables fast sequencing compared to current methods such as fluorescent dyes.


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