Patricia de Winter1, David Sugden2
1UCL, United Kingdom; 2KCL, United Kingdom
High throughput qPCR is achievable by automation of sample processing and preparation of reactions, but the rate-limiting factor is the long cycling time required by most chemistries. Although hardware capable of very fast cycling has been available for a number of years, the DNA polymerase in the master mix, typically requires more than 10 seconds to anneal to the target template and extend the product (amplicon). Previously we routinely used a 3-step qPCR protocol in a Rotor-Gene 6000, an air-driven instrument generally capable of shorter cycling times compared with block-based machines. We recently tested a new mutant Taq (formulated as Brilliant III Ultra-Fast SYBR green qPCR mix, Agilent) reported to allow an extremely short combined annealing and extension step. Available data performed on plate-based real-time cyclers examined only two target genes. In this presentation we report the use of Brilliant III to amplify over 150 targets with a wide range of amplicon sizes from various species, and its resistance to PCR inhibitors. Using 5 sec denaturation (95oC) and 1 sec combined annealing/extension (57oC) for 40 cycles, standards of known copy number prepared from purified PCR products (qStandard) exhibited high efficiency (³98%), and excellent linearity between 107 to 101 copies (r2 >0.999). Serially diluted cDNA and gDNA amplified with similarly good efficiency and linearity. Even challenging templates such as NGS libraries, which contain multiple amplicons were readily amplified. The effect of amplicon length on amplification efficiency and linearity was determined. Four common PCR inhibitors did not diminish Brilliant III performance even at high concentrations. The very rapid amplification enabled by the remarkably short annealing/extension step reduced total cycling time to ~39 min without compromising assay efficiency and linearity, indicating that excellent performance can be achieved routinely with Brilliant III.
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