Integrated real-time PCR formats: Methodological analysis and comparison of two available industry options

Abstract

The real-time PCR methodology improved profoundly the basic amplification technology; not only by permitting the digital tracking of the reaction from the very first minutes, but also by significantly compressing the time for an amplification program, through technical improvements in expendables and hardware. Two of the integrated, end-to-end solutions available in the market, the Roche and the Applied Biosystems products, also capitalize on the advances at the multi-color dyes and the melting-curve analysis to perform products discrimination and dispose of postamplification steps, such as electrophoresis, thus further compressing time and logistics footprint of the integrated assay. They also focus on the well-proven ability of the methodology to provide accurately quantitative results, which was always a priority throughout the field of biosciences, and one rarely tackled efficiently. Electrophoresis, though, remains popular with low-end users, who find it robust, reliable, flexible and relatively cheap since expendables are low-cost and know-how is well-established. Given that high-end users prefer true high-throughput methods (such as arrays) for simultaneous assaying, which vastly outperform real-time PCR, the ease and rapidity of the latter’s results, which both are of capital diagnostic concern, remain the strongest points of the method, coupled with its quantification potential