Detects and quantifies nucleic acids in real time through monitoring of fluorescence emitted by a reporter molecule.
Quantitative real-time PCR (qPCR) enables the detection and quantification of PCR products in real time as opposed to the end-point analysis used in conventional PCR. This technique uses double-stranded DNA (dsDNA)-binding dyes or target-specific fluorescently labeled primers and probes that hybridize with the target sequence and measures the amount of DNA by calculating the proportional increase in fluorescence detected after PCR amplification. In reverse-transcription quantitative PCR (RT-qPCR), RNA is reverse transcribed into cDNA, which is then used as the starting material for amplification. qPCR is used in several applications, including gene expression analysis, genotyping, microbiology, forensic sciences, and in next generation sequencing (NGS) workflows for library quantification and sample QC.
Most commercially available qPCR reagents contain wild-type unmodified enzymes isolated from nature, which were never intended as tools for use in molecular biology. The performance differences among commercial qPCR kits are the result of differences in factors like buffer formulation and/or enzyme concentration.
Roche offers qPCR reagents containing DNA polymerase and other enzymes selected through our directed evolution technology to deliver high-quality results for specific qPCR applications.
What are the advantages of using PCR reagents selected through directed evolution?
- Contain novel enzymes that are optimized specifically for qPCR application
- Confer significant improvements to reaction efficiency, sensitivity, speed and signal-to-noise ratio compared to wild-type reagents
How does directed evolution work?
Directed evolution exploits the principle of natural selection. Mutagens are used to introduce random variations in the gene encoding DNA polymerase, which results in a library consisting of millions of genes coding for unique variants of the enzyme. Next, a selection pressure is applied to identify the genes that survive the pressure, and those genes are then selected. The process is repeated until the enzymes exhibiting the highest improvements are obtained.