Real-Time PCR

Overview

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.

Roche offers reagents that are optimally suited for qPCR applications
Application Products Features and Benifits

Gene Expression

KAPA SYBR FAST qPCR Kits

KAPA SYBR FAST One-Step qRT-PCR Kits

KAPA PROBE FAST qPCR Kits

KAPA PROBE FORCE qPCR Kits

 

 

 

  • Higher sensitivity and improved reaction efficiency across a broad range of amplicon sizes and GC contents*
  • Reduced experimental variation and contamination with a convenient one-step qRT-PCR protocol*
  • Optimized for versatility and speed, providing highly reproducible results for gene expression assays
  • Direct qPCR from crude blood, tissue, and plant extracts
  • Sample-to-Cq workflows in <1 hour
  • High efficiency for accurate, reproducible, and sensitive results*

Genotyping

KAPA SYBR FAST qPCR Kits

KAPA PROBE FAST qPCR Kits

KAPA HRM FAST qPCR Kits

KAPA PROBE FORCE qPCR Kits

 

 

 

  • Higher sensitivity and improved reaction efficiency across a broad range of amplicon sizes and GC contents*
  • Optimized for versatility and speed, compatible with all probe-based SNP detection applications
  • Engineered to maximize differences in melting behavior between sequence variants resulting in accurate SNP genotyping with maximum sensitivity and speed
  • Direct qPCR from crude blood, tissue, and plant extracts
  • Sample-to-Cq workflows in <1 hour
  • High efficiency for accurate, reproducible, and sensitive results

Food and Water Safety

KAPA PROBE FORCE qPCR Kits

 

 
  • Direct qPCR from crude blood, tissue, and plant extracts
  • Sample-to-Cq workflows in <1 hour
  • High efficiency for accurate, reproducible, and sensitive results

*Data on file.