qPCR

 

 

 

Real-Time PCR
Since its introduction in 1993, real-time PCR – also known as quantitative PCR (qPCR) – has become one of the fastest growing PCR applications. Based on the principles of conventional or end-point PCR, qPCR utilizes fluorophores to continuously collect data in the form of fluorescent signals from one or more primer-template interactions over a range of PCR cycles. Non-sequence specific assays employ intercalating fluorescent dyes such SYBR® Green I to detect the accumulation of double-stranded DNA PCR product, whereas sequence specific assays are based on the use of fluorescently labeled probes (e.g. TaqMan® probes used in 5’-nuclease assays, FRET probes or molecular beacons). Fluorescent signals from each reaction are converted into a numerical value for each sample. qPCR has become the most sensitive and specific quantitative method for quantitative gene expression, genotyping, SNP analysis, pathogen detection and drug target validation. In addition to the extremely broad dynamic range and high specificity, the closed-tube qPCR format is ideally suited to high sample throughput and limits the risk of sample contamination associated with post-amplification handing

Kem-En-Tec offers a suite of reagents for real-time PCR both for sequence non-specific (intercalating dye) and sequence specific assays (probe).

From PCRBiosystems:

For sequence non-specific assays using an intercalating dye such as SYBR green or SyGreen is the industry gold-standard for quantification and using polymerases optimized for SYBR/SyGreen has several advantages:

 

  • Superior sensitivity, linearity and reaction efficiency across a wide range of template types and assays.
  • Rapid extension rate for early Ct values.
  • Compatible on all real-time PCR platforms – standard and fast cycling conditions.
  • Reduced cycling times, which reduce overall turnaround times.
  • Increased flexibility in primer design due to the ability to amplify longer targets.

For the majority of applications we recommend the use of an intercalating dye technology rather than costly dual labelled probes. Using an intercalating dye, such as SyGreen, rather than a hydrolysis probe (such as Taqman®) has the added benefit of post amplification melt profile analysis. Intercalating dye real-time PCR also has the advantage of being able to cycle faster than with hydrolysis probes, this is due to the slower rate of 5′ to 3′ exonuclease activity of taq DNA polymerase compared to its polymerisation rate.

Kem-En-Tec nordic provides two main categories of SYBR/SyGreen polymerases. So when would you choose one over the other?

Choose KAPA SYBR® FAST qPCR Kits  for superior signal intensities, due to the inclusion of higher concentrations of SYBR® Green I dye.

Choose qPCRBIO SyGreen Mix because it uses a proprietary intercalating dye which does not inhibit PCR, unlike other popular fluorescent dyes including SYBR® Green.

 

Sequence specific assays probe based technologies has one principel advantage in that multiple amplicons can be detected in the same tube. The number of amplicons that can be detected in real-time PCR is typically limited by the number of light channels found in the real-time PCR instrument. Other advantages of probe optimized polymerases are:

 

  • Compatibility with all probe-based applications and instruments.
  • Fast, reproducible and precise quantification.
  • Discrete clusters in SNP genotyping assays.
  • Suitability for multiplex qPCR.
  • Broad dynamic range.
  • Highly stable master mixes for high throughput workflows.