Real-time PCR
Quantitative "real time" PCR (qPCR) measures the accumulation of fluorescent DNA amplification products as they are generated during each cycling step of a polymerase chain reaction.
This produces a "real-time" plot of fluorescence versus cycle number, where the fluorescence is proportional to the amount of amplified product.
Quantitative data are obtained by measuring the cycle threshold (Ct) - the cycle number at which fluorescent products are first detected, rather than the final amount of fluorescent product accumulated after a fixed number of cycles as in standard PCR.
The higher the amount of initial nucleic acid template in a reaction, the sooner fluorescence is detected and the lower the Ct value that is measured.
This enables us to quantify the relative abundance of various DNA target sequences in a sample, or absolute abundance of a target by comparison with appropriate standards.
The kinetic data produced during the reaction are useful for quality control without having to open reaction tubes and analyse their contents.
For example, the PCR amplification efficiency can be determined during the initial (exponential) phase of the reaction, and frequently approaches the theoretical maximum of 2n when amplifying small DNA target sequences.
Fluorescent "melt curves" obtained by heating the final product can determine if the expected PCR amplicon is contaminated by non-specific amplification products.
Contact us
Contact Dr Vitaliya Sagulenko for more information.
