Real-Time PCR
Real-Time PCR is identical to a standard PCR except that the progress of the reaction in monitored by a camera during each PCR cycle or detector in “real-time”. There are a number of techniques that are used to allow the progress of a PCR to be monitored in this way. Each technique uses some kind of fluorescent marker which binds to the DNA. Hence as the number of gene copies increases during the reaction so the fluorescence increases. This is advantageous because the efficiency and rate of the reaction can be seen. There is also no need to run the PCR product out on a gel after the reaction.
Methods of monitoring DNA amplification in “real-time”:
Fluorescent dyes
Intercalating fluorescent dyes (e.g. SYBR green) are the simplest and cheapest way to monitor a PCR in real-time. These dyes fluoresce only when bound to double-stranded DNA. So as the number of copies of DNA increases during the reaction so the fluorescence increases. The major disadvantage of using a dye such as this is the lack of specificity. This dye will report, not on the amplification of the target sequences, but also any ‘miss-primed’ DNA, that may have been amplified by accident.
Fluorescent probes
Fluorescent probes are pieces of DNA complimentary to your gene of interest that are labeled with a fluorescent dye.
The simplest and most commonly used type of probe is the Taqman-type probe. These probes are labeled with a fluorescent reporter molecule at one end and a quencher molecule (capable of quenching the fluorescence of the reporter) at the other. Hence under normal circumstances the fluorescent emission from the probe is low. However
during the PCR the probe binds to the gene of interest and becomes cleaved by the polymerase. Hence the reporter and quencher are physically separated and the fluorescence increases.
Another commonly used type of probe is the “molecular beacon”. Again these are small pieces of DNA complimentary to your gene of interest labeled with a fluorescent reporter and a quencher molecule on opposite ends. These probes are designed to fold on to themselves to bring the reporter and quencher in to closer proximity and minimize fluorescent emission. However, when the probe binds to the gene of interest the probe takes up a linear confirmation and the reporter and quencher are separated.
This results in the desired increase in fluorescence. Molecular beacon probes are not cleaved by the polymerase but are simply “knocked off” again.
