The purpose of real-time PCR is to perform efficient amplification of a target sequence and quantify the PCR products in “real time” by employing the use of a fluorescent reporter. Fluorescent reporters can found in the form of DNA-binding dyes or fluorescently labeled primers or probes. It is extremely important to understand the difference between DNA-binding dyes, and the various fluorescent primer and probe based chemistries. The best way to grasp these theories is often to have a visual illustration of each of the different chemistries.
- SYBR Green Dye – SYBR Green I is a fluorescent DNA binding dye that is commonly used as it binds to all double-stranded DNA.
- SYBR Green is detected by quantifying the increase in fluorescence during PCR.
- Advantages to using SYBR Green are that it is inexpensive, easy to use, and easily incorporated into the PCR reaction.
- Disadvantages of using SYBR Green are that there is usually an increase in background and non-specific binding that can lead to detection of false positive results.
Fluorescent PCR Primer and Probe Based Chemistries
- Taqman Chemistry – Utilizes 5’ – 3’ exonuclease activity of Taq Polymerase (enzyme that copies DNA and necessary for PCR) to generate a signal.
- The probe is composed of a single stranded DNA oligonucleotide which is complementary to the specific target sequence of the PCR template.
- The probe has a modification to the 3’ end so that the polymerase cannot extend the sequence.
- The 5’ end has the fluorescent dye and the 3’ end contains the quencher
- During DNA synthesis, the exonuclease activity of the Taq Polymerase will degrade the probe, thus resulting in release of the reporter from the quencher.
- Fluorescent Resonance Energy Transfer (FRET) – Energy is transferred between two light sensitive molecules.
- Increase in target à More probes bind à Increase in fluorescence
- The 5’ end is the donor (catalyst) and the 3’ end is the acceptor (fluorophore)
- The energy is detected in the form of heat or fluorescence emission.
- If probes bind, energy is transferred from donor to acceptor and generates the signal.
- Molecular Beacon – This type of chemistry measures the accumulation of product during the annealing phase of PCR.
- Signal is detected only when probes are bound to the template before displacement by the polymerase.
- A chemical modification prevents degradation during the extension step of PCR.
- The 5’ end contains the reporter fluorophore and the 3’ end contains the quencher.
- The amount of fluorescence is directly related to the amount of initial template available for binding and inversely proportional to the cycle threshold (CT) value.
- During extension, the probe is displaced by Taq Polymerase and the hair-pin (non-fluorescent) structure is restored.
- Unbound molecular beacon probe à reporter is too close to quencher à no signal is generated.
- Beacon probe binds to target à reporter is separated à signal is generated.
- Scorpion – Scorpion probes use two PCR primers, where one serves as a probe and once contains a stem-loop structure.
- The stem-loop structure contains a 5’ fluorescent reporter and a 3’ quencher.
- The loop of the scorpion probe contains complementary sequence to the internal portion of the target sequence.
- If the primer binds and extends, the reporter is separated from the quencher and a signal is given off.
Understanding the various primer-probe chemistries including the interactions between the reporters and quenchers will provide some basic groundwork for those interested in pursuing a career in molecular biology.
-LeAnne Noll, BS, MB(ASCP)CM is a molecular technologist in Wisconsin and was recognized as one of ASCP’s Top Five from the 40 Under Forty Program in 2015.