Protocols and Resources

Designing Real Time PCR Experiments (pdf)

CFX Touch Protocol (pdf)

Tetrad Protocol (pdf)

Choosing a Reaction Chemistry:

There are several different techniques available that detect amplified product with similar sensitivity (Wittwer et al., 1997). In each method, a fluorescent dye is included in the PCR reaction mix that allows for the detection of the PCR product as it accumulates through each cycle.

DNA-binding dyes

This is the simplest and cheapest chemistry and is based on detection of the binding of a fluorescent dye (SYBR Green) to DNA. The unbound dye exhibits little fluorescence in solution, but during elongation increasing amounts of dye bind to the minor-grove of the nascent double-stranded DNA. When monitored in real-time, this results in an increase in the fluorescence signal during the polymerization step.

The specificity of this method is determined entirely by the primer set that is being used, as in conventional RT-PCR. The formation of primer dimers and non-specific PCR products will also result in a fluorescent signal. However, the specificity can be verified by plotting fluorescence as a function of temperature to generate a melting curve of the amplicon. The resulting melting curve can be used to determine the composition of the reaction product, but will not correct the results. Therefore, good primer design and optimizing the PCR conditions is essential.

Many vendors sell SYBR Green-containing PCR mastermixes, which contain the dye, buffer, dNTPs, and polymerase enzyme. This mix can also be home-made using SYBR Green Dye from Molecular Probes.

We have found that genes that are expressed at very low quantities can be hard to detect using SYBR Green, mainly because it is difficult to completely eliminate the unspecific signal coming from the formation of primer dimers. Therefore, many researchers use one of the other chemistries for quantification of low abundance transcripts.

Taqman

The Taqman assay utilizes a short oligonucleotide probe that is labeled with a reporter dye on the 5' terminus and a quenching dye on the 3' terminus. The probe sequence is homologous to an internal target sequence present in the PCR amplicon so that it will bind specifically to the target. When the probe is intact, energy transfer occurs between the two fluorophors and emission from the reporter is quenched. During the extension phase of PCR, the probe is cleaved by 5' nuclease activity of Taq polymerase, thereby releasing the reporter from the probe and quencher and producing an increase in reporter emission.

This assay is more specific than SYBR green but the dual-labeled fluorogenic oligonucleotide probe is quite expensive. Also, TaqMan PCR works best on small amplicons and it is sometimes difficult to find gene-specific primer and probe sequences.

Molecular Beacons

Molecular beacons are hybridization probes that form a stem-and-loop structure in which the loop portion binds to the target. A fluorescent marker is attached to the end of one arm and a quencher is attached to the end of the other arm. In solution, free molecular beacons adopt a hairpin structure which quenches the fluorophore. When molecular beacons bind to a complementary target, they undergo a conformational transition that separates the fluorophore and the quencher, leading to the fluorescence.

The main drawback with molecular beacons is the difficulty of designing the probe. Badly designed probes may fold into alternate conformations that do not place the fluorophore in the immediate vicinity of the quencher resulting in large background signals. If the stem of a molecular beacon is too strong, it can interfere with annealing to the target sequence. Therefore, thermal denaturation profiles must be established for each molecular beacon to determine their melting characteristics.

Reference:

Wittwer CT, Herrmann MG, Moss AA & Rasmussen RP. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques (1997) 22: 130–138.