Fig. 1 Sketch of the principle of TaqMan PCR for the quantitation of gene expression. By measuring the amplicon concentration in the early exponential phase of the PCR reaction, the exhaustion of reagents is avoided. (From Ref. [3].)

''hybridization probes.''[4,5] Like exonuclease probes, hybridization probes are used in addition to PCR primers. But unlike the first, hybridization probes combine two different fluorescent labels to allow resonance energy transfer. One of them is activated by external light. When both probes bind very closely at the DNA molecules generated by the PCR amplification process, the emitted light from the first dye activates the fluorescent dye of the second probe. This second dye emits light with a longer wavelength, which is measured every cycle. Thus, the fluorescence intensity is directly correlated to the extent of probe hybridization and, subsequently, is directly related to the amount of PCR product. A major advantage of the LightCycler is the very short time of a PCR run. However, the TaqMan system allows to analyze a higher number of samples at one time and, at least theoretically, might be more accurate as there is an internal reference dye to monitor minor variations in sample preparation.

Apart from exonuclease and hybridization methods for real-time PCR, there are other options, including hairpin probes, hairpin primers, and intercalating fluorescent dyes. Hairpin probes, also known as molecular beacons, contain reverse complement sequences at both ends that bind together, whereas the rest of the strand remains single-stranded, creating a panhandle-like structure. In addition, there are fluorescent dyes at both ends of the molecule, a reporter and a quencher similar to the TaqMan probes. In the panhandle-like conformation, there is no fluorescence because the fluorescent reporter at one end and the quencher at the other end of the probe are very close to one another.[6] As the central part of a molecular beacon consists of a target-specific sequence, both ends are separated from each other when this part of the molecule is bound to the PCR product and a fluorescence signal can be emitted from the reporter dye. Hairpin primers, also named ''Amplifluor PrimersĀ®'' (Intergen), are similar to molecular beacons, but fluorescence is generated as they become incorporated into the double-stranded PCR product during amplification. Another very simple technique to monitor the generation of PCR product in a real-time fashion is the use of intercalating dyes, such as ethidium bromide and SYBR Green IĀ®, which do not bind to single-stranded DNA but to the double-stranded PCR product.[5,7] However, hairpin primers and intercalating dyes do not offer the high specificity of the probe-based techniques and a positive signal might even be generated by primer dimers.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook

Post a comment