Probing Polymerase Chain Reaction

In 1992, Higuchi et al.[25] showed that it was possible to follow the amplification of DNA in real time by adding ethidium bromide to the reaction tube and monitor the increase in fluorescence when the dye binds to the accumulating DNA product. A variety of dyes and probes have been developed for the detection of DNA in homogeneous solution since then. To be useful, the dyes should bind DNA with little sequence selectivity in one dominant-binding mode. They should have strong preference for double-stranded DNA and exhibit large fluorescence enhancement upon binding. Binding should be strong enough to give a signal proportional to the amount of DNA present, but without interfering with the amplification reaction. For the same reason, dissociation kinetics should be fast. Although YO-PRO was used in some early assays,[26] the most common dye is SYBR Green I from Molecular Probes.[27] Its chemical structure has not been published in scientific reports, but its spectral characteristics suggest that it is based on the TO-chromophore, probably with a cyclic substituent (U.S. patents 5,436,134 and 5,658,751). Recently, Bengtsson et al.[28] tested the minor-groove binder BEBO as reporter in real-time PCR (Fig. 6) and found it to compare well with SYBR Green I in all important aspects.

Dyes are excellent reporters in real-time PCR, but they do not distinguish between products. Taqman,[29] Molecular Beacons,[30] Scorpion Primers,[31] and LightCycler

Table 1 Fluorescence and absorbance properties of BETO and BOXTO compared with other minor groove binders and with TO
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