A number of alternative fluorescence chemistries have been devised to detect DNA molecules produced during PCR.
All can be used with our instruments.
The two chemistries used most frequently in our Facility are:
- SYBR Green dye. This dye binds to the minor groove of double-stranded DNA, and the SYBR-dsDNA complex emits green fluorescence when excited with light of lower wavelength. This chemistry is relatively cheap, but is not specific and produces fluorescence from all dsDNA amplified including non-target sequences and primer dimers if present.
- TaqMan fluorogenic probes. TaqMan (named after the computer game PacMan) uses a single-stranded DNA probe of ~15-30 nucleotides (labeled with a fluorogenic dye at the 5' end and a fluorescence quencher at the 3' end) homologous to a target sequence between the PCR priming sites.
During the extension phase of PCR, the Taq DNA polymerase encounters the bound probe and cleaves it, hence releasing the detection dye from the effect of the quencher.
This results in enhanced fluorescence when the dye is excited with incident light.
The extra specificity conferred by the detection probe has advantages for many applications eg. for diagnostic applications where false positives are a problem.