Can you do melting curve analysis in Real-Time PCR after using TaqMan probes?
The fluorophore in a TaqMan probe is not incorporated in the new PCR product. Therefore, in a melting curve analysis, there is no change in fluorescence whether the PCR product is single-stranded (after melting) or double-stranded (before melting). On the other hand, SYBR Green I allows melting curve analysis because it fluoresces when bound to the minor groove of double-stranded DNA. When PCR products melt, they release the SYBR Green I into solution and it no longer fluoresces. PCR products made using LUX primers from Invitrogen can also be subjected to melting curve analysis because the fluorophore only fluoresces when it is incorporated into a double stranded DNA amplicon.
