Large flanking sequence effects in single nucleotide mismatch detection using fluorescent nucleoside Çf

The first systematic study of flanking sequence effects on mismatch detection by a fluorescent nucleotide is described, using fluoroside Çf. Although a high degree of variance was observed in fluorescence intensity of mismatched duplexes between different flanking sequences, Çf was able to distinguish a mismatch from the fully base-paired duplex in 13 out of 16 sequences, and even identify each mismatch in 10 of those flanking sequences. For the flanking sequences where fluoroside Çf did not unambiguously determine its base-pairing partner, the experimental conditions were varied in an attempt to facilitate mismatch identification. No beneficial effect on the relative fluorescence intensities was achieved by changing the temperature, adding organic co-solvents or potassium iodide. In contrast, mercuric ions selectively quenched the fluorescence intensity of the Çf·T mismatch, effectively resolving the overlap of all emission spectra and thereby facilitating identification of all base-pairing partners in any flanking sequence by Çf. This is the first time mercuric ions have been used to selectively quench the fluorescence of a single mismatch. A noticeable characteristic of Çf is that, unlike most fluorosides, the fluorescence intensity of Çf was not quenched to a discernable degree by a flanking G–C pair.

Çf covers all bases. The fluorescent nucleoside Çf can, with occasional assistance from Hg2+, uniquely identify all its base-pairing partners independent of the adjacent base-pairs.Figure optionsDownload as PowerPoint slide