Universal detection of single-base mismatches in duplex DNA using fluorescent nucleoside Çf

Abstract

The fluorescent nucleoside (fluoroside) Çf is a cytosine-analogue that forms a stable base pair with deoxyguanosine in duplex DNA. Upon incorporation of the fluoroside into double stranded DNA, it retains most of its fluorescence. Furthermore, the fluorescent signal is different depending on the nucleotide that Çf is base-paired to, making it able to identify its base-pairing partner. These abilities make it a promising candidate for single nucleotide polymorphism (SNP) genotyping. A preliminary study showed that the flanking sequence, i.e. the nucleotides immediately flanking the 3´ and 5´ side of the fluoroside, affected the emission of Çf (Cekan P and Sigurdsson ST (2008), Chem. Comm., 29, 3393-3395). We were therefore interested in examining the effects of all possible flanking sequences on the fluorescence intensity of Çf. We found that the flanking sequence does induce changes in the emission, but to a different degree depending on the flanking sequence in question. In fact, there were three distinct levels of mismatch detection. First, in the majority of flanking sequences (10 of 16), Çf was capable of identifying its base-pairing partner. In the second category (3 of 16), Çf was able to discriminate the fully base paired duplex from the mismatches. In the last category (3 of 16), the fluoroside was not able to distinguish the fully base paired duplex from a mismatch. However in the sequences where distinction between all base-pairing partners was not possible, we were able to alter conditions, such as temperature, solvent polarity and ionic conditions, so that full discrimination was facilitated. In particular, addition of Hg2+ proved useful as it resulted in the selective quenching of the T-mismatched duplexes. The ability to use Çf in any flanking sequence makes it one of the most versatile SNP genotyping probes available today.