Sequence verification by hybridisation with fluorescent octanucleotides as a first step to a fluorescent sequencing...

Three sets of partly overlapping octanucleotides are 5' labelled with derivates of the fluorescence dyes fluorescein-, coumarine- and rhodamine, respectively. Hybridisation conditions are determined, under which all octanucleotides hybridise correctly against complementary target sequences bound on nylon membranes. Target sequences are three synthetic 48-mer oligonucleotides and herring sperm DNA, a positive control containing almost all possible octanucleotides. None of the octanucleotides hybridised to incorrect target sequences. Analysing these results, a given sequence could be unambiguously verified. A feature critical for the accuracy of the hybridisation is the temperature during the last washing step. This temperature can be estimated using the equation T = 19 - 0.4(G + C) + 0.15(G + C)2. Using octanucleotides labelled with three different colors, three hybridisations can be performed simultaneously.