title: Sequence verification by hybridisation with fluorescent octanucleotides as a first step to a fluorescent sequencing by hybridisation protocol.
abstract: 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.
authors.affiliation: Institut für Molekulare Biotechnologie e. V. Jena, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Eickhoff
authors.forename: E
authors.initials: E
authors.lastname: Birch-Hirschfeld
authors.forename: J
authors.initials: J
authors.lastname: Scheef
authors.forename: C
authors.initials: C
authors.lastname: Hoyer
authors.forename: K H
authors.initials: KH
authors.lastname: Drexhage
authors.forename: K O
authors.initials: KO
authors.lastname: Greulich
mesh_terms: D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D003374:Coumarins; D004247:DNA / Q000737:chemistry*; D005399:Fishes; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008297:Male; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D012235:Rhodamines; D017422:Sequence Analysis, DNA / Q000379:methods*; D013094:Spermatozoa / Q000737:chemistry; D013696:Temperature
chemical_list: D003374:Coumarins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D012235:Rhodamines; D004247:DNA