Sequence verification by hybridisation with fluorescent octanucleotides as a first step to a fluorescent sequencing by hybridisation protocol.
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.
Eickhoff
H
H
Institut für Molekulare Biotechnologie e. V. Jena, Germany.
Birch-Hirschfeld
E
E
Scheef
J
J
Hoyer
C
C
Drexhage
K H
KH
Greulich
K O
KO
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
D003374:Coumarins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D012235:Rhodamines; D004247:DNA