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Preparation of fluorescent-labeled DNA and its use as a probe in molecular hybridization
Authors:L A Frumgarts  S M Kipriianov  S M Kalachikov  N A Dudareva  G M Dymshits
Abstract:A method of the fluorescent-labeled DNA preparation for visualization of the complementary nucleotide sequences has been developed. Polynucleotide probes were alkylated randomly by 4-(N-methylamino-N-2-chloroethyl)-benzylamine followed by modification with such fluorochromes as dansyl chloride or fluorescein isothiocyanate (FITC). It was found that the FITC but not dansyl-labeled polynucleotides could serve as efficient probes when about 4% of nitrogen bases were modified. The conditions minimizing the loss of the alkylated bases from DNA were determined. The procedure for hybridization with FITC-labeled DNA as a probe is described, concentration of DNA probe being about 4 ng/mm2 of the nitrocellulose filter.
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