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.
Subject(s)
DNA , Fluorescent Dyes , Nucleic Acid Hybridization , Alkylation , Spectrometry, FluorescenceABSTRACT
The possibility of site-specific scission of DNA in polytene chromosomes in situ by means of the method of complementarily addressed fragmentation is demonstrated. The fragmentation of polytene chromosomes of Chironomus thummi resulting from the alkylation of the denatured chromosomes with oligoadenylate derivatives, followed by scission of the DNA in specific sites and enlargement of nicks with exonucleases was investigated. Single-stranded regions were registered by means of luminescence microscopy after staining the chromosomes with acridine orange. The pattern of degradation of the chromosomes depends on the length of the oligoadenylate part of the reagents and is different from that obtained with uridine-5'-methylphosphate alkylating derivative which does not form any complexes with DNA. Oligoadenylates inhibit the action of the alkylating derivatives on the chromosomes.
