title: Biotinylation of denatured double-stranded DNA after transamination of cytidines: molecular biology applications.
abstract: Transamination at 100 degrees C of cytosines in denatured double-strand DNA is a rapid and reliable method to obtain DNA molecules containing N4-aminoethylcytosine (4aeC), which can be quantitatively conjugated to biotinyl-N-hydroxysuccinimide ester (BHS) at 37 degrees C, yielding chemically labelled probes for molecular hybridization. The adopted transamination reaction temperature allows for a ten-fold reduction of the time required for labelling at 42 degrees C, and probes obtained by this procedure are equally effective for general use in molecular biology. Dot-blots with 1-5 pg of target lambda DNA were detected by streptavidin-acid phosphatase complex after hybridization with its homologous sequences. Chemically biotinylated mouse satellite DNA has been used in combination with avidin-horseradish peroxidase to detect metaphase and interphase centromeres via in situ hybridization. Moreover probes labelled with differentially spaced linker arms were prepared by this method.
authors.affiliation: Laboratorio di Cancerogenesi Chimica, IST, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Avignolo
authors.forename: R
authors.initials: R
authors.lastname: Roner
authors.forename: S
authors.initials: S
authors.lastname: Cai
authors.forename: F A
authors.initials: FA
authors.lastname: Bignone
mesh_terms: D000586:Amination; D000818:Animals; D001710:Biotin / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002875:Chromosomes; D003562:Cytidine / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D005399:Fishes / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009691:Nucleic Acid Denaturation / Q000502:physiology*; D009693:Nucleic Acid Hybridization
chemical_list: D003562:Cytidine; D001710:Biotin; D004247:DNA