Biotinylation of denatured double-stranded DNA after transamination of cytidines: molecular biology applications.
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.
Avignolo
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Laboratorio di Cancerogenesi Chimica, IST, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy.
Roner
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Cai
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Bignone
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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
D003562:Cytidine; D001710:Biotin; D004247:DNA