Abstract
Transamination at 100°C of cytosines in denatured double-strand DNA is a rapid and reliable method to obtain DNA molecules containing N 4-aminoethylcytosine (4aeC), which can be quantitatively conjugated to biotinyl- N-hydroxysuccinimide ester (BHS) at 37°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°C, and probes obtained by this procedure are equally effective for general use in molecular biology. Dot-blots with 1–5 pg of target λ 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.
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