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.

Biotinylation of double stranded DNA after transamination.

The bisulfite catalyzed transamination of cytidine and cytosine has been reported to be single strand specific, but local thermal instabilities of the DNA double helix, coupled with the extreme sensitivity of the Biotin-Avidin revelation methods, allows the random labelling of cytosines in d.s. DNA to detectable levels for those purposes where the overall label can be very low. We have evaluated the use of this reaction to prepare double stranded DNA molecules containing N4-aminoethyl-cytosine (4-aeC). After this step 4-aeC residues can be conjugated to biotinyl-n-hydroxysuccinimide ester yielding biotinylated DNA. This reaction allows the massive production of biotinylated probes. Labelled DNA can serve as molecular weight marker and positive control in Southern-blots. Moreover it can be useful in the study of DNA-protein interaction and in the isolation of d.s. DNA-binding proteins through chromatographic procedures.

Detection of DNA damage induced in vivo by a cross-linking agent with a circular channel crucible oscillating viscometer.

DNA damage induced in vivo by the cross-linking agent mitomycin C (MMC) was investigated with a new oscillating crucible viscometer. Viscosity was measured by lysing rat liver nuclei in an alkaline lysing solution (pH 12.5; 25 degrees C). In control samples the viscosity increased very slowly with time, reaching a plateau only after 10-12 h. The process was accelerated and the maximum viscosity was decreased by alkaline single-stranded breaks arising from methylation and subsequent depurination of DNA in vitro with dimethylsulphate (DMS). MMC, when given alone, had no evident effect on the time needed for reaching plateau viscosity but it induced a small increase in maximum viscosity. When MMC was given in association with DMS, the time of disentanglement remained unchanged (accelerated) but maximum viscosity was increased in a dose dependent way. We conclude that these data clearly confirm that the slow steady increase of the viscosity of control DNA with time reflects mainly the process of unwinding of the two strands. The speed of this process seems to depend only from the number of unwinding points in DNA (breaks).