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.
Avignolo
C
C
Laboratorio di Cancerogenesi Chimica, Istituto Scientifico Tumori di Genova, Italy.
Valente
P
P
Cai
S
S
Roner
R
R
Fulle
A
A
Pizzorno
G
G
Bignone
F A
FA
D000586:Amination; D001710:Biotin; D055598:Chemical Phenomena; D002621:Chemistry; D003562:Cytidine; D003596:Cytosine; D004247:DNA / Q000032:analysis*
D003562:Cytidine; D001710:Biotin; D003596:Cytosine; D004247:DNA