Localisation of satellite DNA sequences on human metaphase chromosomes using bromodeoxyuridine-labelled probes.
Human highly repeated satellite sequences, cloned into M13, were used as templates to prepare single-stranded DNA probes containing bromodeoxyuridine (BrdUrd) in place of thymine. The probes were hybridised to human metaphase chromosomes and visualised using an indirect immunological detection procedure. The sensitivity and accuracy of the technique were tested using a BrdUrd-labelled probe of known copy number and location: a segment from the 2.5 kb Y chromosome repeat. The procedure proved to be reliable and fast, with a sensitivity similar to that of other in situ hybridisation techniques. The technique was then used to determine the chromosomal locations of a 100 bp repeat from human satellite 3. The satellite 3 probe hybridised to a large number of chromosomes and, surprisingly, the intensity of label at all locations remained unchanged when the slides were washed at a higher stringency. The resolution of the technique was very high and allowed accurate localisation of the satellite sequence. Hybridisation was observed in two regions of the subcentromeric heterochromatin of chromosome 9, in two locations at the centromere and short arm of all the acrocentric autosomes, and at the centromere and long arm of the Y chromosome. In addition the probe hybridised to centromeric heterochromatin in chromosomes 1, 16, 17 and 20. We believe that single-stranded BrdUrd-labelled probes should be very useful for detecting RNA transcripts in cells, and discuss ways by which the procedure could be modified to locate single copy DNA on chromosomes.
Frommer
M
M
Kanematsu Laboratories, Royal Prince Alfred Hospital, Camperdown, N.S.W., Australia.
Paul
C
C
Vincent
P C
PC
D001483:Base Sequence; D015139:Blotting, Southern; D001973:Bromodeoxyuridine; D002877:Chromosomes, Human; D015342:DNA Probes; D004276:DNA, Satellite / Q000032:analysis* / Q000302:isolation & purification; D006801:Humans; D008677:Metaphase; D009693:Nucleic Acid Hybridization; D014998:Y Chromosome
D015342:DNA Probes; D004276:DNA, Satellite; D001973:Bromodeoxyuridine