Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes.
A novel polymerase chain reaction (PCR) technique has been combined with chromosome flow sorting to characterise two lymphoblastoid cell lines and one medullary thyroid carcinoma cell line carrying translocations close to the locus for multiple endocrine neoplasia type 2A (MEN 2A). Five hundred copies of the derivative chromosome(s) were flow sorted from each cell line and amplified by degenerate oligonucleotide-primed-polymerase chain reaction (DOP-PCR). This generated pools of DNA sequences corresponding to the abnormal chromosomes, which were then used as probes in fluorescence in situ hybridisation (FISH) experiments on normal metaphase cells. The resultant chromosome paints revealed the portions of the normal chromosomes related to those involved in the translocations. By this technique, translocation breakpoints in bands p15, q11.2, and q21 of chromosome 10 were defined in the above cell lines, in two cases refining previous cytogenetic data. This study shows that flow sorting of aberrant chromosomes and chromosome painting can be used as a rapid aid to cytogenetic analysis, particularly in cases of difficult karyotypes, such as tumours. Furthermore, the DOP-PCR technique described here will have applications to other areas of genome analysis, such as cloning of new markers; its design will allow a general and representative amplification to occur from any starting DNA in any species.
Telenius
H
H
CRC Human Cancer Genetics Research Group, Department of Pathology, University of Cambridge, United Kingdom.
Pelmear
A H
AH
Tunnacliffe
A
A
Carter
N P
NP
Behmel
A
A
Ferguson-Smith
M A
MA
Nordenskjöld
M
M
Pfragner
R
R
Ponder
B A
BA
D002460:Cell Line; D002879:Chromosomes, Human, Pair 10; D002892:Chromosomes, Human, Pair 22; D002893:Chromosomes, Human, Pair 3; D005260:Female; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D007834:Lasers; D008297:Male; D008853:Microscopy; D016133:Polymerase Chain Reaction / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D014178:Translocation, Genetic / Q000235:genetics
D016650:Fluorescein-5-isothiocyanate