DELINEATION OF MARKER CHROMOSOMES BY REVERSE CHROMOSOME PAINTING USING ONLY A SMALL NUMBER OF DOP-PCR AMPLIFIED MICRODISSECTED CHROMOSOMES

A new procedure for determining the chromosomal origin of marker chrom osomes has been carried out. The origin of marker chromosomes that wer e unidentifiable by standard banding techniques could be verified by r everse chromosome painting. This technique includes microdissection, f ollowed by in vitro DNA amplification and fluorescence in situ hybridi zation (FISH). A number of marker chromosomes prepared from unbanded a nd from GTG-banded lymphocyte chromosomes were collected with micronee dles and transferred to a collection drop. The chromosomal material wa s amplified by a degenerate oligonucleotide-primed polymerase chain re action (DOP-PCR). The resulting PCR products were labelled by nick-tra nslation with biotin-11-dUTP and used as probes for FISH. They were hy bridized onto normal metaphase spreads in order to determine the preci se regional chromosomal origin of the markers. Following this approach , we tested 2-14 marker chromosomes in order to determine how many are necessary for reverse chromosome painting. As few as two marker chrom osomes provided sufficient material to paint the appropriate chromosom e of origin, regardless of whether the marker contained heterochromati c or mainly euchromatic material. With this method, it was possible to identify two marker chromosomes of a healthy proband [karyotype: 48,X Y, +mar(1),+mar(2)] and an aberrant Y chromosome of a mentally retarde d boy [karyotype: 46,X, der(Y)].