Viersbach R, Schwanitz G, Nöthen M M
Institut für Humangenetik, Universität Bonn, Germany.
Hum Genet. 1994 Jun;93(6):663-7. doi: 10.1007/BF00201567.
A new procedure for determining the chromosomal origin of marker chromosomes has been carried out. The origin of marker chromosomes that were unidentifiable by standard banding techniques could be verified by reverse chromosome painting. This technique includes microdissection, followed by in vitro DNA amplification and fluorescence in situ hybridization (FISH). A number of marker chromosomes prepared from unbanded and from GTG-banded lymphocyte chromosomes were collected with microneedles and transferred to a collection drop. The chromosomal material was amplified by a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The resulting PCR products were labelled by nick-translation with biotin-11-dUTP and used as probes for FISH. They were hybridized onto normal metaphase spreads in order to determine the precise 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 chromosomes provided sufficient material to paint the appropriate chromosome of origin, regardless of whether the marker contained heterochromatic or mainly euchromatic material. With this method, it was possible to identify two marker chromosomes of a healthy proband [karyotype: 48,XY,+mar1,+mar2] and an aberrant Y chromosome of a mentally retarded boy [karyotype: 46,X, der(Y)].
一种确定标记染色体染色体来源的新方法已经实施。通过标准显带技术无法识别的标记染色体的来源可以通过反向染色体涂染来验证。该技术包括显微切割，随后进行体外DNA扩增和荧光原位杂交（FISH）。用微针收集从未显带和GTG显带的淋巴细胞染色体制备的一些标记染色体，并转移到收集滴中。通过简并寡核苷酸引物聚合酶链反应（DOP-PCR）扩增染色体物质。所得的PCR产物用生物素-11-dUTP进行缺口平移标记，并用作FISH探针。将它们杂交到正常中期染色体铺片上，以确定标记的精确区域染色体来源。按照这种方法，我们检测了2 - 14条标记染色体，以确定反向染色体涂染需要多少条。少至两条标记染色体就能提供足够的材料来涂染相应的起源染色体，无论该标记包含异染色质还是主要为常染色质物质。用这种方法，可以鉴定出一名健康先证者的两条标记染色体[核型：48,XY,+mar1,+mar2]和一名智力发育迟缓男孩的一条异常Y染色体[核型：46,X, der(Y)]。
