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Proceedings Paper

High-performance analysis of single interphase cells with custom DNA probes spanning translocation break points
Author(s): Heinz-Ulli G. Weier; S. Munne; Robert A. Lersch; C. Marquez; J. Wu; Roger A. Pedersen; Jingly Fung
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Paper Abstract

The chromatin organization of interphase cell nuclei, albeit an object of intense investigation, is only poorly understood. In the past, this has hampered the cytogenetic analysis of tissues derived from specimens where only few cells were actively proliferating or a significant number of metaphase cells could be obtained by induction of growth. Typical examples of such hard to analyze cell systems are solid tumors, germ cells and, to a certain extent, fetal cells such as amniocytes, blastomeres or cytotrophoblasts. Balanced reciprocal translocations that do not disrupt essential genes and thus do not led to disease symptoms exit in less than one percent of the general population. Since the presence of translocations interferes with homologue pairing in meiosis, many of these individuals experience problems in their reproduction, such as reduced fertility, infertility or a history of spontaneous abortions. The majority of translocation carriers enrolled in our in vitro fertilization (IVF) programs carry simple translocations involving only two autosomes. While most translocations are relatively easy to spot in metaphase cells, the majority of cells biopsied from embryos produced by IVF are in interphase and thus unsuitable for analysis by chromosome banding or FISH-painting. We therefore set out to analyze single interphase cells for presence or absence of specific translocations. Our assay, based on fluorescence in situ hybridization (FISH) of breakpoint-spanning DNA probes, detects translocations in interphase by visual microscopic inspection of hybridization domains. Probes are prepared so that they span a breakpoint and cover several hundred kb of DNA adjacent to the breakpoint. On normal chromosomes, such probes label a contiguous stretch of DNA and produce a single hybridization domain per chromosome in interphase cells. The translocation disrupts the hybridization domain and the resulting two fragments appear as physically separated hybridization domains in the nucleus. To facilitate the detection, DNA probes for breakpoints on different chromosomes are labeled in different colors, so the translocation event can be detected as a fusion of red and green hybridization domains. We applied this scheme successfully for the analysis of somatic and germ cells from more than 20 translocation patients, each with individual breakpoints, and provide summaries of our experience as well as strategies, cost and time frames to prepare case-specific translocation probes.

Paper Details

Date Published: 1 June 1999
PDF: 10 pages
Proc. SPIE 3604, Optical Diagnostics of Living Cells II, (1 June 1999); doi: 10.1117/12.349204
Show Author Affiliations
Heinz-Ulli G. Weier, Lawrence Berkeley National Lab. (United States)
S. Munne, Institute for Assisted Reproduction and Medicine (United States)
Robert A. Lersch, Univ. of California/Berkeley (United States)
C. Marquez, Institute for Assisted Reproduction and Medicine (United States)
J. Wu, Univ. of California/San Francisco (United States)
Roger A. Pedersen, Univ. of California/San Francisco (United States)
Jingly Fung, Lawrence Berkeley National Lab. and Univ. of California/San Francisco (United States)


Published in SPIE Proceedings Vol. 3604:
Optical Diagnostics of Living Cells II
Daniel L. Farkas; Robert C. Leif; Bruce J. Tromberg, Editor(s)

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