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© 1993 Oxford University Press

RESEARCH-ARTICLE

Generation of band-specific painting probes from a single microdissected chromosome

X.-Y. Guan1, Jeffrey M. Trent1,2,* and Paul S. Meltzer1,3

1Departments of Radiation Oncology, The University of Michigan, Medical Science Research Budding II Room C560, 1150 West Medical Center Drive, Ann Arbor, Ml 48109-0668, USA 2Departments of Human Genetics, The University of Michigan, Medical Science Research Budding II Room C560, 1150 West Medical Center Drive, Ann Arbor, Ml 48109-0668, USA 3Departments of Pediatrics, The University of Michigan, Medical Science Research Budding II Room C560, 1150 West Medical Center Drive, Ann Arbor, Ml 48109-0668, USA

* To whom correspondence should be addressed

Received May 17, 1993; Revised June 14, 1993; Accepted June 14, 1993

We have developed a modified strategy for the generation of regional probes for human chromosomes by microdissection and degenerate oligonucleotide primed PCR. This modification dramatically increases the efficiency of amplification by pretreatment of the dissected chromatin with topoisomerase I (Topo 1) before PCR. This protocol has enabled us to construct region-specific probes for fluorescence in situ hybridization (FISH) from a single microdissected chromosome. Results are presented which convincingly demonstrate that this new method generates high Intensity region-specific FISH probes, while at the same time significantly decreasing the time-consuming and labor-intensive aspects of microdissection. The reduction of the number of copies required to generate a useful probe also significantly decreases the risk of contamination during the microdissection process. We believe this advance will allow microdissection to be more widely used in the cytogenetic analysis of chromosome rearrangements In both cancer and hereditary diseases. In addition, this method now makes It possible to construct a series of non-overlapping band-specific DNA microclone libraries to provide complete coverage of Indivdual chromosomes for physical mapping.


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