Rapid generation of region-specific genomic clones by chromosome microdissection: Isolation of DNA from a region frequently deleted in malignant melanoma

Abstract

Malignant melanoma is frequently characterized by the deletion of the long arm of chromosome 6 (usually encompassing 6q16-q21). In an effort to saturate this region with DNA markers, microdissection and molecular cloning of DNA from banded human metaphases have been performed. This work was facilitated by the recent development of a novel chromosome microdissection scheme that omits microchemical manipulation of DNA. Microdissection was targeted on band 6q21. Direct PCR amplification of dissected DNA was first used as a probe in chromosomal in situ hybridization of normal metaphases to confirm the specificity of material excised for cloning. A genomic library of 20,000 clones, which is highly enriched for sequences encompassing 6q21, was then constructed. Clones from this library have been mapped against a human-rodent somatic cell hybrid mapping panel that divides chromosome 6 into seven regions, confirming the localization of probes within the target region. Direct PCR amplification of DNA excised by microdissection greatly simplifies and facilitates this chromosome band-specific cloning strategy. The isolation of microclones from this region of chromosome 6 should assist in establishing a physical map of the melanoma deletion region.

Senescence of immortal human fibroblasts by the introduction of normal human chromosome 6.

“…Rare immortal segregants obtained in C1 39neo6-1 maintained in selective MX medium are being analyzed for loss of sequences to further localize this gene (unpublished data). A candidate locus for a tumor-suppressor gene has been mapped to 6q in human melanoma cells based on chromosomal rearrangements (34) and suppression of tumor formation in nude mice after MMCT (17). Most recently, transfection of Mn-superoxide dismutase, which is located at 6q25, into melanoma cells has been shown to inhibit tumor transplantation (35).…”

Identification of heterosomes in spermatoza of rams with 54,XX/54,XY chimerism

“…The X probe was amplified and labelled with DOP-PCR using the bio-16-dUTP nucleotide (Goldammer et al, 1996). The Y probe was amplified by PCR as described by Guan et al (1992) and PCR labelled with digoxigenin-11-dUTP (Boehringer Mannheim, Germany) according to the manufacturer's instruction.…”

The phenomenon of cell chimerism in goats

“…This probe was obtained from ETH Zürich Breeding Biology Group. The Y fragments were amplified by PCR as described by Guan et al (1992) and PCR labelled with digoxigenin-11-dUTP (Boehringer Mannheim, Germany) according to the manufacturer's instruction. The chromosome probe was obtained from the Swedish University of Agricultural Sciences (Uppsala).…”