Rapid generation of region-specific genomic clones by chromosome microdissection: isolation of DNA from a region frequently deleted in malignant melanoma.
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 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.
Guan
X Y
XY
Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center, Ann Arbor 48109-0668.
Meltzer
P S
PS
Cao
J
J
Trent
J M
JM
D001483:Base Sequence; D002478:Cells, Cultured; D002872:Chromosome Deletion; D002896:Chromosomes, Human, Pair 6 / Q000648:ultrastructure; D003001:Cloning, Molecular / Q000379:methods*; D004277:DNA, Single-Stranded; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008545:Melanoma / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D004277:DNA, Single-Stranded