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Laser microdissection of the fragile X region: identification of cosmid clones and of conserved sequences in this region.

Abstract
Laser microdissection has been used to dissect material from the X-chromosome region involved in fragile-X-linked mental retardation. After dissection, single chromosome slices corresponding to this fragile site were subjected to DNA amplification using either a vector ligation method (to provide known anchor sequences) or primer oligonucleotides corresponding to the ubiquitous Alu sequences. Amplified material was then cloned or, alternately, used to screen a gridded cosmid library. Eight cosmid clones identified in this way were regionally mapped using a panel of hybrid cell lines and shown to originate from a narrow interval centered on the fragile X site. Two clones are included in the approximately 6-cM interval defined by probes RNI (DXS369, 5 cM proximal) and VK21 (DXS 296, 1-2 cM distal) and which includes the fragile site, and at least one clone contains sequences conserved across species suggestive of a gene. This method combines the focused approach of microdissection and the convenience of obtaining cosmid (rather than small-insert) clones; it may be useful for studies of other defined chromosomal regions.
AuthorsM Djabali, C Nguyen, I Biunno, B A Oostra, M G Mattei, J E Ikeda, B R Jordan
JournalGenomics (Genomics) Vol. 10 Issue 4 Pg. 1053-60 (Aug 1991) ISSN: 0888-7543 [Print] United States
PMID1916812 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Oligodeoxyribonucleotides
  • DNA
Topics
  • Animals
  • Base Sequence
  • Chromosome Banding
  • Cloning, Molecular (methods)
  • Cosmids
  • DNA (genetics)
  • Fragile X Syndrome (genetics)
  • Gene Library
  • Genetic Vectors
  • Humans
  • Lasers
  • Male
  • Molecular Sequence Data
  • Oligodeoxyribonucleotides
  • Polymerase Chain Reaction (methods)
  • Restriction Mapping
  • X Chromosome

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