Laser microdissection of the fragile X region: identification of cosmid clones and of conserved sequences in this region.
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.
Djabali
M
M
CIML INSERM-CNRS, Marseille-Luminy, France.
Nguyen
C
C
Biunno
I
I
Oostra
B A
BA
Mattei
M G
MG
Ikeda
J E
JE
Jordan
B R
BR
D000818:Animals; D001483:Base Sequence; D002871:Chromosome Banding; D003001:Cloning, Molecular / Q000379:methods; D003360:Cosmids; D004247:DNA / Q000235:genetics; D005600:Fragile X Syndrome / Q000235:genetics*; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D007834:Lasers; D008297:Male; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping; D014960:X Chromosome
D009838:Oligodeoxyribonucleotides; D004247:DNA