This proposal calls for defining the limits of the anirdia region according to the translocation breakpoints of three key patients, and sequencing segments of twelve existing clones (and others from a well-characterized microdissection library if necessary) which map in that region. Primers for PCR will be designed and synthesized from these sequences, and will be used to isolate YAC clones throughout the aniridia region. YACs will be mapped back to 11p13 by fluorescent in-situ hybridization, and characterized for their content of single-copy segments and for overlaps among them, by comparison of their inter-Alu PCR products. These inter-Alu segments will be cloned into pUC 18, (alternatively, the YACs will be subcloned directly and single-copy sequences identified by absence of hybridization to a probe of total human DNA), and the single copy subclones will be used as probes against Zoo blots, Northerns and cDNA libraries. Special attention will be given to any clones detecting transcripts in fetal brain, since we believe genes involved in eye development will be expressed in fetal brains. YACs will be placed on the long-range physical map both by mapping the single copy subclones and the existing parental clones, and by comparing the restriction sites for rare-cutting enzymes in the YACs to the sites in genomic DNA, according to the existing map. DNA sequences of the clones, along with their mapping information, will be entered as STS's into GENBANK, and the YAC mapping information will be stored in the database at the Salk Institute for Human Genome Research. These cloned DNA resources will be used to examine the aniridia region of other patients with eye defects, such as coloboma, to investigate the possibility of a genetic relationship between these disorders.
| Hozier, J; Graham, R; Westfall, T et al. (1994) Preparative in situ hybridization: selection of chromosome region-specific libraries on mitotic chromosomes. Genomics 19:441-7 |
