Cosmid linking clones localized to the long arm of human chromosome 11.
Molecular probes that contain DNA flanking CpG-rich restriction sites are extremely valuable in the construction of physical maps of chromosomes and in the identification of genes associated with hypomethylated HTF (HpaII tiny fragment) islands. We describe a new approach to the isolation and characterization of linking clones in arrayed chromosome-specific cosmid libraries through the large-scale semiautomated restriction mapping of cosmid clones. We utilized a cosmid library representing human chromosome 11q12-11qter and carried out automated restriction enzyme analysis, followed by regional localization to chromosome 11q using high-resolution in situ suppression hybridization. Using this approach, 165 cosmid linking clones containing one or more NotI, BssHII, SfiI, or SacII sites were identified among 960 chromosome-specific cosmids. Furthermore, this analysis allowed clones containing a single site to be distinguished from those containing clusters of two or more rare sites. This analysis demonstrated that more than 75% of cosmids containing a rare restriction site also contained a second rare restriction site, suggesting a high degree of CpG-rich restriction site clustering. Thirty chromosome 11q-specific cosmids containing rare CpG-rich restriction sites were regionally localized by high-resolution fluorescence in situ suppression hybridization, demonstrating that all of the CpG-rich sites detected by this method were located in bands 11q13 and 11q23. In addition, the distribution of (CA)n repetitive sequences was determined by hybridization of the arrayed cosmid library with oligonucleotide probes, confirming a random distribution of microsatellites among CpG-rich cosmid clones. This set of reagent cosmid clones will be useful for physical linking of large restriction fragments detected by pulsed-field gel electrophoresis and will provide a new and highly efficient approach to the construction of a physical map of human chromosome 11q.
Hermanson
G G
GG
Molecular Genetics Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037.
Lichter
P
P
Selleri
L
L
Ward
D C
DC
Evans
G A
GA
D002880:Chromosomes, Human, Pair 11; D003001:Cloning, Molecular; D003360:Cosmids / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field; D004706:Endodeoxyribonucleases / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D009693:Nucleic Acid Hybridization; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D015183:Restriction Mapping
D015342:DNA Probes; D004706:Endodeoxyribonucleases