Gary G. Hermanson

Chromosomal in situ hybridization using yeast artificial chromosomes.

Large DNA fragment cloning methods using yeast artificial chromosomes (YACs) have vastly improved the strategies for constructing physical maps of regions of complex genomes, as well as for isolating and cloning genes important for human disease. We present here a simple and rapid method for carrying out in situ hybridization to metaphase chromosomes using isolated YAC clones by labeling DNA directly in agarose gel slices. Nonisotopic labeling and chromosomal in situ hybridization can be used to determine the chromosomal localization of individual YAC clones on human metaphase chromosomes. This method can also be used to characterize YAC clones consisting of single fragments from those that contain concatamerized, and thus artifactual, inserts. This technique also offers a valuable tool to study consistent translocations in neoplastic diseases by identifying YACs that span a specific chromosomal breakpoint.

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.

Localization of the human oncostatin M gene (OSM) to chromosome 22q12, distal to the Ewing’s sarcoma breakpoint

Using fluorescence in situ hybridization, a cosmid clone containing the gene for oncostatin M (OSM) was mapped to human chromosome 22q12, placing the OSM gene in the same chromosome band as the leukemia-inhibitory factor gene (LIF). The location of the OSM gene was determined relative to the t(11;22)(q24;q12) of Ewings sarcoma and found to be distal to the translocation breakpoint on chromosome 22. Analysis of physical distances by pulsed-field gel electrophoresis demonstrated further that the two genes lie within 500 kb of each other.

Use of Cosmids and Arrayed Clone Libraries for Genome Analysis

Publisher Summary This chapter describes some protocols utilized in our laboratory for large-scale genome mapping, including the construction of cosmid libraries, the production and archiving of arrayed cosmid libraries, the use of robots for cosmid manipulation and processing, and the analysis of individual cosmid clones. Genomic cosmid libraries may be used for isolating and characterizing individual genes or gene families, or as the basis for constructing large scale maps of chromosomes or entire genomes. Techniques and strategies for the creation and manipulation of cosmid libraries for these two goals differ substantially. Cosmid libraries for isolating single genes may be stored as pools of clones and replated for each screening. For large-scale mapping, however, where large numbers of clones will be analyzed, it is often much more convenient to produce cosmid libraries that are chromosome or region specific, and which are stored as individual clones archived in high-density microtiter plates. Similar strategies are useful for the production, screening, and distribution of YAC libraries. Methods for replicating, distributing, and analyzing clones from these arrayed libraries also differ in that simplified methods for DNA preparation, requiring a minimal number of steps, the use of automation and robotics for manipulating and distributing clones, and strategies for rapid restriction mapping and production of probes are all essential.

[15] – Use of Cosmids and Arrayed Clone Libraries for Genome Analysis

Publisher Summary This chapter reviews some protocols utilized in the laboratory for large-scale genome mapping, including the construction of cosmid libraries, the production and archiving of arrayed cosmid libraries, the use of robots for cosmid manipulation and processing, and the analysis of individual cosmid clones. Plasmid and bacteriophage cloning vectors have been widely used for a number of years for the isolation and analysis of individual genes or multigene families. More recently, the construction of large-scale maps of complex genomes and the complete physical mapping of genomes of model organisms have depended on the use of DNA fragments cloned in bacteriophage or cosmid vectors. Genomic libraries constructed for large-scale genome analysis differ substantially from those constructed for single-gene isolation in several respects. For large-scale genome mapping, the criteria for representation, completeness, random distribution of clones, and frequency of cloning artifacts are more significant than with single-gene isolation. For single-gene cloning, genomic libraries are generally prepared as a single pool of individual clones that is plated on agar. Membrane filter transfers are then carried out, and the library screened for specific clones by hybridization with a DNA probe. For genomic analysis, it is more convenient, although more time consuming and labor intensive, to prepare libraries as large arrays.