Exceptional Chromosome Regions Workshop I




David C. Schwartz
University of Wisconsin, Madison

Our laboratory has developed Optical Mapping, a single molecule system for the construction of ordered restriction maps. Optical Mapping dispenses with electrophoretic approaches, and uses fluorescence microscopy to directly image individual DNA molecules bound to derivatized glass surfaces, after cleavage by restriction enzymes. Cleaved fragments retain their original order, and cut sites are flagged by small, visible gaps. Optical Mapping solves the problem of determining fragment order, and when working with clones, works with only a handful of molecules. By determining the existence of these sequence-specific cut sites and the distances between them, we can create a landmark map of the DNA sequence. Efforts in our laboratory, over the last two years have been to automate Optical Mapping, with the goal of creating high throughput systems for the analysis of a wide range of sample types. This work has entailed the fusion of advanced optical, and biochemical techniques with novel statistical and algorithmic developments.

Optical Mapping of clones
Optical Mapping is now a robust system for the analysis of cloned DNA samples. We have mapped cosmid and yeast (YAC), or bacterial artificial chromosome (BAC) clones. We have mapped to high resolution BACs derived from the human Y chromosome which contain numerous repeated regions and are considered difficult to map by conventional means.

Optical Mapping of genomic DNA
We are now also able to map genomic DNA directly which enables large stretches of the genome to be mapped, simplifying contig formation. Library construction is obviated enabling mapping of organisms with DNA which is difficult to clone. Also, cloning artifacts are precluded enabling more accurate maps to be generated. Furthermore, small amounts of starting material are required, enabling mapping of microorganisms which are problematic to culture. We benchmarked our genomic mapping system by mapping E. coli K12 strain which has been completely sequenced, and then mapped the microbial genomes Plasmodium falciparum and Deinococcus radiodurans. Such maps have proved invaluable as a scaffold for assembling of sequence contigs and as a means of sequence verification.

We then went on to map the human genome with 0.6-fold coverage. We project that a whole genome map of high accuracy and minimal gaps will be obtained when approximately 5 genome equivalents are mapped and contiged. We expect to link these contigs and close a large portion of gaps by the Optical Mapping of BAC or YAC contigs previously assigned to the physical map. The complete reference map will serve as a database by which inherited genetic aberrations and genomic rearrangements associated with tumors can be characterized at the molecular level.

Software tools for Optical Mapping.
We have developed an integrated microscope control, machine vision and statistical anlysis system to fully automate image collection, processing and map construction (Optical Map Maker). A number of other software tools have been developed to aid in manipulating large pieces of data such as images of long DNA molecules which span multiple microscope fields (Gencol). Marking of restriction site cuts in images is now semi-automated (Visionade) and multiple maps can be aligned and oriented to form contigs (Gentig).

It is our hope that the Optical Mapping system will be broadly used for large scale genomic analysis and a means to perform population-based genomic studies.

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