| The study of genes and gene systems has introduced novel conceptual and experimental approaches into the biological sciences. Optical Mapping is one such experimental approach. Optical Mapping is a methodology for characterizing nucleotide sequence landmarks on single DNA molecules via fluorescence microscopy. The information generated by Optical Mapping is a restriction map. Optical Mapping is the first practical, non-electrophoretic restriction mapping approach. The technology of Optical Mapping is well proven: this laboratory has used Optical Mapping to restriction map yeast chromosomes, lambda bacteriophage clones, cosmid clones, yeast artificial chromosomes, bacterial artificial chromosomes, PCR products and even raw genomic DNA fragments. The largest of these fragments is approximately 1 million base pairs in length. Automation of the component processes of Optical Mapping is necessary in order to use this approach to construct genomic-scale maps ({dollar}sim{dollar}1 million to 1 billion base pairs in length).; Reported here is the development of biochemical, robotic, and machine vision systems that support the automation of Optical Mapping. Yeast artificial chromosomes are visualized in a laminar shear flow to gain an understanding of the molecule-elongation process used in Optical Mapping. Derivatized surfaces are developed which are compatible with robotic sample deposition and "fluid fixation." These surfaces are analyzed with atomic force microscopy and endonuclease cleavage assays. A simple image processing program (known as "Map-o-matic"), and a computer-controlled automatic epifluorescence microscope are developed. A computer-controlled robot, capable of depositing DNA samples onto derivatized glass surfaces in dense arrays is also constructed.; Simple and complex maps of lambda bacteriophage and human adenovirus type 2 DNA, digested with a variety of restriction endonucleases, are determined using a statistical image processing and map construction system, known as Optical Map Maker (OMM). Restriction maps from a number of cosmid clones from the human D13S25 locus, implicated in chronic lymphocytic leukemia, are determined automatically from robot-deposited DNA samples. |
