Molecular microscopy of DNA for genome analysis: optical mapping of restriction digests

Abstract

Optical microscopy of individual DNA molecules has been an interesting technique for the past 15 years, but until recently has not been useful for genome analysis. We have developed Optical Mapping an emerging single molecule approach for the rapid generation of ordered restriction maps. Many identical individual DNA molecules from a single clone are elongated and fixed onto derivatized glass surfaces, digested with a restriction enzyme which cuts the DNA wherever a specific sequence pattern is found, stained with YOYO, and imaged with a cooled CCD camera attached to an automated epi-fluorescence microscope. Images are automatically processed to correct for non-uniform illumination, remove background, locate the DNA fragments, reject objects which do not look like single DNA molecules, recognize which fragments originate from an original uncut molecule, and calculate the relative sizes of the fragments by apparent length and fluorescence intensity. Results from many molecules are combined by clustering to recognize a consistent cutting pattern. Molecules which match the pattern are averaged to improve the sizing accuracy.