AUTOMATED HIGH-RESOLUTION OPTICAL MAPPING USING ARRAYED, FLUID-FIXED DNA-MOLECULES

New mapping approaches construct ordered restriction maps from fluores cence microscope images of individual, endonuclease-digested DNA molec ules. In optical mapping, molecules are elongated and fixed onto deriv atized glass surfaces, preserving biochemical accessibility and fragme nt order after enzymatic digestion. Measurements of relative fluoresce nce intensity and apparent length determine the sizes of restriction f ragments, enabling ordered map construction without electrophoretic an alysis, The optical mapping system reported here is based on our physi cal characterization of an effect using fluid flows developed within t iny, evaporating droplets to elongate and fix DNA molecules onto deriv atized surfaces. Such evaporation-driven molecular fixation produces w ell elongated molecules accessible to restriction endonucleases, and n otably, DNA polymerase I, We then developed the robotic means to grid DNA spots in well defined arrays that are digested and analyzed in par allel. To effectively harness this effect for high-throughput genome m apping, we developed: (i) machine vision and automatic image acquisiti on techniques to work with fixed, digested molecules within gridded sa mples, and (ii) Bayesian inference approaches that are used to analyze machine vision data, automatically producing high resolution restrict ion maps from images of individual DNA molecules, The aggregate signif icance of this work is the development of an integrated system for map ping small insert clones allowing biochemical data obtained from engin eered ensembles of individual molecules to be automatically accumulate d and analyzed for map construction. These approaches are sufficiently general for varied biochemical analyses of individual molecules using statistically meaningful population sizes.