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A simple and efficient method for constructing high resolution physical maps.

This paper describes a simple and efficient walking method for constructing high resolution physical maps and discusses its applications to genome analysis. The method is an integration of three strategies: (1) use of a highly redundant library of 3Kb-long subclones; (2) construction of a multidimensional pool from the library; (3) direct application of a PCR (polymerase chain reaction)-based screening technique to the pooled library, with two PCR primers, one from the end of the subcloning vector and the other from the leading edge of the walk. This technique allows not only detection of each overlapping subclone but simultaneous determination of its orientation and the size of its overlap. The end of the subclone with the smallest overlap is sequenced and a primer is designed for the next step in the walk. Iteration of the screening procedure with minimum overlapping subclones results in completion of the high resolution map. Using this method, a 3Kb-resolution map was constructed from an 80Kb region of the bithorax complex of Drosophila melanogaster. The method is general enough to be applicable to DNA from other species, and simple enough to be automated.