A SIMPLE AND EFFICIENT METHOD FOR CONSTRUCTING HIGH-RESOLUTION PHYSICAL MAPS

This paper describes a simple and efficient walking method for constru cting 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) construc tion of a multidimensional pool from the library; (3) direct applicati on of a PCR (polymerase chain reaction)-based screening technique to t he pooled library, with two PCR primers, one from the end of the subcl oning vector and the other from the leading edge of the walk. This tec hnique allows not only detection of each overlapping subclone but simu ltaneous 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 scr eening procedure with minimum overlapping subclones results in complet ion of the high resolution map. Using this method, a 3Kb-resolution ma p was constructed from an 80Kb region of the bithorax complex of Droso phila melanogaster. The method is general enough to be applicable to D NA from other species, and simple enough to be automated.