Publisher Summary Constructing restriction maps of cloned DNA fragments is often a tedious, but very necessary task frequently encountered in the course of gene isolation or chromosome walking. Currently, two general methods have been developed for constructing maps of this type. The first requires digesting the cloned DNA with two different restriction enzymes, both singly and together. After separating the resulting fragments by agarose gel electrophoresis, one then attempts to determine, usually by trial and error, a uniquely ordered arrangement that accounts for the observed array of bands in each digest. The amount of time required for this process is considerable, increasing as the size of the insert DNA increases. In the case of cosmids, for example, deciphering the complexity of a fragment pattern derived from a 45-kilobase (kb) insert can be formidable. In addition, the trial and error method usually encounters uncertainty in the relative order of adjacent restriction fragments punctuated by the same restriction site when no (or few) other sites are apparent. This is especially true in cases involving small fragments because these may be easily missed.