Universal Fast Walking for direct and versatile determination of flanking sequence.

We report a highly compact system for accelerating direct genome walking. Unlike previous walking techniques, our strategy does not rely on restriction enzymes or ligases, and is therefore unaffected by the availability of useful restriction sites in the flanking region. A complete circumvention of molecular cloning steps qualifies this method for sequencing genome segments that are regarded unclonable, and thus unsequenceable by the traditional methods. A premium was placed on economy of design: the system comprises just four direct reagent additions, in microliter-scale volumes, over the course of a 6-h procedure. The walk range in this method is directly related to the capabilities of the associated polymerase blend, indicating that it can achieve in excess of 35 kilobases per reaction. It also produces a DNA fingerprint that is distinctive to the flanking sequence. Despite the complexity of banding patterns in these fingerprints, we observed that the reaction products were directly sequenceable. In view of its speed, reliability and generality, we term the described method Universal Fast Walking.