Rapid Directional Walk Within DNA Clones by Step-Out PCR

Genomic segments of many model experimental organisms are now available as segments within cosmid, P1, or YAC vectors (1 –3 ). Even cDNA portions of these segments are becoming available. Preliminary analyses with any of these clones would include sequencing and mapping (either on the genome or within a set of clones or subclones). Generally, such analysis would begin with sequences at the termini of inserts and proceed directionally into the internal sequences. PCR, with one specific primer and one nonspecific primer, is an ideal procedure to generate sequential and ordered fragments for use as sequencing templates or probes in nucleic acid hybridizations (4 ). These fragments would extend from a specific position within the known sequences of either the vector arm or the insert (via the specific primer) into different positions within the adjacent unknown sequence (via the nonspecific primer). This “Step-Out PCR” procedure generates fragments in a matter of h and eliminates the need for time-consuming procedures, such as restriction enzyme digestion/analysis, ligations, and transformation into hosts (5 –8 ).