A NEW PCR BASED METHOD FOR THE GENERATION OF NESTED DELETIONS

We have developed a simple, PCR-based protocol, random primed/anchored -PCR (RPA-PCR), that allows the selective amplification and efficient cloning of segments that are adjacent to any known sequence. We demons trate that RPA-PCR can be used to prepare a nested set of evenly space d deletions suitable for DNA sequencing. However, it should also be po ssible to use this technique for a number of other purposes: generatin g deletions for the analysis of eukaryotic promoters, extending cDNA c lones in the 5' direction, cloning the insertion sites of retroviral p roviruses and transposons, and analyzing intron/exon boundaries.