Gene walking by unpredictably primed PCR.

Several methods have been described for gene walking by PCR on uncloned DNA (2 — 13). In general, they suffer from limitations: they either avoid high-complexity whole genomes, require delicate manipulation or give contaminated products. This report describes a reliable and simple PCR (1) adaptation for gene walks from any specific genomic locus: unpredictably primed PCR (UP-PCR). Besides its similarity to some of those predecessors, UP-PCR offers several advantages: quality of products, simplicity, general applicability and a methodical approach to optimization. It employs undigested mammalian genomic DNA as the original template source and defined walking primers of known working range; it yields remarkably clean results, and can produce a gene walk in a single working day. It employs two successive PCRs, for which four oligos are needed: two sequence specific primers (SPs) and two universal walking primers (WPs) (Figure 1 part 1). The SPs are complementary to the known sequence from which one intends to walk. Both have the same orientation: 3' end towards the unknown region. They can be called outer specific primer (SP) and inner specific primer (iSP), respectively (Figure 1 part 1). The latter gives the advantages of a nested primer and it is used at the second reaction. In order to save limited sequence information it has been currently made shorter than the SP (Table