Figure 4

Inverse splinkerette PCR. Genomic DNA of an insertion mutant is first digested with a restriction enzyme that cuts once (or very few times) in the insertion vector and frequently in the genome (EcoR V). The resulting products are ligated to generate DNA circles. Digestion with Sfi I produces one end with partially degraded vector DNA (λ buffer) for ligation of a double-strand splinkerette adaptor. Genomic DNA bordered by the splinkerette and the partial ura4⁺ marker can be amplified by nested splinkerette and ura4⁺ primer sets in two rounds of PCR.