A recently developed and powerful strategy for site directed mutagenesis is to use two successive polymerase chain reactions (1, 2, 3, 4). The first reaction introduces a specific mutation into the target sequence, normally, by using a mutagenic primer and a primer that anneals beyond a bordering restriction site. The second reaction extends the first product beyond a restriction site adjacent to the opposite border. Restriction endonuclease cleavage and DNA splicing is then used to subclone the mutated cassette for further study. In practice we have found that the second PCR is often weak or even fails entirely, particularly when the product of the first reaction is long or forms a very stable secondary structure. Presumably, this is due to poor extension of the first PCR product, possibly because secondary structure in the large primer disrupts initiation. We have isolated this step of the thermal cycle by labelling the first PCR product of an unsuccessful mutagenesis experiment, and have found that it is indeed weak under typical PCR conditions (results not shown). Successful second reactions have been obtained previously by using room temperature annealing, but the use of reduced temperatures increases the incidence of non-specific amplification (5). Without lowering the annealing temperature, we have greatly improved the second PCR by instead lengthening the annealing period at 55°C to 15 minutes. This modification has been used for various different mutagenesis experiments on ribosomal RNA genes, including simple base pair substitutions in the yeast 5S RNA gene (6), larger sequence substitutions in the Schizosaccharomyces pombe internal transcribed spacers (data submitted but not shown) and an insertion (413 bp) of Tetrahymena thermophila ribosomal DNA into the Schizosaccharomycespombe 25S RNA gene (Figure IA). The results of this last experiment (Figure iB) clearly demonstrate an improved reaction efficiency and the nearly complete extension of the first PCR product that can be achieved when the annealing period is lengthened to 15 minutes. In all cases, the mutated DNA fragments were subcloned and confirmed by sequence analysis. The PCR conditions were as follows: The reaction volume was 50 Al containing 50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25°C), 1.5 mM MgCl, 0.1% Triton X-100, 200 itM of each dNTP, 100 /Ag/ml bovine serum albumin, 70 ng of each primer, 50 ng template DNA and 2 units Taq Polymerase, all covered with 50 t1l mineral oil. An initial denaturation step at 94°C for 3 minutes was followed by 30 cycles of denaturation at 940C for 1 minute, annealing at 550C for 1 minute and extension at 72°C for 2 minutes. For the second PCR, the first PCR products were purified using agarose gel electrophoresis and a unidirectional electroeluter (International Biotechnologies, Inc., New Haven, CT) and the annealing period of the thermal cycle was lengthened to 15 minutes.