Fig. 6. Schematic strategy for PCR-based site-directed mutagenesis. We synthesized 2,314 distinct mutant oligonucleotides, each of which consisted of 26 nucleotides with one base mismatch at the 14th nucleotide from the 5' end, and distributed them at a concentration of 100 μM into 25 96-well microtiter plates labeled #1, #2, #5-#20, and #22-#28 (Table 1). The first PCRs were performed (in 20 μl) containing 1´ cloned Pfu buffer (Stratagene), 87.5 μM each of dNTP, 0.1 ng/μl normal p53 cDNA template, 0.5 μM of each of one of the mutant oligonucleotides, 0.5 μM of each of one of the common oligonucleotides (LS5, LS6, LS8, OHK-8, or OHK-3; sequence information is listed in Table 2), and 1 unit of cloned Pfu polymerase (Stratagene) with an ABI9700 thermal cycler (Applied Biosystems) under the following conditions: 94^oC for 2 min; 25 cycles consisting of 94^oC for 30 sec, 60^oC for 30 sec and 72^oC for 1.5 min; and 72^oC for 7 min. The products were purified on 96-well formatted Multiscreen-PCR plates (Millipore). The second PCRs were performed (in 20 μl) containing 1´ cloned Pfu buffer, 87.5 μM each dNTP, 0.1 ng/μl normal p53 cDNA template, 7 μl of purified first PCR product, 0.5 μM of one of the common oligonucleotides (LS5, LS6, OHK-9, or OHK-10; Table 3), and 1 unit of cloned Pfu polymerase in an ABI9700 thermal cycler under the following conditions: 94^oC for 2 min, 5 cycles consisting of 94^oC for 1 min and 72^oC for 2 min; 25 cycles consisting of 94^oC for 30 sec, 60^oC for 1 min and 72^oC for 1.5 min; and 72 ^oC for 7 min. The combination of a mutant oligonucleotide and a common oligonucleotide or the first PCR product and a common oligonucleotide is indicated in a box. Depending on the combination of mutant oligonucleotides and common oligonucleotides (Table 3), three types of second PCR products were generated: full-length p53 cDNA (mutant oligonucleotide plates labeled #1, #2, and #5-#16), 5'-half of the p53 cDNA (mutant oligonucleotide plates 17-25) and 3'-half of the p53 cDNA (mutant oligonucleotide plates 26-28). Red arrow, 96-well plates containing mutant oligonucleotides; black arrow, common oligonucleotides; and red box, specific mutations.