FIGURE 1 Schematic of the method for SDM using PCR. Template DNA is treated by the PCRSDM protocol for a limited number of PCR cycles. The resulting mixture of template, newly synthesized, and hybrid parental/newly synthesized DNA is treated with DpnI (target site 5'Gm6ATC) and Pfu DNA polymerase.(P) 5'phosphate;(B) 3'-terminal extended base (s). The end-polished PCR product is then intramolecularly ligated together and transformed into E. coil Specific Methods: Template DNA (-0.5 pmole) is added to a 25-~ 1 PCR cocktail containing lx SDM buffer, 15 pmoles of each primer, 250 p, M each dNTP, 2.5 units Taq DNA polymerase; and 2.5 units of Taq Extender (Stratagene). The PCR cycling parameters were 1 cycle of 4 min at 94~ 2 min at 50~ and 2 min at 72~ followed by 8 cycles of I min at 94~ 2 rain at 56~ and I min at 72~(step 1). The parental template DNA and the linear, mutagenesis primer incorporating newly synthesized DNA are treated with DpnI (10 units) and Pfu DNA polymerase (2.5 units, Stratagene). This results in the DpnI digestion of the in vivo-methylated parental template and hybrid DNA (14~ and the removal, by Pfu DNA polymerase, of the Taq DNA polymerase-extended base (s) on the linear PCR product. The reaction is incubated at 37~ for 30 min and then transferred to 72~ for an additional 30 min (step 2). SDM buffer (115~ l, containing 0.5 mM ATP) is added to the DpnI-digested, Pfu DNA polymerase-polished PCR products. The solution is mixed, and 10~ l is removed to a sterile microcentrifuge tube and T4 DNA ligase (4 units) is added. The ligation is incubated for> 60 min at 37~(step 3). The ligase-treated DNA is then transformed into competent E. coli (step 4).