SITE-SPECIFIC FORCED MISINCORPORATION MUTAGENESIS USING MODIFIED T7-DNA POLYMERASE

A new method for forced misincorporation site-specific mutagenesis is described. The method uses an exonuclease-deficient modified version of T7 DNA polymerase in the presence of one dNTP to force a misincorporation. Analysis by PAGE is used to monitor the efficiency of such misincorporation reactions. Brief extension of the terminally mismatched primer/template using the same enzyme in the presence of all four dNTPs is followed by chase/ligation using unmodified T7 DNA polymerase and T4 DNA ligase to give heteroduplex DNA. We have applied the method to mutagenesis of the Lac Z region of M13 and found that, using strand selection, efficiencies of mutagenesis at one site are > 50%. When the mutating dNTP is complementary to a neighbouring homopolymeric tract on the template, multiple mutation is observed and efficiencies are lower. The method is more general than internal mismatch mutagenesis and, because of its rapidity, is more expedient than existing methods of forced misincorporation mutagenesis.