Approaches to DNA mutagenesis: an overview.

In the last several years, the use of double-stranded DNA templates together with thermostable-polymerase PCR has essentially replaced the use single-stranded DNA templates using the thermolabile polymerase for in vitro mutagenesis. Numerous PCR methods are now available, such as overlap-extension PCR, megaprimer PCR, and inverse PCR. All these PCR methods are reliable, effective, and convenient, although they are more prone to high rates of spontaneous error in mutant DNAs than are methods using thermolabile polymerases. Some improvements, such as the introduction of methylated templates, have been employed to minimize PCR errors. On the other hand, because of the introduction of many selection measures (e.g., restoration of antibiotic resistance, restoration of replication origin and unique site elimination), both double-stranded and single-stranded DNAs can now be used as templates for mutagenesis using thermolabile polymerase methods. For PCR methods, selection measures such as nested PCR has developed. All these selection measures have greatly improved the efficiency of mutagenesis by removing wild-type templates prior to transformation. Many efficient methods are available for both SDM and REM. Mutations can be introduce in vitro or in vivo, either by mutagenic primers or by erroneous DNA synthesis. Thus, choices largely depend on the experimental needs and resources of the investigator.