Efficient amplification using 'megaprimer' by asymmetric polymerase chain reaction

摘要

Among the procedures for site specific introduction of mutations polymerase chain reaction (PCR) has been increasingly used because of its simplicity over other techniques. For example, KunkePs method involves multiple steps and the yield of the single stranded DNA depends on the orientation of the insert as observed in our earlier work. PCR based mutagenesis, on the other hand, does not require any special treatment of the DNA; plasniid DNA can be used successfully. The technique originally introduced by Kammann et al and later modified by others is cost-effective among the PCR based methods since it uses only one mutant oligorrier for each site-directed mutagenesis. This method involves two steps; the first step is performed using the mutant oligomer containing the desired mutation and the resulting product, termed 'megaprimer', is then used in a second round of PCR. In the author's experience, the yield of final PCR product is often very poor supporting the observations made by other previous workers. This is probably due to the poor efficiency of priming of the megaprimer (300 oligomers or more) to. the template. In this study, it is shown that initial five cycles of asymmetrical PCR using only the megaprimer greatly improves the yield of thefinal product.