AN EFFICIENT PCR MUTAGENESIS STRATEGY WITHOUT GEL PURIFICATION STEP THAT IS AMENABLE TO AUTOMATION

We describe here an improved megaprimer PCR mutagenesis strategy. The cumbersome gel purification step that is usually used can be omitted b y appropriately cleaving the first and second DNA templates with restr iction enzymes and enzymatically removing remaining primers from the f irst PCR reaction. We show that this improved procedure is reproducibl e and highly efficient. Furthermore this method is suitable for automa tion because all the steps are now carried out in reaction tubes.