3-STEP PCR MUTAGENESIS FOR LINKER SCANNING

'Linker scanning' has been used as an efficient method for systematica lly surveying a segment of DNA for functional elements by mutagenesis. A three-step PCR method was developed to simplify this process. In th is method, a set of 'mutation primers' was made with 6 to 8 base subst itutions in the center of the primers. In the first PCR reaction, thes e 'mutation primers' are paired with an 3' primer from the opposite en d of the analyzed sequences to form a 'ladder' of fragments containing the base pair substitutions. These are used as templates in the secon d PCR with the 3' primer as the only primer to generate single strande d sequences, which are used as primers in the third PCR paired with an 5' primer to complete the mutagenesis. We have tested the method in a mutation screen of the steroid sulfatase promoter. Its application to general site specific mutagenesis is discussed.