DEVELOPMENT OF A TECHNIQUE FOR MULTIPLE SITE-DIRECTED MUTAGENESIS OF THE FTF GENE OF STREPTOCOCCUS-SALIVARIUS CONTAINING PALINDROMIC SEQUENCES

Attempts at site-directed mutagenesis of the fructosyltransferase (ftf ) gene of Streptococcus salivarius ATCC 25975 using standard protocols were unsuccessful and resulted in a series of deletions. These deleti ons appeared to commence at points within the ftf gene where there wer e palindromic sequences which were capable of forming closed loop stru ctures that acted as terminators under the conditions of mutagenesis. To overcome this problem, two modified mutagenic techniques were devel oped. They made use of T4 DNA polymerase in conjunction with either T7 DNA polymerase at 37 degrees C or Vent DNA polymerase from Thermococc us litoralis at an elevated temperature. These methods eliminated the need for a single-stranded DNA template and allowed polymerisation thr ough palindromic sequences to rapidly produce multiple site-directed m utations.