C. Rathsam et Na. Jacques, DEVELOPMENT OF A TECHNIQUE FOR MULTIPLE SITE-DIRECTED MUTAGENESIS OF THE FTF GENE OF STREPTOCOCCUS-SALIVARIUS CONTAINING PALINDROMIC SEQUENCES, FEMS microbiology letters, 153(2), 1997, pp. 447-453
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.