Site-directed mutagenesis was performed using the Quikchange II site-directed mutagenesis kit (Stratagene) according to manufacturer's instructions. Mutagenesis to introduce insertions longer than 6 bp was performed in two steps [24] , by first subjecting the mutagenesis reactions (containing either the sense or antisense primer) to three cycles of PCR, then mixing the reactions and performing a further 18 cycles as previously described [11] .