Simplified protocol for pulsed-field gel electrophoresis analysis of Streptococcus pneumoniae

A variety of pulsed-field gel electrophoresis (PFGE) protocols for the mole cular subtyping of Streptococcus pneumoniae have been reported; most are ti me-consuming and complex. We sought to modify reference PFGE protocols to r educe the time required while creating high-quality gels. Only protocol mod ifications that resulted in high-quality banding patterns were considered. The following protocol components were modified. Lysis enzymes (lysozyme, m utanolysin, and RNase A) were deleted in a stepwise fashion, and then the l ysis buffer was deleted. Lysis and digestion mere accomplished in a single step with EDTA and N-lauroyl sarcosine (ES; pH 8.5 to 9.3) incubation at 50 degrees C in the absence of proteinase K, Ail enzymes except the restricti on enzyme were omitted. A minimum incubation time of 6 h was required to ac hieve high-quality gels. All of the reactions were performed within 9 h, an d the total protocol time from lysis to gel completion was reduced from 3 d ays to only 36 h, Combining lysis and digestion into a single step resulted in a substantial reduction in the time required to perform PFGE for S. pne umoniae. The ES solution mag have caused cell lysis by activating N-acetylm uramyl-L-alanine amidase, the pneumococcal autolysin.