Structural and Genetic Diversity of Group B Streptococcus Capsular Polysaccharides (original) (raw)

2005, Infection and Immunity

DNA sequencing. The capsule clusters for serotypes II, VII, and VIII were PCR amplified from genomic DNAs with primers that correspond to the common regions flanking the central serotype-specific glycosyltransferase region. For these PCRs, Hi-Fidelity Supermix (Invitrogen, Carlsbad, Calif.) was used according to the manufacturer's specifications. PCR products were sonicated and end filled with T4 DNA polymerase to create blunt ends. The fragmented DNAs were isolated in 1.0% agarose gels, and DNAs migrating between 1.0 and 1.5 kb were isolated. After gel extraction, purified DNAs were ligated into pUC18 and transformed into XL-10 Gold competent cells. Ninety-six clones from each library were sequenced with both M13 forward and M13 reverse primers and BIG Dye Terminator version 2 chemistry. Sequences were resolved on an ABI 3700 sequencer, and chromatograms were processed by the Phred base caller and assembled by Phrap.