Restriction endonuclease mspi

Approximately 1,500-bp region of bacterial 16S rRNA gene was amplified using single fluorescently labeled bacterial-specific oligonucleotide primers: 27F (FAM-labeled) and 1492R (Table S1). The PCR reaction mixture was prepared as previously described except different primers were used. For each sample, reactions were performed in triplicate and the PCR products were pooled. The PCR procedure consisted of an initial denaturation step of 5 min at 94 °C followed by 30 cycles at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min with a final extension at 72 °C for 7 min. The PCR products were purified as the above described. The purified PCR products were digested with restriction endonuclease Msp I (New England Biolabs, USA) at 37 °C for 2 h followed by an inactivation step at 65 °C for 10 min.
Fragment analysis was conducted by Capillary electrophoresis on an ABI 3730 DNA analyzer (PE Biosystems) with parameters set to exclude fragments shorter than 50-bp or larger than 550-bp and those under 40 fluorescence units.
Fragments were binned into T-RFs at a spacing of 1.0 ± 0.2 bp standard deviation. The relative abundance of each peak was calculated and expressed as a fraction of relative abundance = peak area/∑ peak areas of sample X (Fahy et al. 2005) (link). Fragments with relative abundance values less than 1% were discarded.