RCA-specific oligonucleotides for FISH

Quantification of RCA phylotypes We developed a specific PCR approach to estimate the abundance of the RCA phylotypes relative to total bacterial 16S rRNA genes based on serial dilution of extracted DNA. Alternate 1:5 and 1:2 dilution steps to extinction were applied in triplicates, to 10-fold dilution or higher. PCRs with bacterial and RCA-specific primers were performed simultaneously using 60 8C for annealing. We used a PCR with primer pair 341f–907r for comparison because it amplifies a fragment consisting of approximately 560 bp (RCA-PCR 570 bp), melting temperatures, Tm, are similar (rca418f Tm 1⁄4 56 8C; rca994r Tm 1⁄4 54 8C; 341f Tm 1⁄4 58 8C; 907r Tm 1⁄4 54 8C) and the two fragments overlap in the majority of their sequences. PCR products were analysed on the same agarose gels. Gel images were edited with GelComparII (Applied Maths). Only bands differing distinctly from background noise were treated as positive results. The fraction of RCA-specific relative to total bacterial 16S rRNA genes was determined as follows. Percentage of RCA 16S rRNA genes 1⁄4 dilBacteria/dilRCA £ 100, where dilRCA is the highest dilution step in which RCA-specific 16S rRNA gene fragments were detected and dilBacteria is the highest dilution step in which bacterial 16S rRNA gene fragments were detected. In all experiments, bacterial and RCA-cluster-specific genes were detected in all triplicates of the highest dilution steps in which they occurred.