Design and validation of primers for housekeeping genes of streptomycetes

Multigene-based phylogenetic analyses are becoming more widely used in molecular taxonomy because of its lab-to-lab portability and reproducibility. The first step that needs to be settled for this approach is to amplify and sequence several housekeeping genes. Four housekeeping genes atpD, recA, rpoB and trpB were chosen for streptomycetes, which are representatives of high G+C mol% Gram-positive bacteria. Primer pairs for amplification and sequencing of the gene fragments were designed according to the known genome sequences of two streptomycetes and three mycobacteria, as well as the available recA gene sequences of another two streptomycetes in NCBI database, by using software packages Primer premier 5.0, Oligo 6.0 and SPCR 3.0, and NCBI BLAST program. Performance of the primer sets were validated by specific amplification of all gene fragments from the 55 streptomycete tested strains under optimized PCR reaction conditions, and by successful sequencing of the amplification products. It is concluded that the primer sets designed here are effective, and the primer design procedure and guidelines are valuable for other housekeeping genes of high G+C mol% bacteria.