Homogeneous detection of cyanobacterial DNA via polymerase chain reaction

Abstract

To design a primer set enabling the identification through PCR of high-quality DNA for routine and high-throughput genomic screening of a diverse range of cyanobacteria. A codon-equivalent multiple alignment of the phycocyanin alpha-subunit coding sequence (cpcA) of 22 cyanobacteria was generated and analysed to produce a single degeneracy primer set with virtually uniform product size. Also, an 18S ribosomal RNA detection set is proposed for rejecting false positives. The primer sets were tested against five diverse cyanobacteria, Chlorella vulgaris, Saccharomyces cerevisiae, and Escherichia coli. All five cyanobacteria showed positive amplification of cpcA product with homogeneous fragment length, and no products were observed for any other organism. Additionally, the only product formation observed for the 18S rRNA set was in C.vulgaris and S.cerevisiae. The newly proposed primer set served as effective check primers for cyanobacteria. Cyanobacteria gDNA had a positive, homogenous result, while other bacteria, eukaryotes and alga tested were negative. These novel, broad-spectrum primers will greatly increase the utility of PCR on newly discovered cyanobacterial species.