PNA-RCA-FISH

We developed a robust DNA-targeted assay based on PNA technology that makes it possible to detect signature sequences on bacterial genomic DNA under non-denaturing, isothermal conditions within fixed cells. This method provides us with single copy sensitivity and single base specificity due to a combination of three techniques: First, a peptide nucleic acid (PNA) probe locally opens a chosen target site, which allows a padlock DNA probe to access the site and become ligated. Second, rolling circle amplification (RCA) generates thousands of single-stranded copies of the target sequence. Finally, fluorescent in situ hybridization (FISH) is used to visualize the amplified DNA.

We have successfully used this approach to fluorescently detect single-copy DNA sequences in bacterial genomes and discriminate between similar sites in various bacteria based on single-nucleotide differences. In a collaborative effort with Boston Medical Center, we employed this technique to develope a novel diagnostic that can detect Staphylococcus aureus and simultaneously distinguish between methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) strains.