Target DNA detection and quantitation on a single cell with single base resolution

In this report, we present a new method for sensitive detection of short DNA sites in single cells with single base resolution. The method combines peptide nucleic acid (PNA) openers as the tagging probes, together with isothermal rolling circle amplification (RCA) and fluorescence-based detection, all performed in a cells-in-flow format. Bis-PNAs provide single base resolution, while RCA ensures linear signal amplification. We applied this method to detect the oncoviral DNA inserts in cancer cell lines using a flow-cytometry system. We also demonstrated quantitative detection of the selected signature sites within single cells in microfluidic nano-liter droplets. Our results show single-nucleotide polymorphism (SNP) discrimination and detection of copy-number variations (CNV) under isothermal non-denaturing conditions. This new method is ideal for many applications in which ultra-sensitive DNA characterization with single base resolution is desired on the level of single cells.