Multiplexed Detection of Single Nucleotide Polymorphisms on Single Transcripts in Situ

Advances in single molecule fluorescent in situ hybridization (smFISH) have enabled nanoscale-resolution imaging of RNA throughout cells and tissues. However, detection of single nucleotide polymorphisms (SNP) on individual transcripts has remained challenging due to limitations in signal-to-noise and probe specificity to single base mismatches. By combining selective hybridization of circular probes with the specificity of enzymatic reactions, Krzywkoski and Nilsson¹ achieved high fidelity of individual SNP detection in vitro. Here we present a strategy to use these “iLock” probes for highly multiplexed SNP detection in cells with subcellular resolution. We demonstrate the proof-of-concept on quantification of mitochondrial heteroplasmy as well as SNP genotyping of cells within a diverse cell pool. Ongoing work focuses on applying this technology to intact tissues. In situ SNP genotyping will enable measurement of allele-specific gene expression in single cells and tissues, thus providing a useful tool for studying gene regulation in an allele-specific manner.

¹Krzywkoski, T., and Nilsson, M. Nucleic Acids Res, 2017 vol. 45 (18) p. e161