Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Poster Abstracts

91-POS Board 46 Highly Specific mRNA Transcript Quantification in Budding Yeast via Strand Displacement Induced Hybridization Chain Reaction Gable Wadsworth , Harold Kim. Georgia Institute of Technology, Atlanta, GA, USA. Quantitative determination of mRNA copy number distribution is essential for understanding phenotypic variability of single cells. Long mRNA transcripts (> 500 nt) can be detected using single-molecule Fluorescence In Situ Hybridization (FISH), where the target mRNA is hybridized with a large number of fluorescently labeled DNA probes. One downside to this method is the inability to interrogate short targets and short polymorphisms in sequence. We demonstrate a novel mRNA detection protocol in budding yeast based on strand displacement induced hybridization chain reaction (HCR). In this method we target a short 10 nucleotide sequence with an HCR amplifier which is comprised of a pair of DNA hairpins with an exposed input toehold and a sequestered output toehold. When the target mRNA is present, polymerization of the HCR amplifier is induced. Sensitive discrimination of the input mRNA sequence is demonstrated by placing mismatches in the stem of the hairpin. To our knowledge, this is the first demonstration of using an HCR amplifier to detect mRNA in yeast. Our method represents an efficient means to obtain quantification of either short mRNA transcripts or short polymorphisms in mRNA sequence.

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