Methods and kits for in situ amplification of fixed cells

Abstract

A method that harnesses the cell membrane as a natural reaction chamber for DNA amplification, enabling optional downstream sequencing and optional downstream cell sorting is described herein. Unlike traditional single-cell techniques, methods herein utilize a strand-displacing, isothermal polymerase to amplify DNA within fixed, permeabilized cells while maintaining the cell's structural integrity. This approach overcomes challenges associated with both typical single-cell DNA sequencing and hindrances encountered when previously attempting to sequence genetic material from fixed microbial cells, allowing amplification of genomic regions up to 100 kb and optional downstream sequencing of whole genomes from diverse cell types, including S. cerevisiae, B. subtilis, and E. coli. Additionally, methods herein (e.g., FISA) can optionally be adapted for targeted DNA enrichment using biotinylated primers and for fluorescence-based cell sorting.

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