INgen: Intracellular Genomic DNA Amplification for Downstream Applications in Sequencing and Sorting

Abstract

Here, we introduce intracellular genomic amplification (INgen), a method that harnesses the cell membrane as a natural reaction chamber to amplify DNA within fixed, permeabilized cells. INgen employs a strand-displacing, isothermal polymerase together with biotinylated primers to achieve robust intracellular DNA amplification and efficient recovery of the amplified material for sequencing. This approach overcomes a long-standing barrier that has prevented important advances in single-cell and rare-cell genomics: the inability to amplify and recover sequenceable DNA from fixed cells. By pushing past this barrier, INgen provides a critical and previously inaccessible step toward scalable single-cell DNA sequencing without the need to isolate each cell into a separate reaction vessel. Using INgen, we demonstrate targeted and whole-genome amplification across diverse organisms, including Saccharomyces cerevisiae, Bacillus subtilis, and Escherichia coli. Together, these capabilities position INgen as a foundational advance that paves the way for the next generation of single cell sequencing methods including high-throughput single-cell sequencing without physical isolation, contamination-resistant amplification within intact cells, and rare-cell enrichment prior to genomic analysis.