Digital Polymerase Chain Reaction Paired with High-Speed Atomic Force Microscopy for Quantitation and Length Analysis...

DNA length polymorphisms are found in many serious diseases, and assessment of their length and abundance is often critical for accurate diagnosis. However, measuring their length and frequency in a mostly wild-type background, as occurs in many situations, remains challenging due to their variable and repetitive nature. To overcome these hurdles, we combined two powerful techniques, digital polymerase chain reaction (dPCR) and high-speed atomic force microscopy (HSAFM), to create a simple, rapid, and flexible method for quantifying both the size and proportion of DNA length polymorphisms. In our approach, individual amplicons from each dPCR partition are imaged and sized directly. We focused on internal tandem duplications (ITDs) located within the FLT3 gene, which are associated with acute myeloid leukemia and often indicative of a poor prognosis. In an analysis of over 1.5 million HSAFM-imaged amplicons from cell line and clinical samples containing FLT3-ITDs, dPCR-HSAFM returned the expected variant length and variant allele frequency, down to 5% variant samples. As a high-throughput method with single-molecule resolution, dPCR-HSAFM thus represents an advance in HSAFM analysis and a powerful tool for the diagnosis of length polymorphisms.