Circulating tumor DNAs (ctDNAs) are fragments of DNA shed by tumors into the bloodstream. These fragments harbor tumor-specific aberrations, such as mutations, that drive cancer progression, and are established biomarkers for early detection of cancer and can affect its treatment. However, the fraction of tumor-originating cell free DNA (cfDNA) in early stage cancer can be as low as <0.1% of all cell-free DNA, complicating their accurate identification. Currently, this task relies primarily on next generation sequencing (NGS), but obstacles remain as these technologies struggle with providing both sufficient sensitivity and a multiplexing capability.
Solid-state nanopores (ssNPs) have recently emerged as a highly efficient tool for single-DNA sensing and analysis and can be used to probe low concentrations of DNA molecules from a dilute solution (2); hence they can be potentially applied for ctDNA quantification. In recent work, Squires et al. detected DNA mutations by combining sequence-specific digestions using restriction enzymes, and DNA length classification using ssNPs (1). They showed that probing just tens of DNA copies is sufficient to distinguish between two variants with a confidence level >0.995. However, this detection method relies on the use of a restriction enzyme, making its adjustment for clinical samples and multiple targets difficult.
To enable multiplexed, specific, and sensitive detection of mutations using ssNPs, we have developed an assay in which genetic variations are converted to a molecular form via sequence specific reaction. In our technique, two short single-stranded DNA probes which meet at the mutation point are hybridized to the denatured ctDNA. Then a DNA ligase enzyme catalyzes the formation of a bond between the two probes only if the nucleotides at the junction are correctly base paired to the template. Hence, a ligation product will be formed only if the mutation exists. The product is then collected using magnetic beads, and the sample is analyzed. An electrical voltage is used to focus the biomolecules to the ssNP, and to control their translocation speed through the nano-scale aperture. Importantly, as the ligated probes are threaded through the nanopore, one at a time, optical information is obtained from the fluorescence signals emitted by the molecules. The multi-color optical information uniquely identifies the presence of tested mutations and provide a quantitative measurement of their abundance.
To demonstrate our method’s capabilities towards the goal of cancer amplification-free genotyping directly from blood, we tested a breast cancer model. A library of ligation probes was designed to recognize two clinically important, treatment actionable breast cancer mutations: ERBB2 S310F and PIK3CA H1047R. Blood samples were obtained from a xenograft mouse model. Two types of mouse tumor populations corresponding to the unmutated or mutant gene sequences were used, differing from each other only by the presence of the single nucleotide variation. Detection probes were designed to have different combinations of fluorescent color labeling. Analysis of these samples showed we were able to uniquely identify the presence of the mutation in the blood sample with 96.6% specificity (3). Thus, we have developed a novel method for rapid detection and quantification of actionable cancer genetic alterations from clinical blood samples.
- Squires, A. H., Atas, E. and Meller, A. Genomic Pathogen Typing Using Solid-State Nanopores. PLoS ONE 10, e0142944 (2015).
- Spitzberg, J. D., Kooten, X. F. van, Bercovici, M. & Meller, A. Microfluidic device for coupling isotachophoretic sample focusing with nanopore single-molecule sensing. Nanoscale 12, 17805–17811 (2020).
- Burck, N. et al. Nanopore Identification of Single Nucleotide Mutations in Circulating Tumor DNA by Multiplexed Ligation. Clinical chemistry 14, 836–10 (2021).