title: An electrochemical clamp assay for direct, rapid analysis of circulating nucleic acids in serum.
abstract: The analysis of cell-free nucleic acids (cfNAs), which are present at significant levels in the blood of cancer patients, can reveal the mutational spectrum of a tumour without the need for invasive sampling of the tissue. However, this requires differentiation between the nucleic acids that originate from healthy cells and the mutated sequences shed by tumour cells. Here we report an electrochemical clamp assay that directly detects mutated sequences in patient serum. This is the first successful detection of cfNAs without the need for enzymatic amplification, a step that normally requires extensive sample processing and is prone to interference. The new chip-based assay reads out the presence of mutations within 15 minutes using a collection of oligonucleotides that sequester closely related sequences in solution, and thus allow only the mutated sequence to bind to a chip-based sensor. We demonstrate excellent levels of sensitivity and specificity and show that the clamp assay accurately detects mutated sequences in a collection of samples taken from lung cancer and melanoma patients.
authors.affiliation: Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto M5S 3M2, Canada.
authors.forename: Jagotamoy
authors.initials: J
authors.lastname: Das
authors.affiliation: Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto M5S 3M2, Canada.
authors.forename: Ivaylo
authors.initials: I
authors.lastname: Ivanov
authors.affiliation: The Research Institute of the McGill University Health Centre, Montreal Children's Hospital, 1001 Decarie Blvd, Montreal, Quebec H4A 3J1, Canada.
authors.forename: Laura
authors.initials: L
authors.lastname: Montermini
authors.affiliation: The Research Institute of the McGill University Health Centre, Montreal Children's Hospital, 1001 Decarie Blvd, Montreal, Quebec H4A 3J1, Canada.
authors.forename: Janusz
authors.initials: J
authors.lastname: Rak
authors.affiliation: Department of Electrical and Computer Engineering, Faculty of Engineering, University of Toronto, Toronto, Canada.
authors.forename: Edward H
authors.initials: EH
authors.lastname: Sargent
authors.affiliation: 1] Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto M5S 3M2, Canada [2] Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 3M2, Canada.
authors.forename: Shana O
authors.initials: SO
authors.lastname: Kelley
mesh_terms: D055664:Electrochemical Techniques / Q000379:methods*; D006801:Humans; D009696:Nucleic Acids / Q000097:blood*; D009841:Oligonucleotides
chemical_list: D009696:Nucleic Acids; D009841:Oligonucleotides