title: Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy.
abstract: BACKGROUND
  Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence microscopy and nucleic acid analogues have been proposed so far.
  
  
  RESULTS
  Here we report a novel enzyme-free approach to efficiently detect cancer mutations. This assay includes gene-specific target enrichment followed by annealing to oligonucleotides containing locked nucleic acids (LNAs) and finally, detection by fluorescence microscopy. The LNA containing probes display high binding affinity and specificity to DNA containing mutations, which allows for the detection of mutation abundance with an intercalating EvaGreen dye. We used a second probe, which increases the overall number of base pairs in order to produce a higher fluorescence signal by incorporating more dye molecules. Indeed we show here that using EvaGreen dye and LNA probes, genomic DNA containing BRAF V600E mutation could be detected by fluorescence microscopy at low femtomolar concentrations. Notably, this was at least 1000-fold above the potential detection limit.
  
  
  CONCLUSIONS
  Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay, which allows for an accurate estimation of mutant abundance when the detection limit requirement is met. Using fluorescence microscopy, this approach presents the opportunity to detect DNA at single-molecule resolution and directly in the biological sample of choice.
authors: The Division of Oncology, Stanford School of Medicine, Stanford University, Stanford, California, United States of America.
authors: Laura
authors: L
authors: Miotke
authors: Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark; Dr B C Roy College of Pharmacy and AHS, Durgapur, West Bengal, India.
authors: Arindam
authors: A
authors: Maity
authors: The Division of Oncology, Stanford School of Medicine, Stanford University, Stanford, California, United States of America.
authors: Hanlee
authors: H
authors: Ji
authors: Memphys Center for Biomembrane Physics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
authors: Jonathan
authors: J
authors: Brewer
authors: Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark.
authors: Kira
authors: K
authors: Astakhova
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis / Q000379:methods*; D004798:Enzymes; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000235:genetics*; D015345:Oligonucleotide Probes / Q000235:genetics*; D009841:Oligonucleotides; D020641:Polymorphism, Single Nucleotide; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics
chemical_list: D004798:Enzymes; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; C477371:locked nucleic acid; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf