Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy

Abstract

BackgroundRapid 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.Methods and ResultsHere 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.ConclusionOverall, 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.

Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay

“…Moreover, BRAF V600E‐mutated melanoma tends to have more aggressive biological behaviour than BRAF wild‐type melanoma . The transversion of T into A requires sensitive T m studies to evaluate the mutation or the design of specific nucleic acid detection probes for diagnostic proposes …”

“…The duplex was prepared by using the established conditions for UV and fluorescence‐assisted melting experiments and by using identical experimental procedures (see the Supporting Information). For the fluorescence‐assisted assay, we used the DNA binding dye EvaGreen as a non‐sequence‐specific indicator of duplex hybridisation . Under all the applied conditions, we observed nearly identical T m values for the fluorescence‐assisted and UV melting assays.…”

“…Therefore, we performed a similar series of experiments for a different duplex. We extended the oligonucleotide test system and designed a 21‐mer antisense strand that targets the RNA of the human oncogene BRAF . This genetic mutation is mediated by single‐nucleotide polymorphism (T→A) at gene position 1799 in human DNA that facilitates substitution of valine to glutamic acid at residue 600 of the serine/threonine‐protein kinase B‐Raf.…”

“…Given these advances, the stage has been set to translate the information garnered from sequencing studies into personalised diagnostic and treatment strategies . In particular, human oncogenes have been extensively investigated, and many promising preclinical studies have shown clinical association of the mutations in these oncogenes with disease progression and treatment outcome . We, and others, have demonstrated that targeting cancer driving mutations in oncogenes is feasible by using synthetic DNA probes labelled with fluorophores .…”

“…In particular, human oncogenes have been extensively investigated, and many promising preclinical studies have shown clinical association of the mutations in these oncogenes with disease progression and treatment outcome . We, and others, have demonstrated that targeting cancer driving mutations in oncogenes is feasible by using synthetic DNA probes labelled with fluorophores . However, for multiple oncogenes such as KRAS and EGFR , efficiency of the detection can be low as a result of severe off‐target binding by the probe to other parts of the human genome .…”