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

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.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.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

Metastatic Tumor Evolution and Organoid Modeling Implicate TGFBR2 as a Cancer Driver in Diffuse Gastric Cancer

Background: Gastric cancer is the second-leading cause of global cancer deaths, with metastatic disease representing the primary cause of mortality. To identify candidate drivers involved in oncogenesis and tumor evolution, we conduct an extensive genome sequencing analysis of metastatic progression in a diffuse gastric cancer. This involves a comparison between a primary tumor from a hereditary diffuse gastric cancer syndrome proband and its recurrence as an ovarian metastasis. Results: Both the primary tumor and ovarian metastasis have common biallelic loss-of-function of both the CDH1 and TP53 tumor suppressors, indicating a common genetic origin. While the primary tumor exhibits amplification of the Fibroblast growth factor receptor 2 (FGFR2) gene, the metastasis notably lacks FGFR2 amplification but rather possesses unique biallelic alterations of Transforming growth factor-beta receptor 2 (TGFBR2), indicating the divergent in vivo evolution of a TGFBR2-mutant metastatic clonal population in this patient. As TGFBR2 mutations have not previously been functionally validated in gastric cancer, we modeled the metastatic potential of TGFBR2 loss in a murine three-dimensional primary gastric organoid culture. The Tgfbr2 shRNA knockdown within Cdh1-/-; Tp53-/- organoids generates invasion in vitro and robust metastatic tumorigenicity in vivo, confirming Tgfbr2 metastasis suppressor activity. Conclusions: We document the metastatic differentiation and genetic heterogeneity of diffuse gastric cancer and reveal the potential metastatic role of TGFBR2 loss-of-function. In support of this study, we apply a murine primary organoid culture method capable of recapitulating in vivo metastatic gastric cancer. Overall, we describe an integrated approach to identify and functionally validate putative cancer drivers involved in metastasi

Sequences and thermal denaturation temperatures of LNA/DNA capture probes prepared in this study.^a

<p>^a Thermal denaturation temperatures <i>T</i>_m (°C)/change in <i>T</i>_m relative to corresponding reference duplex, Δ<i>T</i>_m (°C). <i>T</i>_m values measured as the maximum of the first derivatives of the melting curves (A₂₆₀ vs temperature). Reported Tm values were obtained in medium salt buffer and are averages of at least two measurements with resulting <i>T</i>_m ± 0.5°C. LNA nucleotides are marked with plus before the corresponding letter. Allele-specific nucleotide is underlined. 63Mer <i>BRAF</i> target sequences (SNP position is underlined): 5’-CATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGC TACAG<u>T</u>GAAATCTCGATGGAGT-3’ (<b>T1</b>); 5’-CATGAAGACCTCACAGTAAAAATAGGTGATT TTGGTCTAGCTACAG<u>A</u>GAAATCTCGATGGAGT-3’ (<b>T2</b>).</p><p>Sequences and thermal denaturation temperatures of LNA/DNA capture probes prepared in this study.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136720#t001fn001" target="_blank">^a</a></p

Main principle of DNA detection by short LNA/DNA capture probes on solid support.

<p>Target binding specificity results from the difference in melting temperature (<i>T</i>_m) between fully-matched and mismatched capture probe:target complexes. CPG = controlled pore glass.</p

High Sensitivity Detection and Quantitation of DNA Copy Number and Single Nucleotide Variants with Single Color Droplet Digital PCR

In this study, we present a highly customizable method for quantifying copy number and point mutations utilizing a single-color, droplet digital PCR platform. Droplet digital polymerase chain reaction (ddPCR) is rapidly replacing real-time quantitative PCR (qRT-PCR) as an efficient method of independent DNA quantification. Compared to quantative PCR, ddPCR eliminates the needs for traditional standards; instead, it measures target and reference DNA within the same well. The applications for ddPCR are widespread including targeted quantitation of genetic aberrations, which is commonly achieved with a two-color fluorescent oligonucleotide probe (TaqMan) design. However, the overall cost and need for optimization can be greatly reduced with an alternative method of distinguishing between target and reference products using the nonspecific DNA binding properties of EvaGreen (EG) dye. By manipulating the length of the target and reference amplicons, we can distinguish between their fluorescent signals and quantify each independently. We demonstrate the effectiveness of this method by examining copy number in the proto-oncogene <i>FLT3</i> and the common V600E point mutation in <i>BRAF</i>. Using a series of well-characterized control samples and cancer cell lines, we confirmed the accuracy of our method in quantifying mutation percentage and integer value copy number changes. As another novel feature, our assay was able to detect a mutation comprising less than 1% of an otherwise wild-type sample, as well as copy number changes from cancers even in the context of significant dilution with normal DNA. This flexible and cost-effective method of independent DNA quantification proves to be a robust alternative to the commercialized TaqMan assay

Detection of cancer DNA by fluorescence.

<p>(A) Visualization of <i>BRAF</i> V600E mutation on solid-support containing capture probe <b>CP2m</b>: <b>CP2m</b>:HT29 (2.5 pM, tube 1), <b>CP2m</b>:LS411N (2.5 pM, tube 6). Signal is obtained under laboratory UV-vis lamp (excitation at 365 nm) at 19°C using 10 pM signal-enhancing probe and 0.6X EvaGreen dye. (B) Quantification of genomic <i>BRAF</i> targets by fluorometry in solution. Target titration curves were obtained for fully complementary and mismatched complexes (blue and red lines, respectively) of LNA/DNA capture probes <b>CP2w</b> and <b>CP2m</b> with corresponding targets and signal-enhancing probe <b>P1</b>: 5’-GCT A+GA CCA +AAA TCA CCT A+TT TTT ACT GTG AG+G TCT TCA TGA AGA +AAT AT-3’. LNA nucleotides are marked with plus before the corresponding letter.</p

Ultra-fast detection and quantification of nucleic acids by amplification-free fluorescence assay

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