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

Miotke L, Maity A, Ji H, Brewer J, Astakhova K - PLoS ONE (2015)

Bottom Line: 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.Notably, this was at least 1000-fold above the potential detection limit.Using fluorescence microscopy, this approach presents the opportunity to detect DNA at single-molecule resolution and directly in the biological sample of choice.

View Article: PubMed Central - PubMed

Affiliation: The Division of Oncology, Stanford School of Medicine, Stanford University, Stanford, California, United States of America.

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.

Methods and 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

Main principle of DNA detection by short LNA/DNA capture probes on solid support.Target binding specificity results from the difference in melting temperature (Tm) between fully-matched and mismatched capture probe:target complexes. CPG = controlled pore glass.
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pone.0136720.g002: Main principle of DNA detection by short LNA/DNA capture probes on solid support.Target binding specificity results from the difference in melting temperature (Tm) between fully-matched and mismatched capture probe:target complexes. CPG = controlled pore glass.

Mentions: Having studied hybridization properties of LNA/DNA capture probes, we applied them to the detection of the BRAF V600E mutation in human genomic DNA (Figs 2 and 3, Table 2). We determined, using digital PCR (data not shown), that the human cell lines HT29 and LS411N had a 25.0% and 66.7% abundance of the target mutation, respectively [18]. HMC-1 DNA was used as a 100% wild-type control as it possessed no mutation. First, we pre-enriched the DNA using a 120mer BRAF specific probe labeled with streptavidin that did not overlap the region of the capture probe and thus was universal for both the wt and mut targets (Fig 1 and Materials and Methods;). This step increased concentration of the target genome fragment and simultaneously improved specificity of our assay in the same way as when being applied prior to next-generation sequencing [7–8]. After multiple washing steps and detachment from the biotinylated magnetic beads, single-stranded BRAF fragments (7000 nt) were recovered into the solution.


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

Miotke L, Maity A, Ji H, Brewer J, Astakhova K - PLoS ONE (2015)

Main principle of DNA detection by short LNA/DNA capture probes on solid support.Target binding specificity results from the difference in melting temperature (Tm) between fully-matched and mismatched capture probe:target complexes. CPG = controlled pore glass.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4552304&req=5

pone.0136720.g002: Main principle of DNA detection by short LNA/DNA capture probes on solid support.Target binding specificity results from the difference in melting temperature (Tm) between fully-matched and mismatched capture probe:target complexes. CPG = controlled pore glass.
Mentions: Having studied hybridization properties of LNA/DNA capture probes, we applied them to the detection of the BRAF V600E mutation in human genomic DNA (Figs 2 and 3, Table 2). We determined, using digital PCR (data not shown), that the human cell lines HT29 and LS411N had a 25.0% and 66.7% abundance of the target mutation, respectively [18]. HMC-1 DNA was used as a 100% wild-type control as it possessed no mutation. First, we pre-enriched the DNA using a 120mer BRAF specific probe labeled with streptavidin that did not overlap the region of the capture probe and thus was universal for both the wt and mut targets (Fig 1 and Materials and Methods;). This step increased concentration of the target genome fragment and simultaneously improved specificity of our assay in the same way as when being applied prior to next-generation sequencing [7–8]. After multiple washing steps and detachment from the biotinylated magnetic beads, single-stranded BRAF fragments (7000 nt) were recovered into the solution.

Bottom Line: 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.Notably, this was at least 1000-fold above the potential detection limit.Using fluorescence microscopy, this approach presents the opportunity to detect DNA at single-molecule resolution and directly in the biological sample of choice.

View Article: PubMed Central - PubMed

Affiliation: The Division of Oncology, Stanford School of Medicine, Stanford University, Stanford, California, United States of America.

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.

Methods and 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus