Improvement of the Mutation-Discrimination Threshold for Rare Point Mutations by a Separation-Free Ligase Detection Reaction Assay Based on Fluorescence Resonance Energy Transfer

Hagihara, Kenta; Tsukagoshi, Kazuhiko; Nakajima, Chinami; Esaki, Shinsuke; Hashimoto, Masahiko

doi:10.2116/analsci.32.367

Cited by 7 publications

(12 citation statements)

References 27 publications

(25 reference statements)

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“…The mutation-discrimination threshold obtained in the present study (5%) was in between those reported in two previous studies (1% 22 and 10% 23 ) that used a quantum dot FRET donor. Although the study that used Cy5 as a FRET acceptor featured a slightly lower threshold (1%) than that of the current study, 22 the streptavidin-coated quantum dots needed to be incubated within the post-LDR solution for 30 min to allow the quantum dots to conjugate to the 3′-biotinylated LDR product labeled with Cy5 at the 5′-end through biotin-streptavidin interactions. This post-LDR treatment was much simpler than the sequential treatments involved in the above-mentioned assay using the microbeads.…”

Section: Comparison With Previous Studiessupporting

confidence: 82%

“…This mutation-discrimination threshold is moderate, compared with our recent studies (0.25 -10%), which also used FRET for selective detection of the LDR products. [22][23][24] However, in previous studies, streptavidin-coated microbeads 24 and quantum dots, 22,23 which served as the target concentrator and FRET donor, respectively, needed to be incubated within post-LDR solution to capture the 3′-biotinylated LDR product through biotin-streptavidin interactions. Therefore, the current method can be used to examine the presence of sporadic mutations more rapidly and conveniently than previous methods.…”

Section: Discussionmentioning

confidence: 99%

“…LDR assays LDR assays were carried out under similar conditions to those described elsewhere, 22 with slight modifications. A mixture of the PCR amplicons (wild-type and mutant DNA) was used as template DNA for the LDR, together with a discriminating primer, a common primer, and a thermostable DNA ligase.…”

Section: Pcr Amplification Of Genomic Dnamentioning

confidence: 99%

“…[22][23][24] These previous studies also used micro/nano particles for isolation/concentration or FRET-based detection of the generated LDR products after completion of LDRs. Table 1 summarizes the results from the current study and from previous assays.…”

Section: Comparison With Previous Studiesmentioning

confidence: 99%

See 3 more Smart Citations

A Fluorescence Quenching Assay Based on Molecular Beacon Formation through a Ligase Detection Reaction for Facile and Rapid Detection of Point Mutations

Sawamura

Hashimoto

2017

ANAL. SCI.

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A fluorescence quenching assay based on a ligase detection reaction was developed for facile and rapid detection of point mutations present in a mixed population of non-variant DNA. If the test DNA carried a targeted mutation, then the two allele-specific primers were ligated to form a molecular beacon resulting in the expected fluorescence quenching signatures. Using this method, we successfully detected as low as 5% mutant DNA in a mixture of wild-type DNA (t test at 99% confidence level).

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Section: Comparison With Previous Studiessupporting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Section: Pcr Amplification Of Genomic Dnamentioning

confidence: 99%

Section: Comparison With Previous Studiesmentioning

confidence: 99%

See 2 more Smart Citations

A Fluorescence Quenching Assay Based on Molecular Beacon Formation through a Ligase Detection Reaction for Facile and Rapid Detection of Point Mutations

Sawamura

Hashimoto

2017

ANAL. SCI.

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“…Hagihara et al reported a separationfree ligase detection reaction assay based on fluorescence resonance energy transfer from a donor quantum dot to an acceptor fluorescent dye. 5 This assay could successfully detect one cancer mutation among 10 wild-type templates. The mutation-discrimination threshold was improved by one order of magnitude by replacing the original acceptor dye (Alexa Fluor 647) with another fluorescent dye (Cyanine 5).…”

mentioning

confidence: 99%

Quantum Dot-based Fluorescent Sensing

Kawasaki

2017

ANAL. SCI.

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Quantum dots (QDs) have been developed for sensing applications, such as biosensing and bioimaging, owing to their excellent fluorescence properties and advantages over traditional organic dyes, such as high stability, facile surface modification for target analytes, and size-controlled optoelectronic properties ( Fig. 1) concentration over the range of 2 to 4000 nM, with a detection limit of 1 nM. Moreover, the system was resistant to interference from a number of co-existing metal ions, promising development for the detection of Hg 2+ in environmental samples. The utilization of fluorescence resonance energy transfer is attractive in QD-based fluorescence sensing, where the QDs act as the donor chromophore. Hagihara et al. reported a separationfree ligase detection reaction assay based on fluorescence resonance energy transfer from a donor quantum dot to an acceptor fluorescent dye.5 This assay could successfully detect one cancer mutation among 10 wild-type templates. The mutation-discrimination threshold was improved by one order of magnitude by replacing the original acceptor dye (Alexa Fluor 647) with another fluorescent dye (Cyanine 5). Pillai et al. investigated the steady state and time-resolved photoluminescence quenching of streptavidin modified CdSe/ZnS QDs instigated by biotin-peptide-BHQ-1 (biotin-pep-BHQ-1) molecule. 6 They found that the self-assembled QDs-(pep-BHQ)n conjugates could detect matrix metalloproteinases-2 (MMP-2) produced by cancer cells at a detection limit of 1 ng/mL. QDs have also been recognized as being a promising fluorescent probe for bioimaging.7 Ogihara et al. investigated the transduction function of a cationic dextran hydroxypropyltrimethyl ammonium chloride-coated magnetic iron oxide nanoparticle (TMADM-03) for transducing quantum dots (QDs) into adipose tissue-derived stem cells (ASCs). 8 The fluorescence intensity of ASCs labeled with QDs using TMADM-03 was much higher than that of QDs-only labeling.In cadmium-based QDs, released cadmium ions become problematic because of their cellular toxicity. More recently, cadmium-free quantum dots were introduced, such as carbon dots, graphene QDs, silicon QDs, and gold nanoclusters, in an effort to produce more biocompatible QDs. 9,10Oskoei et al. reported on the selective determination of trinitrotoluene (TNT) based on energy transfer between carbon dots and gold nanoparticles. 11Compared to previous methods, this method has advantages of relatively high sensitivity, low cost, easier operation and requires a short analysis time. Gong et al. also developed an energy transfer-based biosensor using nitrogendoped carbon dots and gold nanoparticles for the highly sensitive detection of organophosphorus pesticides.12 Glutathione is an important antioxidant in body fluids and tissues, which inhibit damage to essential cellular constituents caused by reactive oxygen species. Sivasankaran et al. reported on the fluorescence determination of glutathione using tissue paper-derived carbon dots as fluorophores with a detection limit of 1.74 ...

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Separation‐free single‐base extension assay with fluorescence resonance energy transfer for rapid and convenient determination of DNA methylation status at specific cytosine and guanine dinucleotide sites

Fujita

Hashimoto

2018

Electrophoresis

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A separation‐free single‐base extension (SBE) assay utilizing fluorescence resonance energy transfer (FRET) was developed for rapid and convenient interrogation of DNA methylation status at specific cytosine and guanine dinucleotide sites. In this assay, the SBE was performed in a tube using an allele‐specific oligonucleotide primer (i.e., extension primer) labeled with Cy3 as a FRET donor fluorophore at the 5′‐end, a nucleotide terminator (dideoxynucleotide triphosphate) labeled with Cy5 as a FRET acceptor, a PCR amplicon derived from bisulfite‐converted genomic DNA, and a DNA polymerase. A single base‐extended primer (i.e., SBE product) that was 5′‐Cy3‐ and 3′‐Cy5‐tagged was formed by incorporation of the Cy5‐labeled terminator into the 3′‐end of the extension primer, but only if the terminator added was complementary to the target nucleotide. The resulting SBE product brought the Cy3 donor and the Cy5 acceptor into close proximity. Illumination of the Cy3 donor resulted in successful FRET and excitation of the Cy5 acceptor, generating fluorescence emission from the acceptor. The capacity of the developed assay to discriminate as low as 10% methylation from a mixture of methylated and unmethylated DNA was demonstrated at multiple cytosine and guanine dinucleotide sites.

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Improvement of the Mutation-Discrimination Threshold for Rare Point Mutations by a Separation-Free Ligase Detection Reaction Assay Based on Fluorescence Resonance Energy Transfer

Cited by 7 publications

References 27 publications

A Fluorescence Quenching Assay Based on Molecular Beacon Formation through a Ligase Detection Reaction for Facile and Rapid Detection of Point Mutations

A Fluorescence Quenching Assay Based on Molecular Beacon Formation through a Ligase Detection Reaction for Facile and Rapid Detection of Point Mutations

Quantum Dot-based Fluorescent Sensing

Separation‐free single‐base extension assay with fluorescence resonance energy transfer for rapid and convenient determination of DNA methylation status at specific cytosine and guanine dinucleotide sites

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