Enzymatic Incorporation of Multiple Dyes for Increased Sensitivity in QD‐FRET Sensing for DNA Methylation Detection
2009
DNA methylation is an epigenetic modification that occurs at 5′ cytosine in CpG dinucleotides and is observed in high frequency in the transcription promoter regions of tumor-suppressor genes.[1,2] Such changes account for the loss of function of tumor-suppressor genes for almost every type of cancer;[3] and more genes are inactivated by promoter hyper-methylation than by genetic mutations.[4] These abnormal epigenetic changes appear to be an early event that precedes the occurrence of genetic mutations.[5-8] Hence, detecting DNA promoter methylation has significant clinical and prognostic implications for early cancer detection. Methylation-specific PCR (MSP) remains one of the most widely used methods for the sensitive detection of DNA methylation,[9] but requires gel electrophoresis and is limited to qualitative analysis. Fluorescence-based MSP variants[10,11] utilize fluorescent probes for real-time detection, thereby facilitating high-throughput and quantitative detection of DNA methylation. In recent years, quantum dots (QDs) have emerged as promising fluorophores for biological sensing due to their unique spectroscopic properties, such as broad excitation spectra, narrow size-tunable emission wavelengths, and high photostability. QDs have also been used as FRET donors to detect biomolecular targets.[12-15] The large Stokes shift and narrow emission spectra of QDs allow for FRET with a significantly reduced acceptor excitation and spectral crosstalk, and thus permits the design of QD-FRET biosensors with the minimal background necessary for detecting targets at low-concentration.[13,16] Recently, this feature of low intrinsic background of QD-FRET facilitated the development of a methylation-detection method that achieves greater sensitivity over conventional MSP methods, as exemplified by analyzing sputum samples from lung cancer patients.[16]
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