Cost-Effective Method for High-Throughput and Quantitative Analysis of DNA Methylation

Abstract (Set) Investigators at Johns Hopkins University developed a simple and cost-effective approach to detect and quantify the status of DNA methylation, termed MSqFRET. This method couples the well established methylation-specific PCR (MSP) technology with quantum dot-mediated fluorescence resonance energy transfer (FRET) DNA sensing. The methylation status of genomic DNA present in a specimen is analysed by treating the specimen with sodium bisulfite that modifies unmethylated cytosine, amplifying the modified sequences, and detecting amplicons based on a fluorescence resonance energy transfer (FRET) process. Specifically, this FRET process is mediated by a semiconductor quantum dot that is used both to capture target amplicons and to function as a FRET donor pairing with an organic acceptor associated to the amplicons. The level of methylation of a sample can be determined according to the extent of FRET between the quantum dot donor and the organic acceptor using a broad range of routine laboratory fluorescence readouts. The sensitivity afforded by the separation distance between donor and acceptor fluorophores allows both reaction and detection in a single sample preparation and eliminates the need for gel-based separation. By avoiding post PCR manipulations, FRET-based assays enable a high throughput detection format with the capability of automation. In addition, multiplexed MS-qFRET can be implemented to simultaneously quantify both methylated and unmethylated templates in a single reaction. The high fluorescence signal-to-noise ratio of MS-qFRET allows detection of PCR products at very early amplification cycles (8 cycles) and provides the means to rapidly analyze small quantities of samples without the need for costly real-time PCR techniques. The high sensitivity and quantitative capability of our new method have been exemplified by analysis of methylation with myelodysplastic syndrome (MDS) and sputum samples. Description (Set) This method, termed methylation-specific detection with Quantum dots-mediated fluorescence resonance energy transfer (MS-qFRET), detects PCR products of bisulfite treated DNA to reveal the methylation status of a specific loci. MS-qFRET is a homogeneous detection method that detects PCR products in solution without the need for gel separation. MS-qFRET is characterized by an extremely low level of background fluorescence (or a high signal-to-noise ratio) when compared to other fluorescence based detection methods, such as Molecular Beacon or TaqMan assays. This feature eliminates the need for two-stage PCR to analyze clinical samples that contain rare methylated DNA. MS-qFRET can be carried out using three different methylation-specific primer/probe designs for PCR reactions including (i) a set of methylation-specific primers of which one is conjugated with a quantum dot-capturing label (e.g. biotin) and the other conjugated with a qFRET acceptor (e.g. Cy5 fluorophore), and (ii) a set of methylation-specific primer of which one is conjugated with a quantum dot-capturing label (e.g. biotin) and the other non-labeled, together with a set of nucleotides (dNTP) of which one is labeled with a qFRET acceptor and (iii) a set of non-methylation-specific primer in which one is conjugated with a quantum dot-capturing label and the other non-labeled, together with a methylation-specific probe labeled with a qFRET acceptor. Proposed Use (Set) Accurate, rapid and cost-effective analysis of DNA methylation in a homogenous, separation-free format.

Inventor(s): Inventor (Set) Wang, Tza-Huei ,Bailey, Vasudev,Baylin, Stephen B.,Carraway, Hetty,Easwaran, Hariharan,Herman, James G.

Type of Offer: Licensing

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