Highly sensitive detection of DNA methylation levels by using a quantum dot-based FRET method

Abstract

DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers.

A panel of promoter methylation markers for invasive and noninvasive early detection of NSCLC using a quantum dots-based FRET approach

“…Furthermore, in order to confirm the levels of methylation detected by QDs-based FRET method, two known control samples with high and low methylation in PCDHGB6 gene detected by pyrosequencing method were mixed to define varying levels of methylation. Pyrosequencing is considered as the gold standard for methylation detection and the experimental process was described previously (Ma et al, 2015). With the known methylation levels, a standard curve was obtained versus the E value and is shown in Fig.…”

“…Recently, our group has established a novel strategy for the optical detection of DNA methylation based on fluorescence resonance energy transfer (FRET) of quantum dots (QDs) (Ma et al, 2015). This method relies on a methylation-sensitive restriction enzyme for the differential digestion of methylated and unmethylated DNAs.…”

Two-dimensional nanomaterials for Förster resonance energy transfer–based sensing applications

“…The development of nanomaterials would be beneficial for the fabrication of FRET sensors to achieve better performance. Compared with traditional dyes and fluorescent proteins, the fluorescent nanomaterials, for instance, upconversion nanoparticles (UCNPs) [4,5], semiconductor quantum dots (QDs) [6,7], metal nanoclusters (NCs) [8], polymer dots [9,10], and carbon dots [11,12], have higher quantum yield, stronger photostability, and longer fluorescence lifetime, so they have been extensively utilized as donors in the FRET sensors. Moreover, some nanomaterials, for instance, gold nanoparticles (AuNPs) [13,14], gold nanorods [15], and carbon nanotubes (CNTs) [16,17], have been widely employed as efficient fluorescence quenchers in the FRET sensors owing to their excellent optical quenching capabilities.…”

Electrochemical biosensing strategies for DNA methylation analysis

“…These include combined bisulfite restriction analysis (COBRA) (Xiong and Laird, 1997), microarray based DNA methylation profiling (Gao et al, 2005;Yan et al, 2001), surface enhanced Raman spectroscopy based assays (Wang et al, 2015a(Wang et al, , 2016a, fluorescence based biosensors (Duan et al, 2010;Ma et al, 2015;Taleat et al, 2015;Wang et al, 2009), colorimetric assays (Ge et al, 2012;Wee et al, 2015a), surface plsmon resonance based assays (Carrascosa et al, 2014;Sina et al, 2014a) and quartz crystal microbalance (QCM) assay (Taleat et al, 2015).These techniques are also disadvantaged by their multi-step timeconsuming analytical procedures, the requirement for large amounts of input DNA, hazardous radiolabeling and/or expensive biological molecules, such as antibodies. Hence the development of a cost-effective, convenient and accurate method for analyzing DNA methylation status is important for medical research.…”