Abstract
BackgroundDNA methylation detection is indispensable for the diagnosis and prognosis of various diseases including malignancies. Hence, it is crucial to develop a simple, sensitive, and specific detection strategy.MethodsA novel fluorescent biosensor was developed based on a simple dual signal amplification strategy using functional dendritic DNA nanostructure and signal-enriching polystyrene microbeads in combination with ligase detection reaction (LDR). Dendritic DNA self-assembled from Y-DNA and X-DNA through enzyme-free DNA catalysis of a hairpin structure, which was prevented from unwinding at high temperature by adding psoralen. Then dendritic DNA polymer labeled with fluorescent dye Cy5 was ligated with reporter probe into a conjugate. Avidin-labeled polystyrene microbeads were specifically bound to biotin-labeled capture probe, and hybridized with target sequence and dendritic DNA. LDR was triggered by adding Taq ligase. When methylated cytosine existed, the capture probe and reporter probe labeled with fluorescent dye perfectly matched the target sequence, forming a stable duplex to generate a fluorescence signal. However, after bisulfite treatment, unmethylated cytosine was converted into uracil, resulting in a single base mismatch. No fluorescence signal was detected due to the absence of duplex.ResultsThe obtained dendritic DNA polymer had a large volume. This method was time-saving and low-cost. Under the optimal experimental conditions using avidin-labeled polystyrene microbeads, the fluorescence signal was amplified more obviously, and DNA methylation was quantified ultrasensitively and selectively. The detection range of this sensor was 10−15 to 10−7 M, and the limit of detection reached as low as 0.4 fM. The constructed biosensor was also successfully used to analyze actual samples.ConclusionThis strategy has ultrasensitivity and high specificity for DNA methylation quantification, without requiring complex processes such as PCR and enzymatic digestion, which is thus of great value in tumor diagnosis and biomedical research.
Highlights
DNA methylation detection is indispensable for the diagnosis and prognosis of various diseases including malignancies
Methylation is detected mainly depending on three methods, i.e. methylation sensitive restriction endonuclease analysis established by restriction endonuclease-specific cleavage [14,15,16,17], genomic methylation sequencing established on the basis of bisulfite conversion [18,19,20,21,22] and methylation array analysis [23,24,25,26]
Mechanism for synthesis of dendritic DNA polymer The DNA hairpin structures designed in this study had partially complementary sequences with overhanging sticky ends
Summary
DNA methylation detection is indispensable for the diagnosis and prognosis of various diseases including malignancies. The tumor suppressor genes of breast cancer are inactivated by abnormal methylation of the promoter region. Detecting the DNA methylation level has become a new strategy for the clinical diagnosis, early warning and susceptibility analysis of cancers. Based on a dual signal amplification strategy combining terminal deoxynucleotidyl transferase (TdT)-assisted enzymatic amplification with Ru(III) redox cycling, Cui et al detected DNA methylation by using a label- and immobilization-free electrochemical magnetobiosensor. 5-methylcytosine and 5-hydroxymethylcytosine, as three major epigenetic variants in DNA bases, were accurately quantified and distinguished [28]. Wu et al selectively labeled 5-hydroxymethylcytosine in aqueous solution using various sulfonate reagents under nonenzymatic reaction conditions This method allowed an efficient single binding step of biotin to hydroxymethylcytosine in DNA for accurate identification [30]. Yotani et al [31] developed an anion column to accurately distinguish methylated and unmethylated
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