Novel DNA Binding Domain-Based Assays For Detection of Methylated And Nonmethylated DNA

Abstract

Methylation of cytosine in the context of CpG dinucleotides is an epigenetic phenomenon in eukaryotes that plays important roles in genome function and transcription regulation. Aberrant changes in DNA methylation is an important feature of several human diseases such as cancer and neurological disorders. These discoveries have opened a new field of new therapies and diagnostics. During recent years, there has been a revolution in DNA methylation analysis technologies. This article focuses on methods with which to study DNA methylation that employ protein domains that specifically recognize either 5-methyl-cytosine in the CpG context or nonmethylated DNA, and methods developed for the detection of 5-hydroxymethylcytosine, the recently described epigenetic mark known as the sixth base of the epigenome.

Rapid microfluidic solid-phase extraction system for hyper-methylated DNA enrichment and epigenetic analysis

“…28 MBD protein-based hm-DNA extraction is particularly promising as it facilitates both enrichment and purification of hm-DNA. 6,[28][29][30] In this article, we present a passive hm-DNA lSPE system, where the captured hm-DNA is eluted into a reduced sample volume, thus resulting in enrichment and purification. The lSPE system is comprised of a high surface area microfluidic chip that is functionalized with MBD proteins that serve as the capture agents.…”

Direct Electrocatalytic Oxidation and Simultaneous Determination of 5‐Methylcytosine and Cytosine at Electrochemically Reduced Graphene Modified Glassy Carbon Electrode

“…Then some more advanced methylcytosine assays that do not require bisulfite treatment, polymerase chain reaction(PCR), or a restriction enzyme, have been proposed for labeling methylcytosine with methyl-CpG binding proteins [9,10], osmium complex [11,12], and vanadium complex [13]. Although these methods have offered useful and powerful tools in studying the phenomenon of DNA methylation, they are still laborious, time-consuming, and less sensitive.…”

Electronic DNA detection and diagnostics

“…Therapeutics, such as Decitabine (5-aza-2´-deoxycytidine, MGI Pharma) and Vidaza™ (5-azacytidine; Pharmion), which are both cytosine analogs that induce DNA hypo-methylation by inhibiting DNA methyltransferase, have been reported to indicate effectiveness for the treatment of myelodysplastic syndromes. 90 Currently, there is tremendous effort to find ways to exploit the diagnostic and therapeutic implications of DNA methylation abnormalities. 91,92 Genes, such as APC, SYNE1, GPNMB and MMP2, are normally expressed in the colon, and in CRC are primarily methylated higher than 76% and mutated.…”

“…Commercial kits such as Methyl-Miner (Invitrogen), Epimark (New England Biolabs) and Methyl collector (Active motif) involving specific methyl-binding domain (MBD) proteins that can capture symmetrically methylated CpGs in double stranded DNA are available and are being used in favor of the antibody based CpG analysis methods. 90 Methylated DNA enrichment using these protein based solution assays are then routinely used with conventional bisulfite sequencing or methylation specific PCR for further analysis. 90 Several new PCR-based methods for detecting DNA methylation using bisulphite-treated DNA are now complementing traditional PCR-based techniques by increasing the analytical sensitivity and providing quantitative information.…”

“…90 Methylated DNA enrichment using these protein based solution assays are then routinely used with conventional bisulfite sequencing or methylation specific PCR for further analysis. 90 Several new PCR-based methods for detecting DNA methylation using bisulphite-treated DNA are now complementing traditional PCR-based techniques by increasing the analytical sensitivity and providing quantitative information. For instance, methylation sensitive melting-curve analysis (MS-MCA) takes advantage of the differential resistance of DNA to melting, depending on the relative GC content.…”