Rapid screening of the heterogeneity of DNA methylation by single‐strand conformation polymorphism and CE‐LIF in the presence of electro‐osmotic flow

Abstract

DNA methylation is a complex event in epigenetic studies because of both the large CpG islands present upstream of the promoter region and the different distribution of DNA methylation despite similar methylation levels. For this reason, we proposed a fast, cost-effective method for the screening of DNA methylation based on SSCP and CE-LIF. In this study, the PCR products that were amplified from bisulfite-treated genomic DNA were denatured at 94°C, followed by immediate chilling in ice water to form the ssDNA. The ssDNA were separated by 1.5% poly(ethylene oxide) (Mavg 8 000 000 Da) in the presence of EOF according to the different conformations represented by their unique methylation states. This result demonstrated that four hepatocellular carcinoma cell lines represented a different heterogeneity of DNA methylation and could be distinguished by SSCP-CE. The results obtained from SSCP-CE also corresponded with those obtained from combined bisulfide restriction analysis and methylation-sensitive high-resolution melting analysis. Therefore, the proposed SSCP-CE method may potentially be used for rapid screening for determination of the heterogeneity of DNA methylation in further epigenetic studies and clinical diagnosis.

Microfluidic platforms for DNA methylation analysis

“…Chen and Chang et al 69 reported the combination of COBRA and electrophoresis with laserinduced fluorescence for determining the heterogeneity of DNA methylation. Chang's group further reported 70 a Fig. 4 (A) Sample pre-processing module for on-chip DNA bisulfite conversion consisting of a microchamber, 3D micromixer, and microchannel.…”

Separation‐free single‐base extension assay with fluorescence resonance energy transfer for rapid and convenient determination of DNA methylation status at specific cytosine and guanine dinucleotide sites

“…CpG sites are converted to TpG sites and mCpG (5‐methyl cytosine and guanine dinucleotide) sites are converted to CpG sites in the resultant PCR amplicons. These base changes can be detected by several methods, such as hybridization‐based techniques , electrophoretic sorting , pyrosequencing , gold/DNA affinity‐based techniques , and single‐base extension (SBE) assays.…”

“…DNA polymerase incorporates a single dideoxynucleotide triphosphate (ddNTP) that is complementary to the target base. Single base‐extended primer (i.e., SBE product) can be detected by various methods, including CE , MALDI‐TOF MS , surface enhanced Raman spectroscopy , and BeadChip array . However, all of these techniques are expensive, use high‐precision instruments, and require time‐consuming separation or cleanup/concentration of the SBE products.…”