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. 2015 Oct 13;5:15028.
doi: 10.1038/srep15028.

A Simple Bridging Flocculation Assay for Rapid, Sensitive and Stringent Detection of Gene Specific DNA Methylation

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Free PMC article

A Simple Bridging Flocculation Assay for Rapid, Sensitive and Stringent Detection of Gene Specific DNA Methylation

Eugene J H Wee et al. Sci Rep. .
Free PMC article

Abstract

The challenge of bringing DNA methylation biomarkers into clinic is the lack of simple methodologies as most current assays have been developed for research purposes. To address the limitations of current methods, we describe herein a novel methyl-protein domain (MBD) enrichment protocol for simple yet rapid and highly stringent selection of highly methylated DNA from limiting input samples. We then coupled this with a DNA-mediated flocculation assay for rapid and low cost naked-eye binary evaluation of highly methylated genes in cell line and blood DNA. The low resource requirements of our method may enable widespread adoption of DNA methylation-based diagnostics in clinic and may be useful for small-scale research.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Schematic of the assay.
(1) Genomic DNA is enzymatically fragmented to sizes compatible with MBD enrichment. (2) Rapid high stringency enrichment of methylated DNA is performed on ice (4 °C). (3) Regions of interests are amplified via an isothermal method. (4) The presence of long amplified DNA induces a bridging flocculation which indicates the presence of the HDMR of interest.
Figure 2
Figure 2
(A) Gel electrophoresis image of RPA products using ESR1 primers demonstrating improved stringency of MDB enrichment at low temperatures (4 °C) without a lost in performance as compared to the assay performed at room temperature (RT). M: methylated control, U: unmethylated control. (B) Top: Gel electrophoresis image of RPA reactions using ESR1 primers for DNA inputs at various levels of methylation. Bottom: Photos showing flocculation occurring from as little as 10% methylated samples.
Figure 3
Figure 3. Methylation profiles of 7 human cancer cell lines for (A) ESR1, (B) GSTP1 and (C) NPY.
Unmethylated controls (U-Ctrl) was included to validate assay stringency. (D) Whole blood methylation profiles of ESR1, GSTP1 and NYP from two prostate cancer samples (PC1 and PC2) and normal blood (NB) DNA pooled from 25 female donors. Top: Gel electrophoresis image of RPA reactions. Bottom: Photos showing flocculation as proxy for methylation states.

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