A high-throughput and sensitive method to measure Global DNA Methylation: Application in Lung Cancer

Abstract

Background: Genome-wide changes in DNA methylation are an epigenetic phenomenon that can lead to the development of disease. The study of global DNA methylation utilizes technology that requires both expensive equipment and highly specialized skill sets.

Extra-cellular matrix suppresses expression of the apoptosis mediator Fas by epigenetic DNA methylation

“…Thereafter, DNA and RNA were isolated from about 5 9 10 5 cells with a DNA/RNA isolation Kit (Quiagen). Global methylation was addressed [20] from 100 ng DNA digested in multi core buffer (Promega) with 5 U MspI or HpaII or incubated without enzyme (background) for 3 h at 37°C in a total volume of 30 ll. The restriction of the DNA was followed by an endfill reaction where to the restriction digest 20 ll of a mixture containing 0.1 lM Biotin-11-dCTP and 0.1 lM Biotin-11-dGTP (Perkin Elmer, Boston, MA) and 0.5 U Klenow-fragment of DNA-polymerase III (Promega) in 500 mM Tris-HCl, pH 7.2, 100 mM MgSO 4 and 1 mM dithiothreitol have been added and incubated at room temperature for half an hour.…”

Global DNA hypomethylation in cancer: review of validated methods and clinical significance

“…Examples include methods based on the digestion of DNA with restriction enzymes, such as methylation CpG island amplifi cation (MCA) (98) , differential methylation hybridization (DMH) (99) , restrictionlandmark genomic scanning (RLGS) (100) , amplifi cation of inter-methylated sites (AIMS) (101) , methylation-specifi c digital karyotyping (MSDK) (102) , HPAII tiny fragment enrichment by ligation-mediated PCR (HELP) assay (103) and CpG lobal (104) . All of which have been used in cancer studies, e.g., the application of CpG lobal to measure the changes in global DNA methylation in lung cancer resulted in substantial DNA methylation differences between healthy and tumor tissues (104) .…”

A simple method for high-throughput quantification of genome-wide DNA methylation by fluorescence polarization

“…In assessing restriction by HpaII relative to MspI, the FPDM procedure avoids the need for radioactivity, 16 or complex protocols such as separation of methylated from unmethylated cytosine by HPCE, 11,17,18 washings to remove unincorporated biotinylated nucleotide, 17 four-step pyrosequencing, 18 or prelabeling of PCR amplicons to allow FRET measurement. 32 Thus the entire FPDM can be readily performed on a 96 or 384-well microtiter plate.…”

“…13 The methylsensitive HpaII and methyl-insensitive MspI isoschizomer pair are most widely used for screening DNA methylation status of the ~2.3 million CCGG sites in the human genome. 14,15 Since the target sequence for both of these enzymes is CCGG, but only MspI and not HpaII can cut the methylated Cm 5 CGG, the fraction of unmethylated sites defined by the HpaII/MspI cleavage ratio can be determined by cytosine extension analysis with radioactivelabeled dCTP 16 or biotinylated dCTP, 17 and pyrosequencing. 18 However, some of these techniques require a large amount of to rapidly determine DNA methylation levels from a large number of biological or clinical samples, we have developed an accurate and sensitive method for high-throughput quantification of global methylation of 5'-Cm 5 CGG-3' sites in the genome, visualized by fluorescence polarization (fp) based measurement of DNA methylation (fpDM).…”