This page contains a Flash digital edition of a book.
Benchmarks


A simple modification to the luminometric methylation assay to control for the effects of DNA


fragmentation Elif Aysimi Duman1,2


Department of Psychology, Bogazici University, Istanbul, Turkey, 2Center for Life Sciences and Technologies, Bogazici Ludwig Institute for Cancer Research Biology Department,


Eli Hatchwell5 1


University, Istanbul, Turkey, 3 Ltd, University of Oxford, Oxford, U.K., 4 Brookhaven National Laboratory, Upton, NY, and 5 Department of


Pathology, Stony Brook University, Stony Brook, NY ¥Author deceased in 2012.


BioTechniques 58:262-264 (May 2015) doi 10.2144/000114290 Keywords: epigenetics, global DNA methylation, luminometric methylation assay, DNA integrity.


Supplementary material for this article is available at www.BioTechniques.com/article/114290.


Variations in DNA methylation have been implicated in a number of disorders. Changes in global DNA methylation levels have long been associated with various types of cancer. One of the recently de- scribed methods for determining global DNA methylation levels is the LUminometric Methylation Assay (LUMA), which utilizes methylation sensitive and insensitive restriction endonucleases and pyrosequenc- ing technology for quantification. Here we provide evidence suggest- ing that the global methylation level reported by LUMA is affected by the integrity of the DNA being analyzed. The less intact the DNA, the lower the global methylation levels reported by LUMA. In order to overcome this problem, we propose the use of undigested DNA alongside digested samples. Finally, we demonstrate that this results in a more accurate assessment of global DNA methylation levels.


One of the most studied epigenetic modifications is DNA methylation, which involves methylation at cytosine nucleotides, mostly in the context of CpG dinucleotides. Methylation of DNA


METHOD SUMMARY


Our modification to LUMA involves quantifying background incorporation of nucleotides in an undigested reaction, in parallel with samples digested with methylation-specific endonucleases, and correction for incor- poration due to fragmentation rather than endonuclease digestion.


This method corrects for the effects of DNA


fragmentation in the calculation of global methylation levels quantified by LUMA and yields more accurate data. Vol. 58 | No. 5 | 2015


262 www.BioTechniques.com


is involved in transcriptional regulation, genomic


imprinting, and genomic


stability (1). Changes in DNA methyl- ation levels both throughout the genome and in specific genes have been impli-


, Skirmantas Kriaucionis3 , John J. Dunn4¥ ,


cated in multiple disorders, especially cancer (2). Several


techniques have


been used to determine global DNA methylation levels, including HPLC (3), LINE repetitive element methylation (4) and restriction-based techniques such as the cytosine extension assay (5,6). The LUminometric Methylation


Assay (LUMA) (7) is a method for deter- mining global DNA methylation that involves the digestion of DNA with the restriction endonucleases HpaII and MspI, which are isoschizomers that are methylation sensitive and insensitive, respectively. Both cleave at CCGG sites and leave 5´ GC overhangs. HpaII is unable to cleave when the internal C is methylated, while MspI cleaves independent of methylation. Genomic DNA is digested separately with these enzymes, and the number of 5´ GC overhangs is quantified after the incor- poration of nucleotides during pyrose- quencing. In order to control for the amount


of DNA in each digestion, another restriction endonuclease, EcoRI, which cleaves at GAATTC sites leaving 5´ TTAA overhangs, is used. Double digestions (EcoRI+HpaII and EcoRI+MspI) are performed, and nucleotide incorpo- ration is quantified. The HpaII/MspI ratio is calculated as HpaII/EcoRI divided by MspI/EcoRI for each sample. LUMA assumes that methylation at the internal cytosine in the CCGG site is represen- tative of global methylation levels such that, as the HpaII/MspI ratio varies from 0 to 1, the global methylation level varies from 100 to 0%. LUMA is a powerful tool for estimating global methylation levels. It is easy, rapid, and requires small amounts of DNA (250–500 ng). Since the method relies on the


quantification of nucleotide incorpo- ration into the overhangs generated by the restriction enzymes, it is important that the starting DNA is intact and that all of the overhangs being quantified are solely the result of enzyme digestion. Any signal due to fragmentation of the DNA will influence both the HpaII and


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68