Assessing the DNA methylation status of single cells with the comet assay.
The comet assay (single cell gel electrophoresis) is a cost-effective, sensitive, and simple technique that is traditionally used for analyzing and quantifying DNA damage in individual cells. The aim of this study was to determine whether the comet assay could be modified to detect changes in the levels of DNA methylation in single cells. We used the difference in methylation sensitivity of the isoschizomeric restriction endonucleases HpaII and MspI to demonstrate the feasibility of the comet assay to measure the global DNA methylation level of individual cells. The results were verified with the well-established cytosine extension assay. We were able to show variations in DNA methylation after treatment of cultured cells with 5-azacytidine and succinylacetone, an accumulating metabolite in human tyrosinemia type I.
Wentzel
Johannes F
JF
Division for Biochemistry, School of Physical and Chemical Sciences, North-West University, Potchefstroom 2520, South Africa. 20134045@puknet.ac.za
Gouws
Chrisna
C
Huysamen
Cristal
C
Dyk
Etresia van
Ev
Koekemoer
Gerhard
G
Pretorius
Pieter J
PJ
D001374:Azacitidine / Q000737:chemistry / Q000494:pharmacology; D020552:Comet Assay / Q000379:methods*; D003596:Cytosine / Q000378:metabolism; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D019003:Deoxyribonuclease HpaII / Q000378:metabolism; D056945:Hep G2 Cells; D006537:Heptanoates / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D020176:Tyrosinemias / Q000378:metabolism
D006537:Heptanoates; C020804:succinylacetone; D003596:Cytosine; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases; D019003:Deoxyribonuclease HpaII; D001374:Azacitidine