Using a Medium-throughput Comet Assay to Evaluate the Global DNA Methylation Status of Single Cells

Overview

Journal Front Genet

Date 2014 Jul 30

PMID 25071840

Citations 5

Authors

Angelique Lewies

Etresia van Dyk

Johannes F Wentzel

Pieter J Pretorius

Affiliations

Soon will be listed here.

Abstract

The comet assay is a simple and cost effective technique, commonly used to analyze and quantify DNA damage in individual cells. The versatility of the comet assay allows introduction of various modifications to the basic technique. The difference in the methylation sensitivity of the isoschizomeric restriction enzymes HpaII and MspI are used to demonstrate the ability of the comet assay to measure the global DNA methylation level of individual cells when using cell cultures. In the experiments described here, a medium-throughput comet assay and methylation sensitive comet assay are combined to produce a methylation sensitive medium-throughput comet assay to measure changes in the global DNA methylation pattern in individual cells under various growth conditions.

Citing Articles

Measuring DNA modifications with the comet assay: a compendium of protocols.

Collins A, Moller P, Gajski G, Vodenkova S, Abdulwahed A, Anderson D Nat Protoc. 2023; 18(3):929-989.

PMID: 36707722 PMC: 10281087. DOI: 10.1038/s41596-022-00754-y.

Bi-directional effects of vitamin B and methotrexate on Daphnia magna fitness and genomic methylation.

Kusari F, ODoherty A, Hodges N, Wojewodzic M Sci Rep. 2017; 7(1):11872.

PMID: 28928387 PMC: 5605502. DOI: 10.1038/s41598-017-12148-2.

The development and validation of EpiComet-Chip, a modified high-throughput comet assay for the assessment of DNA methylation status.

Townsend T, Parrish M, Engelward B, Manjanatha M Environ Mol Mutagen. 2017; 58(7):508-521.

PMID: 28755435 PMC: 5839338. DOI: 10.1002/em.22101.

The comet assay: past, present, and future.

Langie S, Azqueta A, Collins A Front Genet. 2015; 6:266.

PMID: 26322077 PMC: 4534839. DOI: 10.3389/fgene.2015.00266.

Estrogen Receptor β Agonists Differentially Affect the Growth of Human Melanoma Cell Lines.

Marzagalli M, Casati L, Moretti R, Montagnani Marelli M, Limonta P PLoS One. 2015; 10(7):e0134396.

PMID: 26225426 PMC: 4520550. DOI: 10.1371/journal.pone.0134396.

References

Espada J, Esteller M . DNA methylation and the functional organization of the nuclear compartment. Semin Cell Dev Biol. 2009; 21(2):238-46. DOI: 10.1016/j.semcdb.2009.10.006. View

Brooks J, Cairns P, Shore R, Klein C, Wirgin I, Afanasyeva Y . DNA methylation in pre-diagnostic serum samples of breast cancer cases: results of a nested case-control study. Cancer Epidemiol. 2010; 34(6):717-23. PMC: 2956002. DOI: 10.1016/j.canep.2010.05.006. View

Azqueta A, Gutzkow K, Priestley C, Meier S, Walker J, Brunborg G . A comparative performance test of standard, medium- and high-throughput comet assays. Toxicol In Vitro. 2012; 27(2):768-73. DOI: 10.1016/j.tiv.2012.12.006. View

Nag R, Smerdon M . Altering the chromatin landscape for nucleotide excision repair. Mutat Res. 2009; 682(1):13-20. DOI: 10.1016/j.mrrev.2009.01.002. View

Collins A . Investigating oxidative DNA damage and its repair using the comet assay. Mutat Res. 2007; 681(1):24-32. DOI: 10.1016/j.mrrev.2007.10.002. View

Fairbairn D, Olive P, ONeill K . The comet assay: a comprehensive review. Mutat Res. 1995; 339(1):37-59. DOI: 10.1016/0165-1110(94)00013-3. View

van Dyk E, Steenkamp A, Koekemoer G, Pretorius P . Hereditary tyrosinemia type 1 metabolites impair DNA excision repair pathways. Biochem Biophys Res Commun. 2010; 401(1):32-6. DOI: 10.1016/j.bbrc.2010.09.002. View

Walsh C, Xu G . Cytosine methylation and DNA repair. Curr Top Microbiol Immunol. 2006; 301:283-315. DOI: 10.1007/3-540-31390-7_11. View

Shen L, Waterland R . Methods of DNA methylation analysis. Curr Opin Clin Nutr Metab Care. 2007; 10(5):576-81. DOI: 10.1097/MCO.0b013e3282bf6f43. View

10.

Singh N, McCoy M, Tice R, Schneider E . A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988; 175(1):184-91. DOI: 10.1016/0014-4827(88)90265-0. View

11.

Dusinska M, Collins A . The comet assay in human biomonitoring: gene-environment interactions. Mutagenesis. 2008; 23(3):191-205. DOI: 10.1093/mutage/gen007. View

12.

Collins A, Dusinska M, Horvathova E, Munro E, Savio M, Stetina R . Inter-individual differences in repair of DNA base oxidation, measured in vitro with the comet assay. Mutagenesis. 2001; 16(4):297-301. DOI: 10.1093/mutage/16.4.297. View

13.

Tice R, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H . Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen. 2000; 35(3):206-21. DOI: 10.1002/(sici)1098-2280(2000)35:3<206::aid-em8>3.0.co;2-j. View

14.

Andersson M, Hellman B . Different roles of Fpg and Endo III on catechol-induced DNA damage in extended-term cultures of human lymphocytes and L5178Y mouse lymphoma cells. Toxicol In Vitro. 2005; 19(6):779-86. DOI: 10.1016/j.tiv.2005.04.011. View

15.

Bird A . DNA methylation patterns and epigenetic memory. Genes Dev. 2002; 16(1):6-21. DOI: 10.1101/gad.947102. View

16.

Epe B, Pflaum M, Haring M, Hegler J, Rudiger H . Use of repair endonucleases to characterize DNA damage induced by reactive oxygen species in cellular and cell-free systems. Toxicol Lett. 1993; 67(1-3):57-72. DOI: 10.1016/0378-4274(93)90046-z. View

17.

Lisanti S, Omar W, Tomaszewski B, De Prins S, Jacobs G, Koppen G . Comparison of methods for quantification of global DNA methylation in human cells and tissues. PLoS One. 2013; 8(11):e79044. PMC: 3832524. DOI: 10.1371/journal.pone.0079044. View

18.

Collins A, Gaivao I . DNA base excision repair as a biomarker in molecular epidemiology studies. Mol Aspects Med. 2007; 28(3-4):307-22. DOI: 10.1016/j.mam.2007.05.005. View

19.

Calini V, Urani C, Camatini M . Comet assay evaluation of DNA single- and double-strand breaks induction and repair in C3H10T1/2 cells. Cell Biol Toxicol. 2002; 18(6):369-79. DOI: 10.1023/a:1020811522100. View

20.

Wentzel J, Gouws C, Huysamen C, van Dyk E, Koekemoer G, Pretorius P . Assessing the DNA methylation status of single cells with the comet assay. Anal Biochem. 2010; 400(2):190-4. DOI: 10.1016/j.ab.2010.02.008. View