Microwave Assisted DNA Hydrolysis for Global Methylation Analysis by Gas Chromatography/Tandem Mass Spectrometry
Keywords:DNA methylation, microwave assisted hydrolysis, nucleobases, derivatization, gas chromatography/tandem mass spectrometry, multiple reaction monitoring
The analysis of the global DNA methylation, calculated as the percentage of 5-methylcytosine (5mC) over the total sum of cytosines, is a well stablished biomarker for monitoring large scale epigenetic events in organisms. DNA purification, hydrolysis, separation and detection methods are critical steps to determine this biomarker. In the present work is proposed a robust procedure for DNA acid-hydrolysis assisted by microwave that provides identical DNA methylation patterns that enzymatic hydrolysis and better release of 5mC than acid classic method. The quantification was performed using a gas chromatographer coupled to a mass spectrometer with triple quadrupole as mass analyzer (GC-TQ-MS/MS) using multiple reaction monitoring (MRM) mode for the trimethylsilyl-derivates of nucleobases; following the transitions of 254→238, 240→170 and 254→238, 254→184 (m/z) for C and 5mC respectively, was achieved a limit of detection of 0.46 fmol for C and 0.41 fmol for 5mC. The proposed procedure is capable of determine 0.004% of 5mC in 50 ng of DNA in a chromatographic time of 10 minutes, being a good alternative to LC-MS/MS analysis.
Cantone I, Fisher AG: Epigenetic programming and reprogramming during development. Nature structural & molecular biology 2013, 20(3):282-289.
Jeon J, Choi J, Lee G-W, Park S-Y, Huh A, Dean RA, Lee Y-H: Genome-wide profiling of DNA methylation provides insights into epigenetic regulation of fungal development in a plant pathogenic fungus, Magnaporthe oryzae. Scientific reports 2015, 5.
Feil R, Fraga MF: Epigenetics and the environment: emerging patterns and implications. Nature reviews Genetics 2012, 13(2):97.
Terry MB, Delgado-Cruzata L, Vin-Raviv N, Wu HC, Santella RM: DNA methylation in white blood cells: association with risk factors in epidemiologic studies. Epigenetics 2011, 6(7):828-837.
Maldonado Santoyo M, Rodriguez Flores C, Lopez Torres A, Wrobel K, Wrobel K: Global DNA methylation in earthworms: A candidate biomarker of epigenetic risks related to the presence of metals/metalloids in terrestrial environments. Environmental pollution 2011, 159(10):2387-2392.
Lam K, Pan K, Linnekamp JF, Medema JP, Kandimalla R: DNA methylation based biomarkers in colorectal cancer: A systematic review. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer 2016, 1866(1):106-120.
Rabinowicz PD, Schutz K, Dedhia N, Yordan C, Parnell LD, Stein L, McCombie WR, Martienssen RA: Differential methylation of genes and retrotransposons facilitates shotgun sequencing of the maize genome. Nature genetics 1999, 23(3).
Colot V, Rossignol JL: Eukaryotic DNA methylation as an evolutionary device. Bioessays 1999, 21(5):402-411.
Tang Y, Gao X-D, Wang Y, Yuan B-F, Feng Y-Q: Widespread existence of cytosine methylation in yeast DNA measured by gas chromatography/mass spectrometry. Analytical chemistry 2012, 84(16):7249-7255.
Bestor TH: The DNA methyltransferases of mammals. Human molecular genetics 2000, 9(16):2395-2402.
Ponger L, Li W-H: Evolutionary diversification of DNA methyltransferases in eukaryotic genomes. Molecular biology and Evolution 2005, 22(4):1119-1128.
Russell PJ, Welsch J, Rachlin E, McCloskey J: Different levels of DNA methylation in yeast and mycelial forms of Candida albicans. Journal of bacteriology 1987, 169(9):4393-4395.
Lisanti S, Omar WA, Tomaszewski B, De Prins S, Jacobs G, Koppen G, Mathers JC, Langie SA: Comparison of methods for quantification of global DNA methylation in human cells and tissues. PLoS One 2013, 8(11):e79044.
Liu J, Hesson L, Ward R: Liquid chromatography tandem mass spectrometry for the measurement of global DNA methylation and hydroxymethylation. J Proteomics Bioinform S 2013, 2:5-10.
Marrubini G, Fattorini P, Previderé C, Goi S, Cigliero SS, Grignani P, Serra M, Biesuz R, Massolini G: Experimental design applied to the optimization of microwave-assisted DNA hydrolysis. Journal of Chromatography A 2012, 1249:8-16.
Zhang JJ, Zhang L, Zhou K, Ye X, Liu C, Zhang L, Kang J, Cai C: Analysis of global DNA methylation by hydrophilic interaction ultra high-pressure liquid chromatography tandem mass spectrometry. Analytical biochemistry 2011, 413(2):164-170.
Rossella F, Polledri E, Bollati V, Baccarelli A, Fustinoni S: Development and validation of a gas chromatography/mass spectrometry method for the assessment of genomic DNA methylation. Rapid Communications in Mass Spectrometry 2009, 23(17):2637-2646.
Singer J, Schnute WC, Shively JE, Todd CW, Riggs AD: Sensitive detection of 5-methylcytosine and quantitation of the 5-methylcytosine/cytosine ratio in DNA by gas chromatography-mass spectrometry using multiple specific ion monitoring. Analytical biochemistry 1979, 94(2):297-301.
San Romerio A, Fiorillo G, Terruzzi I, Senesi P, Testolin G, Battezzati A: Measurement of DNA methylation using stable isotope dilution and gas chromatography-mass spectrometry. Analytical biochemistry 2005, 336(2):158-163.
Crain PF, McCloskey JA: Analysis of modified bases in DNA by stable isotope dilution gas chromatography-mass spectrometry: 5-methylcytosine. Analytical biochemistry 1983, 132(1):124-131.
Wang X, Suo Y, Yin R, Shen H, Wang H: Ultra-performance liquid chromatography/tandem mass spectrometry for accurate quantification of global DNA methylation in human sperms. Journal of chromatography B, Analytical technologies in the biomedical and life sciences 2011, 879(19):1647-1652.
Leghissa A, Hildenbrand ZL, Foss FW, Schug KA: Determination of cannabinoids from a surrogate hops matrix using multiple reaction monitoring gas chromatography with triple quadrupole mass spectrometry. Journal of separation science 2017.
Lopez-Torres A, Yanez-Barrientos E, Wrobel K, Wrobel K: Selective derivatization of cytosine and methylcytosine moieties with 2-bromoacetophenone for submicrogram DNA methylation analysis by reversed phase HPLC with spectrofluorimetric detection. Anal Chem 2011, 83(20):7999-8005.
Aljanabi SM, Martinez I: Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleic acids research 1997, 25(22):4692-4693.
Yanez Barrientos E, Wrobel K, Lopez Torres A, Gutierrez-Corona F, Wrobel K: Application of reversed-phase high-performance liquid chromatography with fluorimetric detection for simultaneous assessment of global DNA and total RNA methylation in Lepidium sativum: effect of plant exposure to Cd (II) and Se (IV). Analytical and bioanalytical chemistry 2013, 405(7):2397-2404.
Wieslander L: A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels. Analytical biochemistry 1979, 98(2):305-309.
Le T, Kim K-P, Fan G, Faull KF: A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samples. Analytical biochemistry 2011, 412(2):203-209.
Capuano F, Mu?lleder M, Kok R, Blom HJ, Ralser M: Cytosine DNA methylation is found in Drosophila melanogaster but absent in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and other yeast species. Analytical chemistry 2014, 86(8):3697-3702.
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