Abstract
DNA methylation is an important enzymatic covalent modification of DNA that plays an important role in genome regulation. DNA methylation patterns are fashioned during development and could be altered in response to experience and exposure. Aberrations in DNA methylation patterns are noted in cancer and other diseases. It is therefore extremely important to accurately quantify DNA methylation states for studying physiology and disease as well as for using DNA methylation markers in diagnosis. Here, we review the most commonly used methods for quantifying DNA methylation states of single genes: Pyrosequencing, Quantitative Methylated DNA Immunoprecipitation (qMeDIP), and methylation-sensitive high resolution melting (MS-HRM). Each method is described and required steps are detailed. We also discuss the advantages and disadvantages of the different methods.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
Abbreviations
- dsDNA:
-
double-stranded DNA
- FFPE :
-
Formalin-fixed paraffin-embedded tissue
- GWAS:
-
Genome-Wide Association Study
- MeDIP:
-
methylated DNA immunoprecipitation
- RRBS:
-
Reduced representation bisulfite sequencing
References
Ronaghi M et al (1998) PCR-introduced loop structure as primer in DNA sequencing. Biotechniques 25(5):876, -8, 880-2, 884
Worm J, Aggerholm A, Guldberg P (2001) In-tube DNA methylation profiling by fluorescence melting curve analysis. Clin Chem 47(7):1183–1189
Xiong Z, Laird PW (1997) COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res 25(12):2532–2534
Ronaghi M, Uhlen M, Nyren P (1998) A sequencing method based on real-time pyrophosphate. Science 281(5375):363, 365
Langaee T, Ronaghi M (2005) Genetic variation analyses by Pyrosequencing. Mutat Res 573(1-2):96–102
Ogino S et al (2005) Sensitive sequencing method for KRAS mutation detection by Pyrosequencing. J Mol Diagn 7(3):413–421
Ronaghi M (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res 11(1):3–11
Sanger F, Coulson AR (1975) A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol 94(3):441–448
Petropoulos S, Matthews SG, Szyf M (2014) Adult glucocorticoid exposure leads to transcriptional and DNA methylation changes in nuclear steroid receptors in the hippocampus and kidney of mouse male offspring. Biol Reprod 90(2):43
Kirby KS (1956) A new method for the isolation of ribonucleic acids from mammalian tissues. Biochem J 64(3):405–408
Ebeling W et al (1974) Proteinase K from Tritirachium album Limber. Eur J Biochem 47(1):91–97
Manchester KL (1996) Use of UV methods for measurement of protein and nucleic acid concentrations. Biotechniques 20(6):968–970
Glasel JA (1995) Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios. Biotechniques 18(1):62–63
Huberman JA (1995) Importance of measuring nucleic acid absorbance at 240 nm as well as at 260 and 280 nm. Biotechniques 18(4):636
Manchester KL (1995) Value of A260/A280 ratios for measurement of purity of nucleic acids. Biotechniques 19(2):208–210
Hayatsu H et al (1970) Reaction of sodium bisulfite with uracil, cytosine, and their derivatives. Biochemistry 9(14):2858–2865
Robert Shapiro RES, Welcher M (1970) Reactions of uracil and cytosine derivatives with sodium bisulfite. J Am Chem Soc 92(2):422–424
Frommer M et al (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A 89(5):1827–1831
Warnecke PM et al (1997) Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA. Nucleic Acids Res 25(21):4422–4426
Holmes EE et al (2014) Performance evaluation of kits for bisulfite-conversion of DNA from tissues, cell lines, FFPE tissues, aspirates, lavages, effusions, plasma, serum, and urine. PLoS One 9(4):e93933
Leontiou CA et al (2015) Bisulfite conversion of DNA: performance comparison of different kits and methylation quantitation of epigenetic biomarkers that have the potential to be used in non-invasive prenatal testing. PLoS One 10(8):e0135058
Tost J, Gut IG (2007) DNA methylation analysis by pyrosequencing. Nat Protoc 2(9):2265–2275
Li LC, Dahiya R (2002) MethPrimer: designing primers for methylation PCRs. Bioinformatics 18(11):1427–1431
Aranyi T et al (2006) The BiSearch web server. BMC Bioinformatics 7:431
Kampke T, Kieninger M, Mecklenburg M (2001) Efficient primer design algorithms. Bioinformatics 17(3):214–225
Shen L, et al. (2007) Optimizing annealing temperature overcomes bias in bisulfite PCR methylation analysis. Biotechniques 42(1): 48, 50, 52 passim
Korbie DJ, Mattick JS (2008) Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nat Protoc 3(9):1452–1456
Tost J, El abdalaoui H, Gut IG (2006) Serial pyrosequencing for quantitative DNA methylation analysis. Biotechniques 40(6): 721–722, 724, 726
Weber M et al (2005) Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet 37(8):853–862
Jin SG, Kadam S, Pfeifer GP (2010) Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine. Nucleic Acids Res 38(11):e125
Bustin SA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4):611–622
Wrobel G, Kokocinski F, Lichter P (2004) AutoPrime: selecting primers for expressed sequences. Genome Biol 5(5):P11
Petropoulos S et al (2015) Gestational diabetes alters offspring DNA methylation profiles in human and rat: identification of key pathways involved in endocrine system disorders, insulin signaling, diabetes signaling, and ILK signaling. Endocrinology 156(6):2222–2238
Labonte B et al (2012) Genome-wide epigenetic regulation by early-life trauma. Arch Gen Psychiatry 69(7):722–731
Lisanti S, von Zglinicki T, Mathers JC (2012) Standardization and quality controls for the methylated DNA immunoprecipitation technique. Epigenetics 7(6):615–625
Wittwer CT et al (1997) The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. Biotechniques 22(1):176–181
Wojdacz TK, Dobrovic A (2007) Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation. Nucleic Acids Res 35(6), e41
Wojdacz TK, Hansen LL (2006) Reversal of PCR bias for improved sensitivity of the DNA methylation melting curve assay. Biotechniques 41(3):274, 276, 278
Wojdacz TK et al (2010) Limitations and advantages of MS-HRM and bisulfite sequencing for single locus methylation studies. Expert Rev Mol Diagn 10(5):575–580
Rubatino FV et al (2015) Manipulation of primer affinity improves high-resolution melting accuracy for imprinted genes. Genet Mol Res 14(3):7864–7872
Wojdacz TK, Dobrovic A, Hansen LL (2008) Methylation-sensitive high-resolution melting. Nat Protoc 3(12):1903–1908
Wittwer CT et al (2003) High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49(6 Pt 1):853–860
Monis PT, Giglio S, Saint CP (2005) Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis. Anal Biochem 340(1):24–34
Radvanszky J et al (2015) Comparison of different DNA binding fluorescent dyes for applications of high-resolution melting analysis. Clin Biochem 48(9):609–616
Candiloro IL et al (2008) Rapid analysis of heterogeneously methylated DNA using digital methylation-sensitive high resolution melting: application to the CDKN2B (p15) gene. Epigenetics Chromatin 1(1):7
Candiloro IL, Mikeska T, Dobrovic A (2011) Assessing combined methylation-sensitive high resolution melting and pyrosequencing for the analysis of heterogeneous DNA methylation. Epigenetics 6(4):500–507
Ronaghi M et al (1996) Real-time DNA sequencing using detection of pyrophosphate release. Anal Biochem 242(1):84–89
Amornpisutt R, Sriraksa R, Limpaiboon T (2012) Validation of methylation-sensitive high resolution melting for the detection of DNA methylation in cholangiocarcinoma. Clin Biochem 45(13–14):1092–1094
Migheli F et al (2013) Comparison study of MS-HRM and pyrosequencing techniques for quantification of APC and CDKN2A gene methylation. PLoS One 8(1):e52501
Acknowledgments
D.C. is supported by fellowship from the Israel Cancer Research Foundation. S.P. is supported by the Mats Sundin Fellowship in Developmental Health.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Cheishvili, D., Petropoulos, S., Christiansen, S., Szyf, M. (2017). Targeted DNA Methylation Analysis Methods. In: Stefanska, B., MacEwan, D. (eds) Epigenetics and Gene Expression in Cancer, Inflammatory and Immune Diseases. Methods in Pharmacology and Toxicology. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6743-8_3
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6743-8_3
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6741-4
Online ISBN: 978-1-4939-6743-8
eBook Packages: Springer Protocols