Advertisement

Targeted DNA Methylation Analysis Methods

  • David Cheishvili
  • Sophie Petropoulos
  • Steffan Christiansen
  • Moshe Szyf
Protocol
Part of the Methods in Pharmacology and Toxicology book series (MIPT)

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.

Key words

DNA methylation Sodium bisulfite Quantitative Methylated DNA Immunoprecipitation (qMeDIP) Methylation-sensitive high resolution melting (MS-HRM) Pyrosequencing 

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

Notes

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.

References

  1. 1.
    Ronaghi M et al (1998) PCR-introduced loop structure as primer in DNA sequencing. Biotechniques 25(5):876, -8, 880-2, 884PubMedGoogle Scholar
  2. 2.
    Worm J, Aggerholm A, Guldberg P (2001) In-tube DNA methylation profiling by fluorescence melting curve analysis. Clin Chem 47(7):1183–1189PubMedGoogle Scholar
  3. 3.
    Xiong Z, Laird PW (1997) COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res 25(12):2532–2534CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Ronaghi M, Uhlen M, Nyren P (1998) A sequencing method based on real-time pyrophosphate. Science 281(5375):363, 365CrossRefPubMedGoogle Scholar
  5. 5.
    Langaee T, Ronaghi M (2005) Genetic variation analyses by Pyrosequencing. Mutat Res 573(1-2):96–102CrossRefPubMedGoogle Scholar
  6. 6.
    Ogino S et al (2005) Sensitive sequencing method for KRAS mutation detection by Pyrosequencing. J Mol Diagn 7(3):413–421CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Ronaghi M (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res 11(1):3–11CrossRefPubMedGoogle Scholar
  8. 8.
    Sanger F, Coulson AR (1975) A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol 94(3):441–448CrossRefPubMedGoogle Scholar
  9. 9.
    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):43CrossRefPubMedGoogle Scholar
  10. 10.
    Kirby KS (1956) A new method for the isolation of ribonucleic acids from mammalian tissues. Biochem J 64(3):405–408CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ebeling W et al (1974) Proteinase K from Tritirachium album Limber. Eur J Biochem 47(1):91–97CrossRefPubMedGoogle Scholar
  12. 12.
    Manchester KL (1996) Use of UV methods for measurement of protein and nucleic acid concentrations. Biotechniques 20(6):968–970PubMedGoogle Scholar
  13. 13.
    Glasel JA (1995) Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios. Biotechniques 18(1):62–63PubMedGoogle Scholar
  14. 14.
    Huberman JA (1995) Importance of measuring nucleic acid absorbance at 240 nm as well as at 260 and 280 nm. Biotechniques 18(4):636PubMedGoogle Scholar
  15. 15.
    Manchester KL (1995) Value of A260/A280 ratios for measurement of purity of nucleic acids. Biotechniques 19(2):208–210PubMedGoogle Scholar
  16. 16.
    Hayatsu H et al (1970) Reaction of sodium bisulfite with uracil, cytosine, and their derivatives. Biochemistry 9(14):2858–2865CrossRefPubMedGoogle Scholar
  17. 17.
    Robert Shapiro RES, Welcher M (1970) Reactions of uracil and cytosine derivatives with sodium bisulfite. J Am Chem Soc 92(2):422–424CrossRefGoogle Scholar
  18. 18.
    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):1827–1831CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Warnecke PM et al (1997) Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA. Nucleic Acids Res 25(21):4422–4426CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    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):e93933CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    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):e0135058CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Tost J, Gut IG (2007) DNA methylation analysis by pyrosequencing. Nat Protoc 2(9):2265–2275CrossRefPubMedGoogle Scholar
  23. 23.
    Li LC, Dahiya R (2002) MethPrimer: designing primers for methylation PCRs. Bioinformatics 18(11):1427–1431CrossRefPubMedGoogle Scholar
  24. 24.
    Aranyi T et al (2006) The BiSearch web server. BMC Bioinformatics 7:431CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Kampke T, Kieninger M, Mecklenburg M (2001) Efficient primer design algorithms. Bioinformatics 17(3):214–225CrossRefPubMedGoogle Scholar
  26. 26.
    Shen L, et al. (2007) Optimizing annealing temperature overcomes bias in bisulfite PCR methylation analysis. Biotechniques 42(1): 48, 50, 52 passimGoogle Scholar
  27. 27.
    Korbie DJ, Mattick JS (2008) Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nat Protoc 3(9):1452–1456CrossRefPubMedGoogle Scholar
  28. 28.
    Tost J, El abdalaoui H, Gut IG (2006) Serial pyrosequencing for quantitative DNA methylation analysis. Biotechniques 40(6): 721–722, 724, 726Google Scholar
  29. 29.
    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):853–862CrossRefPubMedGoogle Scholar
  30. 30.
    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):e125CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Bustin SA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4):611–622CrossRefPubMedGoogle Scholar
  32. 32.
    Wrobel G, Kokocinski F, Lichter P (2004) AutoPrime: selecting primers for expressed sequences. Genome Biol 5(5):P11CrossRefGoogle Scholar
  33. 33.
    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):2222–2238CrossRefPubMedGoogle Scholar
  34. 34.
    Labonte B et al (2012) Genome-wide epigenetic regulation by early-life trauma. Arch Gen Psychiatry 69(7):722–731CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Lisanti S, von Zglinicki T, Mathers JC (2012) Standardization and quality controls for the methylated DNA immunoprecipitation technique. Epigenetics 7(6):615–625CrossRefPubMedGoogle Scholar
  36. 36.
    Wittwer CT et al (1997) The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. Biotechniques 22(1):176–181PubMedGoogle Scholar
  37. 37.
    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), e41CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Wojdacz TK, Hansen LL (2006) Reversal of PCR bias for improved sensitivity of the DNA methylation melting curve assay. Biotechniques 41(3):274, 276, 278CrossRefPubMedGoogle Scholar
  39. 39.
    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):575–580CrossRefPubMedGoogle Scholar
  40. 40.
    Rubatino FV et al (2015) Manipulation of primer affinity improves high-resolution melting accuracy for imprinted genes. Genet Mol Res 14(3):7864–7872CrossRefPubMedGoogle Scholar
  41. 41.
    Wojdacz TK, Dobrovic A, Hansen LL (2008) Methylation-sensitive high-resolution melting. Nat Protoc 3(12):1903–1908CrossRefPubMedGoogle Scholar
  42. 42.
    Wittwer CT et al (2003) High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49(6 Pt 1):853–860CrossRefPubMedGoogle Scholar
  43. 43.
    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):24–34CrossRefPubMedGoogle Scholar
  44. 44.
    Radvanszky J et al (2015) Comparison of different DNA binding fluorescent dyes for applications of high-resolution melting analysis. Clin Biochem 48(9):609–616CrossRefPubMedGoogle Scholar
  45. 45.
    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):7CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    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):500–507CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Ronaghi M et al (1996) Real-time DNA sequencing using detection of pyrophosphate release. Anal Biochem 242(1):84–89CrossRefPubMedGoogle Scholar
  48. 48.
    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):1092–1094CrossRefPubMedGoogle Scholar
  49. 49.
    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):e52501CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • David Cheishvili
    • 1
  • Sophie Petropoulos
    • 2
  • Steffan Christiansen
    • 3
  • Moshe Szyf
    • 1
  1. 1.Department of Pharmacology and TherapeuticsMcGill University Medical SchoolMontrealCanada
  2. 2.Department of Clinical Science, Intervention and Technology (CLINTEC)Karolinska InstitutetStockholmSweden
  3. 3.Department of BiomedicineAarhus UniversityAarhusDenmark

Personalised recommendations