Advertisement

Pyrosequencing pp 291-313 | Cite as

SNP-Based Quantification of Allele-Specific DNA Methylation Patterns by Pyrosequencing®

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1315)

Abstract

The analysis of allele-specific DNA methylation patterns has recently attracted much interest as loci of allele-specific DNA methylation overlap with known risk loci for complex diseases and the analysis might contribute to the fine-mapping and interpretation of non-coding genetic variants associated with complex diseases and improve the understanding between genotype and phenotype. In the presented protocol, we present a method for the analysis of DNA methylation patterns on both alleles separately using heterozygous Single Nucleotide Polymorphisms (SNPs) as anchor for allele-specific PCR amplification followed by analysis of the allele-specific DNA methylation patterns by Pyrosequencing®. Pyrosequencing is an easy-to-handle, quantitative real-time sequencing method that is frequently used for genotyping as well as for the analysis of DNA methylation patterns. The protocol consists of three major steps: (1) identification of individuals heterozygous for a SNP in a region of interest using Pyrosequencing; (2) analysis of the DNA methylation patterns surrounding the SNP on bisulfite-treated DNA to identify regions of potential allele-specific DNA methylation; and (3) the analysis of the DNA methylation patterns associated with each of the two alleles, which are individually amplified using allele-specific PCR. The enrichment of the targeted allele is re-enforced by modification of the allele-specific primers at the allele-discriminating base with Locked Nucleic Acids (LNA). For the proof-of-principle of the developed approach, we provide assay details for three imprinted genes (IGF2, IGF2R, and PEG3) within this chapter. The mean of the DNA methylation patterns derived from the individual alleles corresponds well to the overall DNA methylation patterns and the developed approach proved more reliable compared to other protocols for allele-specific DNA methylation analysis.

Key words

Pyrosequencing® Allele-specific PCR Bisulfite SNP, locked nucleic acid 
This is a preview of subscription content, log in to check access.

References

  1. 1.
    Tost J (2010) DNA methylation: an introduction to the biology and the disease-associated changes of a promising biomarker. Mol Biotechnol 44:71–81PubMedCrossRefGoogle Scholar
  2. 2.
    Kerkel K, Spadola A, Yuan E et al (2008) Genomic surveys by methylation-sensitive SNP analysis identify sequence-dependent allele-specific DNA methylation. Nat Genet 40:904–908PubMedCrossRefGoogle Scholar
  3. 3.
    Schalkwyk LC, Meaburn EL, Smith R et al (2010) Allelic skewing of DNA methylation is widespread across the genome. Am J Hum Genet 86:196–212PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Hutchinson JN, Raj T, Fagerness J et al (2014) Allele-specific methylation occurs at genetic variants associated with complex disease. PLoS One 9:e98464PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Ronaghi M, Uhlen M, Nyren P (1998) A sequencing method based on real-time pyrophosphate. Science 281(363):365Google Scholar
  6. 6.
    Harrington CT, Lin EI, Olson MT et al (2013) Fundamentals of pyrosequencing. Arch Pathol Lab Med 137:1296–1303PubMedCrossRefGoogle Scholar
  7. 7.
    Langaee T, Ronaghi M (2005) Genetic variation analyses by pyrosequencing. Mutat Res 573:96–102PubMedCrossRefGoogle Scholar
  8. 8.
    Tost J, Dunker J, Gut IG (2003) Analysis and quantification of multiple methylation variable positions in CpG islands by pyrosequencing. Biotechniques 35:152–156PubMedGoogle Scholar
  9. 9.
    Dupont JM, Tost J, Jammes H et al (2004) De novo quantitative bisulfite sequencing using the pyrosequencing technology. Anal Biochem 333:119–127PubMedCrossRefGoogle Scholar
  10. 10.
    Tost J, Gut IG (2007) DNA methylation analysis by pyrosequencing. Nat Protoc 2:2265–2275PubMedCrossRefGoogle Scholar
  11. 11.
    Ronaghi M (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res 11:3–11PubMedCrossRefGoogle Scholar
  12. 12.
    Ahmadian A, Ehn M, Hober S (2006) Pyrosequencing: history, biochemistry and future. Clin Chim Acta 363:83–94PubMedCrossRefGoogle Scholar
  13. 13.
    Ogino S, Kawasaki T, Brahmandam M et al (2005) Sensitive sequencing method for KRAS mutation detection by pyrosequencing. J Mol Diagn 7:413–421PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Lundin KE, Hojland T, Hansen BR et al (2013) Biological activity and biotechnological aspects of locked nucleic acids. Adv Genet 82:47–107PubMedGoogle Scholar
  15. 15.
    How Kit A, Mazaleyrat N, Daunay A et al (2013) Sensitive detection of KRAS mutations using enhanced-ice-COLD-PCR mutation enrichment and direct sequence identification. Hum Mutat 34:1568–1580PubMedCrossRefGoogle Scholar
  16. 16.
    Peters J (2014) The role of genomic imprinting in biology and disease: an expanding view. Nat Rev Genet 15:517–530PubMedCrossRefGoogle Scholar
  17. 17.
    Wong HL, Byun HM, Kwan JM et al (2006) Rapid and quantitative method of allele-specific DNA methylation analysis. Biotechniques 41:734–739PubMedCrossRefGoogle Scholar
  18. 18.
    Shaw RJ, Akufo-Tetteh EK, Risk JM et al (2006) Methylation enrichment pyrosequencing: combining the specificity of MSP with validation by pyrosequencing. Nucleic Acids Res 34:e78PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Koressaar T, Remm M (2007) Enhancements and modifications of primer design program Primer3. Bioinformatics 23:1289–1291PubMedCrossRefGoogle Scholar
  20. 20.
    Untergasser A, Cutcutache I, Koressaar T et al (2012) Primer3: new capabilities and interfaces. Nucleic Acids Res 40:e115PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Li LC, Dahiya R (2002) MethPrimer: designing primers for methylation PCRs. Bioinformatics 18:1427–1431PubMedCrossRefGoogle Scholar
  22. 22.
    Zhou GH, Gotou M, Kajiyama T et al (2005) Multiplex SNP typing by bioluminometric assay coupled with terminator incorporation (BATI). Nucleic Acids Res 33:e133PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Aranyi T, Varadi A, Simon I et al (2006) The BiSearch web server. BMC Bioinformatics 7:431PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Frommer M, McDonald LE, Millar DS 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 89:1827–1831PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Campan M, Weisenberger DJ, Trinh B et al (2009) MethyLight. Methods Mol Biol 507:325–337PubMedGoogle Scholar
  26. 26.
    Okimoto R, Dodgson JB (1996) Improved PCR amplification of multiple specific alleles (PAMSA) using internally mismatched primers. Biotechniques 21:20–22PubMedGoogle Scholar
  27. 27.
    Tost J, El abdalaoui H, Gut IG (2006) Serial pyrosequencing for quantitative DNA methylation analysis. Biotechniques 40:721–726PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Laboratory for Epigenetics and EnvironmentCentre National de Génotypage, CEA-Institut de GénomiqueEvryFrance
  2. 2.Laboratory for Epigenetics and EnvironmentCentre National de Génotypage, CEA-Institut de GénomiqueEvryFrance

Personalised recommendations

Cite protocol

Buy options