Advertisement

The Design and Optimization of DNA Methylation Pyrosequencing Assays Targeting Region-Specific Repeat Elements

  • Gwen Hoad
  • Kristina HarrisonEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1589)

Abstract

Epigenetic modifications, such as DNA methylation, can contribute to gene regulation and chromosomal stability. There are several methods and techniques available for methylation analysis, ranging from global methylation to gene-specific targeted regions. Bisulfite conversion enables numerous methodologies to be used for downstream applications, including pyrosequencing which measures DNA methylation at an individual CpG site level. This allows specific regions of interest to be targeted for DNA methylation analysis. Designing and optimizing pyrosequencing assays correctly is vital for the interpretation of results.

Dysregulation of DNA methylation has been implicated in human diseases, with regions such as repeat elements commonly altered. Human population studies investigating these tend to use consensus sequences to target repeat elements. However, these elements have high mutational rates, particularly Alu sequences, which could lead to assay bias and masking of changes at a regional level. Therefore, it may be more beneficial to target specific repeat elements depending upon their chromosomal location, rather than analyzing overall methylation levels.

Keywords:

DNA methylation Epigenetics Pyrosequencing Bisulfite conversion CpGs Bisulfite sequencing 

References

  1. 1.
    Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, Paul CL (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci 89(5):1827–1831CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Clark SJ, Statham A, Stirzaker C, Molloy PL, Frommer M (2006) DNA methylation: bisulphite modification and analysis. Nat Protoc 1(5):2353–2364CrossRefPubMedGoogle Scholar
  3. 3.
    Ronaghi M, Uhlén M, Nyrén P (1998) A Sequencing Method Based on Real-Time Pyrophosphate. Science 281(5375):363–365CrossRefPubMedGoogle Scholar
  4. 4.
    Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W (2001) Initial sequencing and analysis of the human genome. Nature 409(6822):860–921CrossRefPubMedGoogle Scholar
  5. 5.
    de Koning AJ, Gu W, Castoe TA, Batzer MA, Pollock DD (2011) Repetitive elements may comprise over two-thirds of the human genome. PLoS Genet 7(12), e1002384CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Cordaux R, Batzer MA (2009) The impact of retrotransposons on human genome evolution. Nat Rev Genet 10(10):691–703CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Levin HL, Moran JV (2011) Dynamic interactions between transposable elements and their hosts. Nat Rev Genet 12(9):615–627CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Liu GE, Alkan C, Jiang L, Zhao S, Eichler EE (2009) Comparative analysis of Alu repeats in primate genomes. Genome Res 19(5):876–885CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Natural Products Group, Rowett Institute of Nutrition and HealthUniversity of AberdeenAberdeenUK

Personalised recommendations