Bisulfite Pyrosequencing

  • Christopher F. Bassil
  • Zhiqing Huang
  • Susan K. Murphy
Part of the Methods in Molecular Biology book series (MIMB, volume 1049)


Bisulfite pyrosequencing is a sequencing-by-synthesis method used to quantitatively determine the methylation of individual CG cytosines from PCR amplicons of a region up to 115 bases in length. The procedure relies on prior bisulfite conversion of all potentially methylated CG cytosines to either cytosine (methylated) or thymine (unmethylated) and involves the stepwise incorporation of deoxynucleotide triphosphates into the growing strand of nascent DNA. The incorporation of these dNTPs results in the proportional release of pyrophosphate, which is converted into ATP to aid in a subsequent conversion of luciferin to oxyluciferin. The amount of light released in the process is proportional to the number of nucleotides incorporated, and the procedure provides a quantitative portrait of the methylation profile for the amplicon in question.

Key words

PCR Pyrosequencing Sequencing-by-synthesis Methylation Epigenetics 



We gratefully acknowledge Zack Davenport for his contributions to the artwork. We thank Allison Barratt and Carole Grenier for critical reading of the manuscript. This work was supported by Department of Defense grant W81XWH-11-1-0469, NIH grants R01DK085173 and R01CA142983, and the Gail Parkins Ovarian Cancer Awareness Fund.


  1. 1.
    Shen L, Guo Y, Chen X, Ahmed S, Issa JP (2007) Optimizing annealing temperature overcomes bias in bisulfite PCR methylation analysis. Biotechniques 42:48–58Google Scholar
  2. 2.
    Murphy SK, Huang Z, Hoyo C (2012) Differentially methylated regions of imprinted genes in prenatal, perinatal and postnatal human tissues. PLoS One 7:e40924Google Scholar
  3. 3.
    Coley HM, Safuwan NA, Chivers P, Papacharalbous E, Giannopoulos T et al (2012) The cyclin-dependent kinase inhibitor p57(Kip2) is epigenetically regulated in carboplatin resistance and results in collateral sensitivity to the CDK inhibitor seliciclib in ovarian cancer. Br J Cancer 106:482–489PubMedCrossRefGoogle Scholar
  4. 4.
    Feng W, Marquez RT, Lu Z, Liu J, Lu KH et al (2008) Imprinted tumor suppressor genes ARHI and PEG3 are the most frequently down-regulated in human ovarian cancers by loss of heterozygosity and promoter methylation. Cancer 112:1489–1502PubMedCrossRefGoogle Scholar
  5. 5.
    Mirabello L, Sun C, Ghosh A, Rodriguez AC, Schiffman M et al (2012) Methylation of human papillomavirus type 16 genome and risk of cervical precancer in a Costa Rican population. J Natl Cancer Inst 104:556–565PubMedCrossRefGoogle Scholar
  6. 6.
    Lof-Ohlin ZM, Levanat S, Sabol M, Sorbe B, Nilsson TK (2011) Promoter methylation in the PTCH gene in cervical epithelial cancer and ovarian cancer tissue as studied by eight novel Pyrosequencing® assays. Int J Oncol 38:685–692PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Christopher F. Bassil
    • 1
  • Zhiqing Huang
    • 1
  • Susan K. Murphy
    • 1
  1. 1.Division of Gynecologic OncologyDuke University Medical CenterDurhamUSA

Personalised recommendations