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The Retinoblastoma Protein pp 111–122Cite as

Detection of p16 Promoter Hypermethylation by Methylation-Specific PCR

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1726))

Abstract

DNA methylation plays a decisive role in the regulation and control of gene expression. DNA methylation is a covalent modification, in which a methyl group is attached to the 5th carbon of the cytosine ring of a CpG dinucleotide that is located upstream from the promoter region of a gene. Promoter hypermethylation (gain of DNA methylation) of the p16 gene may cause silencing of gene expression and plays an important role in cancer. Therefore, detection of the methylation status of p16 gene is an important tool in epigenetic studies of various human cancers. The methylation-specific PCR (MSP) is the most commonly used technique for studying DNA methylation. This technique is based on bisulfite modification of DNA, which converts unmethylated cytosine (C) into uracil (U) and leaving methylated cytosine (Cm) unchanged. Here we describe the bisulfite modification of DNA samples and detection of promoter methylation of p16 gene from bisulfite-treated DNA using MSP. In MSP, modified DNA samples are subjected to PCR amplification using methylated and unmethylated specific primers for the p16 gene separately. The PCR amplified products are then analyzed in a 2.5–3% agarose gel containing ethidium bromide. The PCR amplified band generated by specific sets of primers is used to determine the methylation status of the p16 gene.

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References

  1. Ng HH, Bird A (1999) DNA methylation and chromatin modification. Curr Opin Genet Dev 9(2):158–163

    Article  CAS  PubMed  Google Scholar 

  2. Baylin SB (2005) DNA methylation and gene silencing in cancer. Nat Clin Pract Oncol 2(Suppl 1):S4–11. https://doi.org/10.1038/ncponc0354

    Article  CAS  PubMed  Google Scholar 

  3. Asokan GS, Jeelani S, Gnanasundaram N (2014) Promoter hypermethylation profile of tumour suppressor genes in oral leukoplakia and oral squamous cell carcinoma. J Clin Diagn Res 8(10):ZC09–ZC12. https://doi.org/10.7860/JCDR/2014/9251.4949

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Choudhury J, Ghosh S (2015) Promoter hypermethylation profiling identifies subtypes of head and neck cancer with distinct viral, environmental, genetic and survival characteristics. PLoS One 10(6):e0129808

    Article  PubMed  PubMed Central  Google Scholar 

  5. Toyota M, Ahuja N, Ohe-Toyota M, Herman JG, Baylin SB, Issa JP (1999) CpG island methylator phenotype in colorectal cancer. Proc Natl Acad Sci U S A 96(15):8681–8686

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Fraga MF, Esteller M (2002) DNA methylation: a profile of methods and applications. BioTechniques 33(3):632. 634, 636–649

    CAS  PubMed  Google Scholar 

  7. Kristensen LS, Hansen LL (2009) PCR-based methods for detecting single-locus DNA methylation biomarkers in cancer diagnostics, prognostics, and response to treatment. Clin Chem 55(8):1471–1483

    Article  CAS  PubMed  Google Scholar 

  8. Hayatsu H, Wataya Y, Kazushige K (1970) The addition of sodium bisulfite to uracil and to cytosine. J Am Chem Soc 92(3):724–726

    Article  CAS  PubMed  Google Scholar 

  9. Hayatsu H (2008) Discovery of bisulfite-mediated cytosine conversion to uracil, the key reaction for DNA methylation analysis—a personal account. Proc Jpn Acad Ser B Phys Biol Sci 84(8):321–330

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 93(18):9821–9826

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Shen L, Waterland RA (2007) Methods of DNA methylation analysis. Curr Opin Clin Nutr Metab Care 10(5):576–581

    Article  CAS  PubMed  Google Scholar 

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Acknowledgment

The authors are thankful to Cachar Cancer Hospital and Research Centre (CCHRC) and Silchar Medical College and Hospital (SMC), Assam, for providing blood/tissue samples. We are also grateful to the Department of Biotechnology (DBT), Government of India for providing infrastructural facilities.

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Authors and Affiliations

  1. Molecular Medicine Laboratory, Department of Biotechnology, Assam University, Silchar, Assam, India

    Javed Hussain Choudhury, Raima Das, Shaheen Laskar, Sharbadeb Kundu, Manish Kumar, Partha Pratim Das, Yashmin Choudhury & Sankar Kumar Ghosh

  2. Institute of Advanced Study in Science and Technology, Guwahati, Assam, India

    Rosy Mondal

  3. University of Kalyani, Kalyani, West Bengal, India

    Sankar Kumar Ghosh

Authors
  1. Javed Hussain Choudhury
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  2. Raima Das
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  3. Shaheen Laskar
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  4. Sharbadeb Kundu
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  5. Manish Kumar
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  6. Partha Pratim Das
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  7. Yashmin Choudhury
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  8. Rosy Mondal
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  9. Sankar Kumar Ghosh
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Corresponding author

Correspondence to Sankar Kumar Ghosh .

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Editors and Affiliations

  1. Biochemistry and Cancer Biology Divisions Basic Science Department, Ponce Health Sciences University, Ponce, Puerto Rico

    Pedro G. Santiago-Cardona

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Choudhury, J.H. et al. (2018). Detection of p16 Promoter Hypermethylation by Methylation-Specific PCR. In: Santiago-Cardona, P. (eds) The Retinoblastoma Protein. Methods in Molecular Biology, vol 1726. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7565-5_11

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  • DOI: https://doi.org/10.1007/978-1-4939-7565-5_11

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7564-8

  • Online ISBN: 978-1-4939-7565-5

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