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Pyrosequencing Analysis for Breast Cancer DNA Methylome

  • Cem Kuscu
  • Canan Kuscu
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1406)

Abstract

Unraveling DNA methylation profile of tumor is important for the diagnosis and treatment of cancer patients. Because of the heterogeneity of clinical samples, it is very difficult to get methylation profile of only tumor cells. Laser capture Microdissection (LCM) is giving us a chance to isolate the DNA only from the tumor cells without any stroma cell’s DNA contamination. Once we capture the breast tumor cells, we can isolate the genomic DNA which is followed by the bisulfite treatment in which unmethylated cytosines of the CG pairs are converted into uracil; however, methylated cytosine does not go into any chemical change during this reaction. Next, bisulfite treated DNA is used in the regular PCR reaction to get a single band PCR amplicon which will be used as a template for the pyrosequencing. Pyrosequencing is a powerful method to make a quantitative methylation analysis for each specific CG pair.

Key words

Breast cancer Laser capture microdissection (LCM) DNA methylation Bisulfite conversion Pyrosequencing 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular Genetics, School of MedicineUniversity of VirginiaCharlottesvilleUSA

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