Format

Send to

Choose Destination
Methods Mol Biol. 2016;1406:89-104. doi: 10.1007/978-1-4939-3444-7_8.

Pyrosequencing Analysis for Breast Cancer DNA Methylome.

Author information

1
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Charlottesville, VA, 800733, 22908, USA. ck6mr@eservices.virginia.edu.
2
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Charlottesville, VA, 800733, 22908, USA.

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.

KEYWORDS:

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

PMID:
26820948
DOI:
10.1007/978-1-4939-3444-7_8
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Springer
Loading ...
Support Center