title: Pyrosequencing Analysis for Breast Cancer DNA Methylome.
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.
authors.affiliation: Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Charlottesville, VA, 800733, 22908, USA. ck6mr@eservices.virginia.edu.
authors.forename: Cem
authors.initials: C
authors.lastname: Kuscu
authors.affiliation: Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Charlottesville, VA, 800733, 22908, USA.
authors.forename: Canan
authors.initials: C
authors.lastname: Kuscu
mesh_terms: D001943:Breast Neoplasms / Q000473:pathology*; D018899:CpG Islands / Q000235:genetics; D004247:DNA / Q000235:genetics*; D019175:DNA Methylation / Q000187:drug effects; D017931:DNA Primers / Q000235:genetics; D004801:Eosine Yellowish-(YS) / Q000378:metabolism; D005557:Formaldehyde; D015894:Genome, Human / Q000235:genetics; D023281:Genomics / Q000379:methods*; D006416:Hematoxylin / Q000378:metabolism; D006801:Humans; D061346:Laser Capture Microdissection; D061986:MCF-7 Cells; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013194:Staining and Labeling; D013223:Statistics as Topic; D013447:Sulfites / Q000494:pharmacology; D016707:Tissue Fixation
chemical_list: D017931:DNA Primers; D013447:Sulfites; D005557:Formaldehyde; D004247:DNA; C042345:hydrogen sulfite; D004801:Eosine Yellowish-(YS); D006416:Hematoxylin
keywords: Bisulfite conversion; Breast cancer; DNA methylation; Laser capture microdissection (LCM); Pyrosequencing