title: DNA methylation analysis by digital bisulfite genomic sequencing and digital MethyLight.
abstract: Alterations in cytosine-5 DNA methylation are frequently observed in most types of human cancer. Although assays utilizing PCR amplification of bisulfite-converted DNA are widely employed to analyze these DNA methylation alterations, they are generally limited in throughput capacity, detection sensitivity, and or resolution. Digital PCR, in which a DNA sample is analyzed in distributive fashion over multiple reaction chambers, allows for enumeration of discrete template DNA molecules, as well as sequestration of non-specific primer annealing templates into negative chambers, thereby increasing the signal-to-noise ratio in positive chambers. Here, we have applied digital PCR technology to bisulfite-converted DNA for single-molecule high-resolution DNA methylation analysis and for increased sensitivity DNA methylation detection. We developed digital bisulfite genomic DNA sequencing to efficiently determine single-basepair DNA methylation patterns on single-molecule DNA templates without an interim cloning step. We also developed digital MethyLight, which surpasses traditional MethyLight in detection sensitivity and quantitative accuracy for low quantities of DNA. Using digital MethyLight, we identified single-molecule, cancer-specific DNA hypermethylation events in the CpG islands of RUNX3, CLDN5 and FOXE1 present in plasma samples from breast cancer patients.
authors.affiliation: Department of Surgery, University of Southern California/Norris Comprehensive Cancer Center, Los Angeles, CA 90033, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Weisenberger
authors.forename: Binh N
authors.initials: BN
authors.lastname: Trinh
authors.forename: Mihaela
authors.initials: M
authors.lastname: Campan
authors.forename: Shikhar
authors.initials: S
authors.lastname: Sharma
authors.forename: Tiffany I
authors.initials: TI
authors.lastname: Long
authors.forename: Suchitra
authors.initials: S
authors.lastname: Ananthnarayan
authors.forename: Gangning
authors.initials: G
authors.lastname: Liang
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Esteva
authors.forename: Gabriel N
authors.initials: GN
authors.lastname: Hortobagyi
authors.forename: Frank
authors.initials: F
authors.lastname: McCormick
authors.forename: Peter A
authors.initials: PA
authors.lastname: Jones
authors.forename: Peter W
authors.initials: PW
authors.lastname: Laird
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics; D018899:CpG Islands; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000097:blood* / Q000378:metabolism; D023281:Genomics / Q000379:methods*; D006801:Humans; D046210:Microfluidic Analytical Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D013447:Sulfites / Q000737:chemistry*
chemical_list: D004273:DNA, Neoplasm; D013447:Sulfites; C042345:hydrogen sulfite