title: Extraction and processing of circulating DNA from large sample volumes using methylation on beads for the detection of rare epigenetic events.
abstract: The use of methylated tumor-specific circulating DNA has shown great promise as a potential cancer biomarker. Nonetheless, the relative scarcity of tumor-specific circulating DNA presents a challenge for traditional DNA extraction and processing techniques. Here we demonstrate a single tube extraction and processing technique dubbed "methylation on beads" that allows for DNA extraction and bisulfite conversion for up to 2 ml of plasma or serum. In comparison to traditional techniques including phenol chloroform and alcohol extraction, methylation on beads yields a 1.5- to 5-fold improvement in extraction efficiency. The technique results in far less carryover of PCR inhibitors yielding analytical sensitivity improvements of over 25-fold. The combination of improved recovery and sensitivity make possible the detection of rare epigenetic events and the development of high sensitivity epigenetic diagnostic assays.
authors.affiliation: Department of Mechanical Engineering, Johns Hopkins University, Baltimore, MD 21218, United States.
authors.forename: Brian
authors.initials: B
authors.lastname: Keeley
authors.forename: Alejandro
authors.initials: A
authors.lastname: Stark
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Pisanic
authors.forename: Ruby
authors.initials: R
authors.lastname: Kwak
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: John
authors.initials: J
authors.lastname: Wrangle
authors.forename: Stephen
authors.initials: S
authors.lastname: Baylin
authors.forename: James
authors.initials: J
authors.lastname: Herman
authors.forename: Nita
authors.initials: N
authors.lastname: Ahuja
authors.forename: Malcolm V
authors.initials: MV
authors.lastname: Brock
authors.forename: Tza-Huei
authors.initials: TH
authors.lastname: Wang
mesh_terms: D000199:Actins / Q000097:blood / Q000235:genetics; D000368:Aged; D014408:Biomarkers, Tumor / Q000097:blood* / Q000235:genetics; D002289:Carcinoma, Non-Small-Cell Lung / Q000097:blood* / Q000175:diagnosis / Q000235:genetics; D004247:DNA / Q000097:blood*; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood* / Q000175:diagnosis / Q000235:genetics; D008297:Male; D008863:Microspheres; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D013447:Sulfites / Q000737:chemistry; D025521:Tumor Suppressor Proteins / Q000097:blood* / Q000235:genetics
chemical_list: D000199:Actins; D014408:Biomarkers, Tumor; C411762:RASSF1 protein, human; D013447:Sulfites; D025521:Tumor Suppressor Proteins; D004247:DNA; C042345:hydrogen sulfite
keywords: Blood-processing protocols; Cancer epigenetics; Circulating DNA; DNA methylation; Methylation specific PCR; Quantification
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