title: Collection of genomic DNA from adults in epidemiological studies by buccal cytobrush and mouthwash.
abstract: Blood samples are an excellent source of large amounts of genomic DNA. However, alternative sources are often needed in epidemiological studies because of difficulties in obtaining blood samples. This report evaluates the buccal cytobrush and alcohol-containing mouthwash protocols for collecting DNA by mail. Several DNA extraction techniques are also evaluated. The study was conducted in two phases. In phase 1, we compared cytobrush and mouthwash samples collected by mail in two different epidemiological studies: (a) cytobrush samples (n = 120) from a United States case-control study of breast cancer; and (b) mouthwash samples (n = 40) from a prospective cohort of male United States farmers. Findings from phase 1 were confirmed in phase 2, where we randomized cytobrush (n = 28) and mouthwash (n = 25) samples among participants in the breast cancer study to directly compare both collection methods. The median human DNA yield determined by hybridization with a human DNA probe from phenol-chloroform extracts was 1.0 and 1.6 microg/2 brushes for phases 1 and 2, respectively, and 27.5 and 16.6 microg/mouthwash sample for phases 1 and 2, respectively. Most (94-100%) mouthwash extracts contained high molecular weight DNA (>23 kb), in contrast to 55-61% of the brush extracts. PCR success rates for amplification of beta-globin gene fragments (268, 536, and 989 bp) were similar for cytobrush and mouthwash phenol-chloroform extracts (range, 94.4-100%). Also, we obtained high success rates in determining the number of CAG repeats in the androgen receptor gene, characterizing tetranucleotide microsatellites in six gene loci, and screening for mutations in the BRCA1/2 genes in a subset of phenol-chloroform DNA extracts. Relative to DNA extracted by phenol-chloroform from cytobrush samples, DNA extracted by NaOH had lower molecular weight, decreased PCR success rates for most assays performed, and unreliably high spectrophotometer readings for DNA yields. In conclusion, although DNA isolated from either mouthwash or cytobrush samples collected by mail from adults is adequate for a wide range of PCR-based assays, a single mouthwash sample provides substantially larger amounts and higher molecular weight DNA than two cytobrush samples.
authors.affiliation: Division of Cancer Epidemiology and Genetics, National Cancer Institute, Rockville, Maryland 20852-7234, USA.
authors.forename: M
authors.initials: M
authors.lastname: García-Closas
authors.forename: K M
authors.initials: KM
authors.lastname: Egan
authors.forename: J
authors.initials: J
authors.lastname: Abruzzo
authors.forename: P A
authors.initials: PA
authors.lastname: Newcomb
authors.forename: L
authors.initials: L
authors.lastname: Titus-Ernstoff
authors.forename: T
authors.initials: T
authors.lastname: Franklin
authors.forename: P K
authors.initials: PK
authors.lastname: Bender
authors.forename: J C
authors.initials: JC
authors.lastname: Beck
authors.forename: L
authors.initials: L
authors.lastname: Le Marchand
authors.forename: A
authors.initials: A
authors.lastname: Lum
authors.forename: M
authors.initials: M
authors.lastname: Alavanja
authors.forename: R B
authors.initials: RB
authors.lastname: Hayes
authors.forename: J
authors.initials: J
authors.lastname: Rutter
authors.forename: K
authors.initials: K
authors.lastname: Buetow
authors.forename: L A
authors.initials: LA
authors.lastname: Brinton
authors.forename: N
authors.initials: N
authors.lastname: Rothman
mesh_terms: D000328:Adult; D000368:Aged; D001943:Breast Neoplasms; D004247:DNA / Q000032:analysis*; D016021:Epidemiologic Studies; D005260:Female; D006801:Humans; D008875:Middle Aged; D009061:Mouth Mucosa / Q000166:cytology; D009067:Mouthwashes; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D013048:Specimen Handling
chemical_list: D009067:Mouthwashes; D004247:DNA