title: Collection of buccal cell DNA in seventh-grade children using water and a toothbrush.
abstract: We developed a simple and effective method for collecting a large quantity of buccal cell DNA in school-based studies of seventh-grade and older children. Seventh-grade students at schools in Wuhan, China brushed each buccal surface with a soft toothbrush and then rinsed with 10 ml of water. We added 5 ml of 99% ethanol to preserve the sample. Among 1563 samples transported at room temperature over 1 week and then stored for 13-14 months at -70 degrees C before extraction, using a modified Gentra Puregene protocol, the median total DNA yield was 108 microg, range of 14 to 416 microg. We assayed every 20th sample (n = 77) for NAT2 by the PCR, and all samples gave a 1093-bp product. From the 1563 samples, we obtained a result for single nucleotide polymorphisms in the interleukin-13 gene (at +2044) by RFLP-PCR on 98.8% and in the promoter of the myeloperoxidase gene (at -463) by real-time PCR on 99.7%. A water-rinse method, that we used among 12th-grade students in Southern California, gave a lower total DNA yield than the toothbrush rinse (median of 17 microg) and a slightly reduced ability to generate a PCR product. However, 26 of 27 water-rinse samples gave a result for two genes, albumin and CYP1A1, using real-time PCR methods. We did not quantify human, versus bacterial, DNA in our samples. However, given the amounts of total DNA required for genotyping, a sample with the median yield of 108 microg should suffice for approximately 2160 genotypes by RFLP-PCR methods or five times as many by real-time PCR. We recommend the toothbrush-rinse method, combined with a modified Gentra Puregene DNA extraction protocol, for large-scale, in-person collections of buccal cell DNA in children. The method requires only inexpensive, readily available materials and produces a large quantity of high-quality DNA for PCR analyses.
authors.affiliation: Epidemiology Branch and Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: London
authors.forename: J
authors.initials: J
authors.lastname: Xia
authors.forename: T A
authors.initials: TA
authors.lastname: Lehman
authors.forename: J H
authors.initials: JH
authors.lastname: Yang
authors.forename: E
authors.initials: E
authors.lastname: Granada
authors.forename: L
authors.initials: L
authors.lastname: Chunhong
authors.forename: L
authors.initials: L
authors.lastname: Dubeau
authors.forename: T
authors.initials: T
authors.lastname: Li
authors.forename: G L
authors.initials: GL
authors.lastname: David-Beabes
authors.forename: Y
authors.initials: Y
authors.lastname: Li
mesh_terms: D000418:Albumins / Q000235:genetics; D001191:Arylamine N-Acetyltransferase / Q000235:genetics; D002648:Child; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000235:genetics; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics; D004247:DNA / Q000302:isolation & purification*; D006801:Humans; D018793:Interleukin-13 / Q000235:genetics; D009061:Mouth Mucosa / Q000166:cytology*; D009195:Peroxidase / Q000235:genetics; D016133:Polymerase Chain Reaction; D013048:Specimen Handling / Q000295:instrumentation / Q000379:methods
chemical_list: D000418:Albumins; D018997:Chorionic Gonadotropin, beta Subunit, Human; D018793:Interleukin-13; D004247:DNA; D009195:Peroxidase; D019363:Cytochrome P-450 CYP1A1; D001191:Arylamine N-Acetyltransferase; C478900:NAT2 protein, human