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Collection of Buccal Cell DNA in Seventh-Grade Children Using Water and a Toothbrush¹

Epidemiology Branch and Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 [S. L., G. D-B.]; Wuhan Public Health and Anti-Epidemic Station, Wuhan, 430022 People’s Republic of China [J. X., J-H. Y., L. C., T. Li, Y. L.]; BioServe Biotechnologies, Ltd., Laurel, Maryland 20707 [T. Le.]; and Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California 90033 [E. G., L. D.]

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°C before extraction, using a modified Gentra Puregene protocol, the median total DNA yield was 108 µg, range of 14 to 416 µg. 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 µg) 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 µg should suffice for 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.

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