Collection of buccal cell DNA in seventh-grade children using water and a toothbrush

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 child ren. 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 adde d 5 ml of 99% ethanol to preserve the sample. Among 1563 samples transporte d at room temperature over 1 week and then stored for 13-14 months at -70 d egreesC before extraction, using a modified Gentra Puregene protocol, the m edian total DNA yield was 108 mug, range of 14 to 416 mug. We assayed every 20th sample (n = 77) for NAT2 by the PCR, and all samples gave a 1093-bp p roduct. From the 1563 samples, we obtained a result for single nucleotide p olymorphisms 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 Sout hern California, gave a lower total DNA yield than the toothbrush rinse (me dian of 17 mug) and a slightly reduced ability to generate a PCR product. H owever, 26 of 27 water-rinse samples gave a result for two genes, albumin a nd CYP1A1, using real-time PCR methods. We did not quantify human, versus b acterial, DNA in our samples. However, given the amounts of total DNA requi red for genotyping, a sample with the median yield of 108 mug should suffic e for similar to 2160 genotypes by RFLP-PCR methods or five times as many b y real-time PCR. We recommend the toothbrush-rinse method, combined with a modified Gentra Puregene DNA extraction protocol, for large-scale, in-perso n collections of buccal cell DNA in children. The method requires only inex pensive, readily available materials and produces a large quantity of high- quality DNA for PCR analyses.