London S J, Xia J, Lehman T A, Yang J H, Granada E, Chunhong L, Dubeau L, Li T, David-Beabes G L, Li Y
Epidemiology Branch and Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Cancer Epidemiol Biomarkers Prev. 2001 Nov;10(11):1227-30.
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.
我们开发了一种简单有效的方法，用于在以七年级及以上儿童为对象的学校研究中收集大量颊细胞DNA。中国武汉学校的七年级学生用软牙刷刷每个颊面，然后用10毫升水冲洗。我们加入5毫升99%乙醇保存样本。在1563个样本中，室温下运输超过1周，然后在-70摄氏度下储存13 - 14个月后进行提取，使用改良的Gentra Puregene方案，总DNA产量中位数为108微克，范围为14至416微克。我们通过PCR对每20个样本（n = 77）检测NAT2，所有样本均产生1093 bp的产物。从1563个样本中，我们通过RFLP-PCR对98.8%的样本获得了白细胞介素-13基因（+2044处）单核苷酸多态性的结果，通过实时PCR对99.7%的样本获得了髓过氧化物酶基因启动子（-463处）的结果。我们在南加州的十二年级学生中使用的水洗方法，总DNA产量低于牙刷冲洗法（中位数为17微克），产生PCR产物的能力略有降低。然而，27个水洗样本中的26个通过实时PCR方法获得了白蛋白和CYP1A1两个基因的结果。我们没有对样本中的人类DNA与细菌DNA进行定量。然而，考虑到基因分型所需的总DNA量，产量中位数为108微克的样本通过RFLP-PCR方法大约足以进行2160次基因分型，通过实时PCR方法则是其五倍。我们推荐将牙刷冲洗法与改良的Gentra Puregene DNA提取方案相结合，用于大规模亲自收集儿童颊细胞DNA。该方法仅需廉价、容易获得的材料，并能产生大量用于PCR分析的高质量DNA。
