Feigelson H S, Rodriguez C, Robertson A S, Jacobs E J, Calle E E, Reid Y A, Thun M J
Department of Epidemiology and Surveillance Research, American Cancer Society, National Home Office, Atlanta, Georgia 30329-4251, USA.
Cancer Epidemiol Biomarkers Prev. 2001 Sep;10(9):1005-8.
Buccal cells are becoming an important source of genomic DNA in epidemiological studies, but little is known about the effect of different sampling conditions on DNA quality and yield. We used a mouthwash protocol to collect six daily buccal cell samples from 35 healthy volunteers. Twenty-four individuals (six men and 18 women) correctly completed the protocol and were included in paired analyses to determine whether "swish" time (30 s versus 60 s), toothbrushing before collection, or lag time between collection and DNA extraction (1 day versus 5, 10, or 30 days at room temperature) would affect sample quality and yield. Total DNA, human-specific DNA (hDNA), degradation of DNA, and ability to amplify by PCR were determined. hDNA yield did not significantly vary by "swish" time. However, toothbrushing 1 h before sample collection reduced the amount of hDNA by nearly 40% (34 microg versus 21 microg; P = 0.06). Median hDNA yields for samples that were held for 1, 5, 10, and 30 days before extraction were 32 microg (range, 4-196), 32 microg (2-194), 23 microg (3-80), and 21 microg (5-56), respectively. The 10- and 30-day samples had significantly less hDNA than those processed after 1 day (P = 0.01). PCR success rates for beta-globin gene fragments of length 268 bp, 536 bp, and 989 bp were 94% or better, and high molecular weight DNA (>23 kb) was found in all but one sample. These results suggest that buccal cells should be collected before brushing teeth and processed within 5 days of collection to maximize hDNA yield.
颊细胞正成为流行病学研究中基因组DNA的重要来源，但对于不同采样条件对DNA质量和产量的影响却知之甚少。我们采用一种漱口水方案，从35名健康志愿者中每天收集6份颊细胞样本。24名个体（6名男性和18名女性）正确完成了该方案，并被纳入配对分析，以确定“漱口”时间（30秒与60秒）、采集前刷牙或采集与DNA提取之间的延迟时间（室温下1天与5天、10天或30天）是否会影响样本质量和产量。测定了总DNA、人特异性DNA（hDNA）、DNA降解情况以及通过PCR进行扩增的能力。hDNA产量并未因“漱口”时间而有显著差异。然而，在样本采集前1小时刷牙使hDNA量减少了近40%（34微克对21微克；P = 0.06）。在提取前保存1天、5天、10天和30天的样本，其hDNA产量中位数分别为32微克（范围4 - 196）、32微克（2 - 194）、23微克（3 - 80）和21微克（5 - 56）。保存10天和30天的样本其hDNA明显少于采集后1天处理的样本（P = 0.01）。长度为268 bp、536 bp和989 bp的β-珠蛋白基因片段的PCR成功率为94%或更高，并且除了一个样本外，在所有样本中都发现了高分子量DNA（>23 kb）。这些结果表明，颊细胞应在刷牙前采集，并在采集后5天内进行处理，以最大限度提高hDNA产量。
