Sigurdson Alice J, Ha Mina, Cosentino Mark, Franklin Tracie, Haque Kashif A, Qi Ying, Glaser Cynthia, Reid Yvonne, Vaught Jim B, Bergen Andrew W
Radiation Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Department of Health and Human Services, 6120 Executive Boulevard, EPS 7092, MSC 7238, Bethesda, Maryland 20892-7238, USA.
Cancer Epidemiol Biomarkers Prev. 2006 Feb;15(2):385-8. doi: 10.1158/1055-9965.EPI-05-0662.
Economical methods for collecting and storing high-quality DNA are needed for large population-based molecular epidemiology studies. Buccal cell DNA collected via saliva and stored on treated filter paper cards could be an attractive method, but modest DNA yields and the potential for reduced recovery of DNA over time were unresolved impediments. Consequently, buccal cell DNA collection via oral mouthwash rinsing became the method of choice in epidemiologic studies. However, the amount of genomic DNA (gDNA) required for genotyping continues to decrease, and reliable whole genome amplification (WGA) methods further reduced the mass of gDNA needed for WGA to 10 ng, diminishing the obstacle of low DNA yields from cards. However, concerns about yield and DNA quality over time remained. We located and analyzed 42 buccal cell saliva samples collected and stored on treated cards for 7 years at room temperature, -20 degrees C, and -80 degrees C. We recovered DNA from the treated cards, estimated the concentration by a human-specific quantitative real-time PCR assay, and evaluated the quality by PCR amplification of 268-, 536-, and 989-bp fragments of the beta-globin gene and by AmpFlSTR Identifiler assay analysis. Most DNA yields per 3-mm punch were <10 ng, and most PCR amplicons failed to amplify, where size of the amplicon was negatively associated with successful amplification. Using these methods, treated cards did not consistently provide sufficient quantities of buccal cell gDNA after 7 years of storage for genotyping or WGA.
对于基于大规模人群的分子流行病学研究而言，需要经济实惠的方法来收集和储存高质量的DNA。通过唾液收集并储存在经过处理的滤纸卡上的颊细胞DNA可能是一种有吸引力的方法，但DNA产量不高以及随着时间推移DNA回收率可能降低的问题尚未得到解决。因此，通过口腔含漱液冲洗收集颊细胞DNA成为了流行病学研究中的首选方法。然而，基因分型所需的基因组DNA（gDNA）数量持续减少，可靠的全基因组扩增（WGA）方法进一步将WGA所需的gDNA量减少至10 ng，减少了卡片DNA产量低的障碍。然而，对产量和DNA质量随时间变化的担忧依然存在。我们定位并分析了42份颊细胞唾液样本，这些样本在经过处理的卡片上分别于室温、-20℃和-80℃储存了7年。我们从处理过的卡片中回收DNA，通过人特异性定量实时PCR测定法估计浓度，并通过对β-珠蛋白基因的268 bp、536 bp和989 bp片段进行PCR扩增以及通过AmpFlSTR Identifiler测定法分析来评估质量。每3毫米打孔的大多数DNA产量<10 ng，并且大多数PCR扩增子未能扩增，其中扩增子大小与成功扩增呈负相关。使用这些方法，经过处理的卡片在储存7年后并不能始终提供足够数量的颊细胞gDNA用于基因分型或WGA。
