Collection of Buccal Cell DNA Using Treated Cards Lea C. Harty, Montserrat Garcia-Closas, Nathaniel Rothman, Yvonne A. Reid, Margaret A. Tucker, 1 and Patricia Hartge Genetic Epidemiology Branch [L. C. H., M. A. T.], Environmental Epidemiology Branch [M. G-C.], Occupational Epidemiology Branch [N. R.], and Epidemiology and Biostatistics Program [P. H.], National Cancer Institute, Bethesda, Maryland 20892, and American Type Culture Collection, Manassas, Virginia 20110 [Y. A. R.] Abstract We devised a simple, noninvasive, cost-efficient technique for collecting buccal cell DNA for molecular epidemiology studies. Subjects (n  52) brushed their oral mucosa and expectorated the fluid in their mouths, which was applied to “Guthrie” cards pretreated to retard bacterial growth and inhibit nuclease activity (IsoCode, Schleicher and Schuell, Keene, NH). The cards are well-suited for transport and storage because they dry quickly, need no processing, and are compact and lightweight. We stored the samples at room temperature for 5 days to mimic a field situation and then divided them into portions from which DNA was extracted either immediately or after storage for 9 months at room temperature, 20°C, or 70°C. The fresh samples had a median yield of 2.3 g of human DNA (range, 0.2–53.8 g), which was adequate for at least 550 PCR reactions. More than 90% of the samples were amplified in all three -globin gene fragment assays attempted. DNA extract frozen for 1 week at 20°C also performed well. Stored samples had reduced DNA yields, which achieved statistical significance for room temperature and 70°C, but not 20°C, storage. However, because all of the stored samples tested were successfully amplified, the observed reduction may represent tighter DNA fixation to the card over time rather than loss of genetic material. We conclude that treated cards are an alternative to brushes/ swabs and mouth rinses for the collection of buccal cell DNA and offer some advantages over these methods, particularly for large-scale or long-term studies involving stored samples and studies in which samples are collected off-site and transported. Future studies that enable direct comparisons of the various buccal cell collection methods are needed. Introduction Simple methods of collecting DNA samples in large-scale community studies could extend the range of molecular epide- miological studies. Venipuncture, the standard DNA collection method, cannot be used in many situations for medical, logistic, or cultural reasons. In other situations, it is feasible but pro- hibitively expensive. We therefore devised a simple, noninva- sive, cost-efficient technique to obtain DNA samples in large- scale community studies and assessed the quantity and quality of DNA collected. Buccal cells provide an accessible source of germ-line DNA. Buccal cell collection techniques involving swabs, brushes, and scraping instruments (1–9) and oral rinses using water, saline, and mouthwash (4, 10 –15) have been described. Although these methods are generally easy to administer, non- invasive, cost-efficient, well accepted by subjects, and safe for study personnel, the DNA collected using swabs, brushes, and scraping tools may be vulnerable to degradation if the samples are not processed or frozen soon after collection (1, 6 –9). 2 The unfortunate result may be specimens that are inadequate for testing multiple genetic factors. Rinses typically provide ade- quate quantities of good quality DNA, but liquid samples may spill or leak during shipment. In addition, storing large numbers of rinses requires considerable space, some preservatives pose safety hazards to untrained individuals, and extracting DNA from rinses may be labor-intensive. We sought an alternative technique for collecting buccal cell samples, which would elim- inate these potential drawbacks. “Guthrie cards” (903 filter paper, Schleicher & Schuell, Inc., Keene, NH) are used to collect heelstick blood from newborns for metabolic disease screening (16); however, blood spots archived as long as 17 years, sometimes at room temper- ature, have also provided valuable sources of amplifiable DNA (17–20). A modified card (IsoCode, Schleicher and Schuell, Keene, NH) has been developed that is treated to retard bacte- rial and viral growth, inhibit nuclease activities, and release template DNA during processing (21, 22). Treated cards reli- ably yield amplifiable nucleic acid from blood and buccal cell samples, whereas untreated cards do not (22). In one genetic epidemiology study, PCR-based assays were performed on DNA from fingerstick blood samples collected on treated cards from 5300 subjects (23). In the present study, we used such treated cards to collect buccal cell DNA and report the quantity, quality, and stability of human DNA from samples collected in a manner similar to that which would occur in many epidemi- ological studies. Materials and Methods Subjects. Fifty-two healthy employees of the NIH (Bethesda, MD) and Westat, Inc. (Rockville, MD) provided written, in- formed consent to participate in this study. The study protocol was approved by the Institutional Review Boards of the NIH and Westat, Inc. Received 9/30/98; revised 2/2/00; accepted 2/29/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at National Cancer Institute, Executive Plaza South, 7th Floor, 6120 Executive Boulevard, MSC 7236, Be- thesda, MD 20892-7236. 2 J. 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