A Simple Mouthwash Method for Obtaining Genomic DNA in Molecular Epidemiological

Vol. 7, 719-724, August 1998 Cancer Epidemiology, Biomarkers & Prevention 7/9

Short Communication

Annette Lum and LoIc Le Marchand2 blood. Although this method yields large amounts of DNA

Etiology Program. Cancer Research Center of Hawaii, University of Hawaii, N 100 j.g from a l0-ml blood sample), an alternative source of Honolulu, Hawaii 96813 DNA is desirable because venipuncture is invasive and uncom- fortable for the subject and involves risk of exposure to blood- borne pathogens. It is also unacceptable in some individuals for Abstract cultural or religious reasons. As a consequence, it is not un- Genomic DNA for genetic analyses has traditionally been common in molecular epidemiological studies for a substantial derived from blood samples. With the availability of PCR proportion (20-40%) of the participants to refuse the blood techniques requiring only minute amounts of DNA and collection. Furthermore, obtaining blood samples becomes pro- the current demand for high-volume testing, a less hibitively expensive and logistically arduous when the study invasive, simpler to perform, and cheaper method to size is large or when the participants reside across a large obtain DNA is desirable. We developed a method to geographical area, as is typical in family studies, in association obtain high-quality genomic DNA from buccal cells that studies examining gene-gene or gene-environment interactions, has high acceptability and allows for a large number of and in prospective studies of rare diseases. PCR assays from a single sample. Sixty subjects DNA for genetic analysis has also been derived from vigorously swished 10 ml of undiluted commercial finger-stick blood, paraffin-embedded tissue, urine, hair roots, mouthwash in the mouth for 60 s and expelled the liquid saliva, cheek scrapings. buccal brushes, buccal swabs, and oral into a collection container. DNA was isolated from the saline rinse samples (1-8). However, these methods are some- buccal cells with a rapid method using proteinase K what invasive (finger stick, cheek scrapings or brushes, and digestion, phenol-chloroform extraction, and ethanol saline rinse) or do not yield an adequate amount (urine, hair precipitation. Electrophoretic analysis of the extracted roots, and saliva) or quality (paraffin blocks) of DNA. Also, DNA showed detectable levels of high molecular weight some of these methods require the samples to be stored in a genomic DNA in all samples. The DNA yields ranged preservative solution that is toxic, which makes it problematic from 0.2 to 134.0 ,zg, for an average of 49.7 tg. Using for use by mail (buccal brushes and swabs). This work was these samples, all 60 subjects were successfully genotyped aimed at developing a method to obtain high-quality genomic by PCR-based assays for polymorphisms in the CYPJAJ DNA from buccal cells that would have high applicability and (MspI and exon 7), CYP2EJ (RsaI), GSTMJ, GSTTJ, and acceptability and allow for a large number of PCR assays from NQOJ genes, confirming that the quality of DNA isolated a single sample. Our requirements were that, with this method, from mouthwash samples was sufficient to reliably samples could be collected by the participants themselves, support PCR amplification. Storage of the (unprocessed) mailed back to the study center, and stored for months prior to specimens at room temperature or at 37#{176}Cfor 1 week DNA extraction. (temperature conditions that may be encountered when mailing samples) or at -20#{176}C for at least 6 months did Materials and Methods not affect the DNA yield or ability to PCR amplify the Subjects for this feasibility study were recruited among the samples. The results suggest that this mouthwash employees of the Cancer Research Center of Hawaii and their procedure may be suitable for large community-based acquaintances. A total of 64 individuals were given a 30-mi, studies of genetic susceptibility to disease in which wide-mouth, screw-capped jar that contained 10 ml of undi- samples can be collected by the participants themselves, luted mouthwash (FreshBurst Listerine), along with written mailed back to the study center, and stored for months instructions for collecting the sample at home. About 1 h after prior to DNA analysis. they brushed their teeth, the subjects swished the mouthwash vigorously throughout the mouth for 1 mm and expelled it back Introduction into the jar. On the same day or on the following day, the participants returned the specimens to the laboratory. The Almost invariably, genomic DNA for PCR-based genetic anal- mouthwash samples were either processed within 1 week or yses has been derived from leukocytes prepared from whole stored at - 20#{176}Cfor later extraction. Processing consisted of transferring the sample to a 50-mi conical tube for centrifuga- tion at 2700 rpm for 15 mm. The supernatant was decanted, and the pellet was washed in 25 ml of TE buffer [10 mi Tris (pH Received 1/30/98; revised 5/I 1/98: accepted 5/I 9/98. The costs of publication of this article were defrayed in part by the payment of 8.0), 10 mM EDTA (pH 8.0)]. The suspension was centrifuged page charges. This article must therefore be hereby marked advertisement in at 2700 rpm for 15 mm, and the supernatant was discarded. We accordance with 18 U.S.C. Section 1734 solely to indicate this fact. modified the protocol published by Walsh et a!. (9) to extract t This research was supported in part by NIH Grant CA60987. DNA. Briefly, the pellet was resuspended in 700 l of lysis 2 Ta whom requests for reprints should be addressed, at Cancer Research Center of Hawaii, University of Hawaii, 1236 Lauhala Street, Suite 407. Honolulu, HI buffer [10 mM Tris (pH 8.0), 10 mi EDTA (pH 8.0), 0.1 M 968 I 3. NaCI, and 2% SDS} and transferred to a 2-mI microcentrifuge

8 7 1n6 9-. 0 w5

Fig. 1. Distribution of amounts of DNA in .sg ob- Cl) tamed by proteinase K digestion, phenol-chloroform Is- 03 extraction, and ethanol precipitation from single mouthwash samples collected by 60 subjects. The 0 DNA concentration of each sample was determined by z2 spectrophotometer (GeneQuant II: Pharmacia Bio- tech) and ranged from 0.2 to 134 sg. I 0 III I II t, ‘II ( II I III cg $ $ DNA .Lg)

l23 4 5 6 7 8 9 10 11 12 13 l4,,

Fig. 2. Five jzl of each buccal cell DNA sample were resolved by dcc- trophoresis in a I .8% agarose gel and visualized with ethidium bromide, as shown here for the first I 7 samples extracted. Arrow, high molecular weight DNA.

tube containing 35 l of 20 mg/mi proteinase K. The samples DNA template with initial denaturation at 95#{176}Cfor 4 mm, were mixed and digested at 58#{176}Cfor 2 h. The DNA was then followed by 30 cycles with denaturation at 95#{176}Cfor 1 mm, extracted from each sample with equal volumes of phenol- annealing at 65#{176}Cfor 1 mm, and extension at 72#{176}Cfor 1 mm chloroform ( 1: 1) and with an equal volume of chloroform and a final annealing and extension step at 65#{176}Cfor 1 mm and alone, each time vortexing for 10 s and centrifuging at 14,000 72#{176}Cfor 8 mm, respectively. The PCR product was then rpm for 2 mm. The DNA was removed from the supernatant digested with MspI and subjected to electrophoresis on an with 3 M NaOAc (pH 6.0; 1/10 volume of supernatant) and 2 agarose gel. volumes of cold 100% ethanol and precipitated at -20#{176}Cfor The second CYPJA] polymorphism studied was an A- 2 h. The DNA was pelleted at 10,000 rpm for 10 mm, washed to-G transition, which results in the substitution of valine for with 70% ethanol, and dried in a SpeedVac (Savant) for 15 mm. isoleucine at residue 462 in the heme-binding region of the The pellet was resuspended in 200 pA of TB, and the concem- CYP1A1 protein. We assessed this polymorphism by the allele- tratiom of DNA was calculated on a OeneQuant (Pharmacia specific PCR method described by Hirvonen et a!. (11). For this Biotech). The integrity of the genomic DNA was determined by purpose, each of the primers 5’-AAOACCTCCCAGCO- resolving 5 l of the buccal DNA extract on a 1.5% agarose gel GGCAAT-3’ (for the detection of the A allele) and 5’-AAOAC- followed by visualization with ethidium bromide staining. The CTCCCAOC000CAAC-3’ (for the detection of the G allele) DNA samples were then subjected to genotyping following the were used in subsequent PCRs, together with the opposite same PCR-based protocols used in our laboratory for blood- strand primer 5’-OAAAGGCT000TCCACCCTCT-3’, the 5’ derived DNA samples. end of which is located 303 bp upstream of the AJG polymor- The first of the two CYPIA] polymorphisms studied was phic site. Three hundred ng of DNA were used in each reaction. a T-to-C transition 264 bp downstream from the poly(A) signal, The PCR conditions consisted of an initial denaturation step at which creates an MspI restriction site (m2 allele). Oenotyping 94#{176}Cfor 1 mm 30 s, followed by 25 cycles with denaturation of the CYPJA 1 alleles associated with the presence or absence at 94#{176}Cfor 1 mm and annealing and extension at 70#{176}Cfor 1 of this MspI site was carried out by PCR amplification using mm 30 5. The PCR products were then subjected to electro- primers 5’-TAOGAGTC1TOTCTCATGCCT-3’ and 5’-CAO- phoresis on an agarose gel. TGAAGAGGTGTAGCCGCT-3’ (10). Amplification was per- We also genotyped subjects for a polymorphism in the 5’ formed in a thermal cycler (Perkin-Elmer) using 300 ng of flanking region of CYP2EJ, consisting of two distinct base

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

720bp

268bp

215bp - k- ‘ . ..: #{149}.‘. __

Fig. 3. GSJ7’l and GSTMI polymorphisms analyzed by PCR. The deletion polymorphisms of GSTTI and GSTMI were studied by PCR. Lane M. Hinfl-digested X I 74 DNA molecular weight marker: Lanes 1-17. PCR products from the same 17 subjects shown in Fig. 2. each showing positive amplification ofthe internal -glohin control at 268 bp: Lane /8. negative control. The 720- and 2l5-bp fragments indicate the presence of the GS17’1 and GSTM/ genes. respectively.

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

340 bp -

200 bp - 140 bp - .:. ::- I Fig. 4. CYPIAI polymorphism analyzed by PCR. The 3-end polymorphism was studied by PCR. followed by MspI restriction enzyme digestion (6). Lotte M. Hinfl-digessed 4X174 DNA molecular weight marker: Lanes 1-17, same samples as in Fig. 2: Lane 18, negative control: Lane /9. uncut PCR product. Presence of the 200- and 140-bp fragments indicates the variant allele.

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

195bp-F 151 bp -

Fig. 5. NAD(P)H:quinane axidoreductase (NQOJ) gene polymorphism analyzed by PCR. The point mutation at position 609 was studied by PCR. kllowed by Hinfl restriction enzyme digestion (8). Lane M, Hinfl-digested 4Xl74 DNA molecular weight marker: Lanes 1-1 7. PCR products for the same I 7 subjects as in Fig. 2: Lane 18, uncut PCR product: Lane 19. negative control. The I51-bp fragment indicates the variant allele.

substitutions that are in genetic disequilibrium with each other for 15 5, annealing at 69#{176}Cfor 15 s, and extension at 72#{176}Cfor and create RsaI and PstI restriction sites (12). We used the 30 s; 2 cycles of denaturation at 94#{176}Cfor 15 s, annealing at primers 5’-TFCATFCTGTCTFCTAACTGG-3’ and 5’- 67#{176}Cfor 15 s, and extension at 72#{176}Cfor 30 s; 31 cycles of CCAOTCOAOTCTACATFOTCA-3’ to amplify a region con- denaturation at 94#{176}Cfor 30 s, annealing at 64#{176}Cfor 30 s, and taming the two distinct base substitutions. Five hundred ng of extension at 72#{176}Cfor 1 mm; and a final extension at 72#{176}Cfor DNA were used for each PCR. Cycling conditions included an S mm. initial denaturation at 94#{176}Cfor4 mm, followed by 30 cycles of In a separate experiment, multiple mouthwash samples denaturation at 94#{176}Cfor 1 mm, annealing at 55#{176}Cfor 1 mm, were collected from four individuals I day apart and either and extension at 72#{176}Cfor 1 mm and a final annealing and stored at room temperature or 37#{176}Cfor 1 week to mimic extension step at 55#{176}Cfor 1 mm and 72#{176}Cfor 7 mm, respec- conditions that may be encountered when samples are mailed or tively. RsaI digestion of the PCR products followed by reso- stored at -20#{176}Cfor 3 or 6 months to test the effect of long-term lution on an agarose gel helped identify the genotypes. storage. PCR assays were performed following the same pro- To detect deletion of the GSTMJ and/or GS77’l gene loci, tocols on these stored samples. we used the multiplex PCR method described by Deakin et al. (13), using primers 5’-OAACTCCCTGAAAAGCTAAAOC-3’ and 5’-GTF0000TCAAATATACGGTGG-3’ for GSTMJ Results and 5’-UCC’VfACTOOTCCTCACATCTC-3’ and 5’-TCAC- Sixty subjects returned a mouthwash sample, giving a partici- CGGATCATGGCCAGCA-3’ for GS7TJ. We coamplified a pation rate of 94%. These included 23 males and 37 females, 268-bp fragment of the f3-globin gene as an internal standard ages 26-68 years, and they were of various ethnic backgrounds using the primers 5’-CAAC’VfCATCCACOTTCACC-3’ and (19 Caucasian, 18 Japanese, 8 Chinese, 6 Hawaiian/part- 5’-GAAGAOCCAAOGACAGGTAC-3’. Three hundred ng of Hawaiian, 3 Asian Indian, 2 Koreans, 2 Filipinos, and 2 DNA template were used for each reaction. PCR conditions African-American subjects). Sixteen subjects were smokers. included an initial denaturation step at 94#{176}Cfor4 mm, followed Fig. 1 represents the distribution of the amounts of DNA by 30 cycles of denaturation at 94#{176}Cfor 1 mm, annealing at extracted from the mouthwash samples. The DNA yields 63#{176}Cfor 1 mm, and extension at 72#{176}Cfor 1 mm and a final ranged from 0.2 to 134.0 g, for an average of 49.7 g (SD annealing and extension step at 70#{176}Cfor 10 mm. 3 1 .7 pg). Electrophoretic analysis of the extracted buccal cell Genotyping for the NQOJ polymorphism followed a PCR DNA showed detectable levels of high molecular weight method modified from Traver et a!. (14), using primers 5’- genomic DNA in each sample (Fig. 2). The size of the gene TCCTCAGAGTOOCATFCTTGC-3’ and 5’-TCTCCTCATC- regions amplified with the PCR methods used here ranged from CTOTACCTCT-3’. One hundred ng of DNA were used in each 215 bp (for GSTMJ) to 720 bp (for GST7’l). Ethidium bromide reaction. The PCR conditions included an initial denaturation at gels of PCR products after electrophoresis or after digestion 94#{176}Cfor 1 mm, followed by: 2 cycles of denaturation at 94#{176}C with restriction enzymes followed by electrophoresis are shown

M 1 2 3 4 5 6 7 8 9 10 11 12 13 1415 1617 18 19

4lObp - - 360bp - - - -

Fig. 6. CYP2EI polymorphism analyzed by PCR. The 5-end polymorphism was studied by PCR followed by Rsal restriction enzyme digestion (12). Lane M, Hinfl-digested 4X174 DNA molecular weight marker: Lanes 1-17, samples corresponding to the same samples as in Fig. 2: Lane 18, uncut PCR product (410 bp): Lane /9, negative control. Presence of the 410-bp fragment indicates the variant allele.

1 2 3 4 5 6 7 8 9 10 ...... A GAGAGAG AG AG AG A G AG AG Fig. 7. CYPIAI exan 7 polymar- -#{149}‘ phism analyzed by PCR. The point i::. : . mutation at position 462 resulting in 322bp- a change from lIe to Val in CYP1AI [ was studied by PCR (I I). Lane M, ,Ja .t HaeIlI-digested 4X I 74 DNA malec- ular weight marker; Lanes 1-17, allele-specific PCR paired by sam- pIes as in Fig. 2; Lanes A, wild-type specific amplification; Lanes G, mu- tatian-specific amplification: Lane 11 12 13 14 15 16 17 18 19 18, positive control far Ile-Val het- .. .. -...... erozygote: Lane /9, negative can- M AGAGAGAGAGAGAGAGAG trals. Presence of the 322-bp fragment in Lane G indicates the replacement mutation of lIe far Val in exan 7. . a -- - ...- .. . 322 bp - ‘

in Figs. 3-7 for the first 17 samples extracted. Using the Table I DNA yields (in jig) from mouthwash samples by storage condition extracted DNA samples, we were able to unequivocally geno- and duration before extraction type all 60 subjects for the six polymorphisms studied. The PCR failure rates were 2% for CYPJA] MspI, 8% for CYPJAJ Storage Storage Subj ect . Mean duration temperature ( C) J!e-Val, 5% for CYP2EJ RsaI, 12% for NQOJ, and 2% for A B C ID GSTMJ/GSTfl. All subjects were successfully genotyped for Extracted: all genes as the result of a second attempt, except three subjects, Immediately 80 24 31 41 44.0 for whom a third PCR was required for either CYP2EJ RsaI or After I week Room temperature 47 10 24 73 38.5 GSTMI/GSTTI. The null GSTMI and GSTJ’I genotypes were After I week 37 101 10 17 43 42.7 found in 41 and 37% of subjects, respectively. The variant After 3 months -20 65 16 55 41 44.2 allele frequencies for the NQOJ, CYP2EI RsaI, CYPJA] MspI, After 6 months -20 1 10 18 50 34 53.0 and CYP/A1 I!e-Val polymorphisms were 0.22, 0.13, 0.28, and 0. 17, respectively. These alleles frequencies are comparable to past results for a predominantly Asian population (15). Within epidemiology may need reevaluation. Blood collection is inva- the constraints of our sample size, we did not detect any obvious effect of age, sex, race, or smoking status on DNA sive and expensive and is not always accepted or practical. It yield or ability to PCR amplify the samples or on the intensity also requires special handling and storage. By contrast, buccal of the bands on the gels. cell collection is noninvasive and simple to perform and re- Table 1 represents the DNA yields obtained in the storage quires no special equipment or training. The use of such a experiment. There was no apparent change in DNA yield with source of genomic DNA in lieu of, or as an alternative to, blood storage of the mouthwash samples from four individuals either collection is likely to increase participation and reduce costs in molecular epidemiological studies. for I week at room temperature or 37#{176}Cor for 3 or 6 months The validity of using DNA isolated from buccal cells has at - 20#{176}C.Fig. 8 compares the electrophoretic patterns of the PCR products obtained with these samples after amplification been demonstrated in previous studies. Richards et a!. (16), collected buccal cells on cytology brushes or swabs from 533 of GSTMI . No difference could be detected in the intensity of the bands that would have suggested a change in human DNA individuals for the multiplex amplification of five exons within concentration with storage. The genotyping of these samples the CFTR gene. The success rate of PCR multiplex amplification in this study was 99%. In a blind comparison of the for the other polymorphisms led to the same observation. frequency of 12 mutations responsible for cystic fibrosis in products amplified with DNA from both blood and buccal cell Discussion samples collected from 464 individuals, there was 100% agree- With the availability of PCR techniques that require much less ment in the results for the two types of DNA source (16). DNA than traditional Southern blotting, the notion that blood Buccal brushes or swabs may be perceived as invasive, espe- samples are the specimens of choice for molecular genetic cially outside a clinical setting. Additionally, if not analyzed

A B C D

M 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 (-) ;4 :i

340 bp - 200 bp -

l4Obp - L. Fig. 8. GSTM 1 polymorphism far samples from four subjects (A, B, C, and D) using freshly collected samples (Lane 1) and samples that have been stored at room temperature (Lane 2) or at 37CC (Lane 3) far 1 week or at -20CC for 3 months (Lane 4) or 6 months (Lane 5). Lane M, Hinfl-digested 4X174 DNA molecular weight marker: Lane (-), negative control.

shortly after collection, the brushes or swabs should be placed include DNA yields. This is an issue in studies of disease in a preservative solution to optimize DNA yield and PCR susceptibility genes in which a large number of PCR amplifi- amplification (5, 8). The toxicity of this storage solution does cations are typically conducted on the same samples. However, not make the method appropriate for unsupervised collection a few studies reported DNA yields that can be used for com- and transport by mail. parison with this study. Meulenbelt et a!. (5) reported a DNA In contrast to using brushes or swabs, several authors have yield of 1.3 ± 0.05 per buccal swab. Similarly, in the study collected buccal cells by asking subjects to rinse their mouth by Freeman et a!. (8), in which 10 buccal swabs were collected with isotonic saline. This simpler and noninvasive collection per subject, the average total DNA yield was 32 g, with a method requires no supervision by trained personnel, no use of range of 3.2-1 10.8 g. Thus, these results suggest that a single toxic reagents, and the samples can be obtained through the mouthwash sample yields an amount of DNA that is compa- mail. The method has been validated in mass screening with rable to that obtained from 16 to 38 buccal swabs. This amount PCR amplifications for specific mutations with specificity and is sufficient to run several hundred PCR assays and can be sensitivity of 100% (17, 18). In an investigation of the SF508 increased by collecting multiple samples. mutations of the cystic fibrosis gene in mouth rinse samples We believe that this noninvasive method of buccal cell collected in sputum containers from over 1 1,000 blood donors, collection is likely to have a high acceptability, at least in the PCR failure rate was only 5.6% (17). These failures were populations where mouthwash is commonly used in oral hy- thought to be due to insufficient rinsing of the mouth, contain- giene, such as in the United States. The DNA extraction method ers leaking during transportation, or residual food contamina- used is sufficiently short to be practical for the processing of a tion. large number of samples and yields human genomic DNA in a In another study testing the stability of the mouth rinse form that is easily amplified by PCR. Thus, this mouthwash specimens when exposed to a variety of temperature conditions procedure appears suitable for large community-based studies (7 days at -20#{176}C,4#{176}C,25#{176}C,or 37#{176}C)possibly encountered of genetic susceptibility to disease in which samples can be when samples are obtained by mail, it was noted that the collected by the participants themselves, kept at room temper- specimens stored at 25#{176}Cand 37#{176}Cwere more likely to yield ature for several days during transportation back to the study increased amount of high molecular weight DNA, possibly of center, and stored for months prior to DNA extraction. bacterial origin (19). Foreign DNA is unlikely to interfere with PCR amplification of specific alleles, and the subsequent visualization of the amplification products on the agarose gel. Acknowledgements However, it was also noted in this study that “samples stored at We thank the subjects for their participation. Hangwei Chen far his work on the higher temperatures resulted in slightly less robust PCR reac- NQOI protocol, and Ann Seifried far helpful comments. tions” (19). The method we propose here minimizes the chance of substantial bacterial contamination by collecting samples References after subjects brushed their teeth and by using an alcoholic solution. The alcohol content of the mouthwash brand that we 1. Lench, N., Stanier, P., and Wiliamsan, R. Simple non-invasive method to obtain DNA for gene analysis. Lancet, i: 1356-1358. 1988. used was 21.6%. Indeed, we did not observe greater DNA 2. Tabal, K.. Layton, D. M., and Mufti, G. J. Non-invasive isolation of consti- yields in samples stored at room temperature or at 37#{176}Cfor 1 tutianal DNA far genetic analysis. Lancet. ii: 1281-1282. 1989. week, and the PCRs worked well. Moreover, mint-flavored 3. Martin, M., Carringtan. M.. and Mann, D. A method for using serum or plasma mouthwash presented in a store-bought, sealed bottle of a as a source of DNA far HLA typing. Hum. Immunal., 33: 108-1 13, 1992. familiar brand is likely to be more acceptable to study subjects, 4. Hagerman, R. J., Wilson, P., Staley. L. W.. Lang. K. A.. Fan. T.. Uhlhom. C.. as part of a sample collection kit, than the saline solution used Jewell-Smart, S., Hull, C., Driska, J., Flom, K., and Taylor. A. K. Evaluation of in previous studies. Possibility of leakage during transport can school children at high risk far fragile X syndrome utilizing buccal cell FMR- I be minimized with the use of an appropriate container. testing. Am. J. Med. Genet.. 51: 474-481. 1994. Our studies thus far have also shown that the suitability for 5. Meulenbelt, I., Droog. S.. Trammelen, G. J. M., Baomsma, D. I., and Slag- PCR amplification of the DNA obtained by this method is not boom, P. E. High-yield naninvasive human genamic DNA isolation method for affected by freezing the mouthwash samples at -20#{176}Cfor 6 genetic studies in geographically dispersed families and populations. Am. J. Hum. Genet., 57: 1252-1254, 1995. months before DNA isolation. Although we have not yet tested longer storage durations, the samples are likely to remain stable 6. Thomson. D. M.. Brown, N. N., and Clague. A. E. Routine use of hair root or buccal swab specimens for PCR analysis: advantages aver using blood. Clin. for a longer period of time. Chim. Acta, 207: 169-174, 1992. In previous studies, samples were apparently collected for 7. Bltimeke, B., Bennett, W. P., Hams, C. C., and Shields. P. G. Serum. plasma a single genetic test. Thus, these previous reports focused on the and paraffin-embedded tissues as sources of DNA far studying cancer suscepti- successful amplification of a particular sequence and did not bility genes. Carcinagenesis (Land.), 18: 1271-1275, 1997.

8. Freeman, B., Powell, J., Ball, D., Hill, L., Craig, I., and Plomin, R. DNA by 14. Traver. R. ID., Siegel. ID., Beall, H. ID., Phillips, R. M., Gibson, N. W., mail: an inexpensive and noninvasive method far collecting DNA samples from Franklin, W. A., and Ross, ID. Characterization of a polymorphism in NAID(P)H: widely dispersed populations. Behav. Genet.. 27: 251-257. 1997. quinone oxidoreductase (DT-diaphorase). Br. J. Cancer. 75: 69-75, 1997.

9. Walsh. D. J., Corey. A. C., Cotton. R. W.. Forman, L., Herrin, G. L.. Jr., Word, 15. Sivaraman, L., Lau, A. F.. and Le Marchand, L. Frequency of CYPIAI, C. J., and Gamer, D. ID. Isolation of DNA farm saliva and forensic science CYP2D6, CYP2EI, GSTMJ and p53 polymorphisms in ethnic populations of samples containing saliva. J. Forensic Sci.. 37: 387-395, 1992. Hawaii. In: R. S. Rao, M. 0. Deo, L. ID. Sanghvi, and I. Mittra (eds.), Proceedings of the XVI UICC International Cancer Congress, New Delhi, India, pp. 17 1-175. 10. Sivaraman, L., Leatham, M. P., Yee, J., Wilkens, L. R., Lou, A. F., and Le Bologna. Italy: Monduzzi Editore, 1994. Marchand. L. CYPIAJ genetic polymorphisms and in situ colorectal cancer. 16. Richards, B., Skoletsky, J.. Shuber, A. P., Balfour, R., Stern, R. C., Dorkin, Cancer Res., 54: 3692-3695. 1994. H. L., Parad, R. B., Witt, ID.. and Klinger, K. W. Multiplex PCR amplification for I I. Hirvonen, A., Husgafvel-Pursiainen. K., Karjalainen. A., Anttila. S.. and the CFTR gene using DNA prepared from buccal brushes/swabs. Hum. Mol. Vainia, H. Point-mutational Mspl. and Ile-Val polymorphisms closely linked in Genet., 2: 159-163, 1993. the CYPJAI gene: lack of association with susceptibility to lung cancer in a 17. IDe Vries, H. G., Coll#{233}e,J. M., van Veldhuizen, M. H. R., Achterhof, L., Smit Finnish study population. Cancer Epidemiol. Biomark. Prey., 1: 485-489, 1992. Sibinga. C. T., Scheffer, H., Buys, C. H. C., and ten Kate, L. P. Validation of the 12. Hayashi. S., Watanabe, J., and Kawajiri. K. Genetic polymorphisms in the determination of F508 mutations of the cystic fibrosis gene in over I 1,000 5-flanking region change transcriptional regulation of the human cytochrome mouthwashes. Hum. Genet., 97: 334-336, 1996. P45OIIEI gene. J. Biochem., 110: 559-565, 1991. 18. Bolla, M. K., Haddad, L., Humphries, S. E., Winder, A. F., and Day, N. M. 13. Deakin, M., Elder, J.. Hendrickse, C., Peckham, ID., Baldwin, ID., Pantin, C., High-throughput methods for determination of Apolipoprotein E genotypes with Wild, N., Leopard. P., Bell, D. A., Jones, P., Duncan, H., Branningan, K., use of restriction digestion analysis by microplate array diagonal gel electro- Alldersea, J., Fryer. A. A., and Strange, R. C. Glutathione S-transferase GSTT/ phoresis. Clin. Chem., 41: 1599-1604, 1995. genotypes and susceptibility to cancer: studies of interactions with GSTMI in 19. Hayney, M. S., Dirnanlig, P., Lipsky, J. J., and Poland, G. A. Utility of a lung, oral. gastric and colorectal cancers. Carcinogenesis (Land.), 17: 881-884, “swish and spit” technique for the collection of buccal cells for TAT haplotype 1996. determination. Mayo CIin. Proc., 70: 951-954. 1995.

A Lum and L Le Marchand

Cancer Epidemiol Biomarkers Prev 1998;7:719-724.

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