a simple mouthwash method for obtaining genomic dna in ... · a simple mouthwash method for...

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

Short Communication

A Simple Mouthwash Method for Obtaining Genomic DNA in

Molecular Epidemiological

Annette Lum and LoIc Le Marchand2

Etiology Program. Cancer Research Center of Hawaii, University of Hawaii,

Honolulu, Hawaii 96813

Abstract

Genomic DNA for genetic analyses has traditionally beenderived from blood samples. With the availability of PCRtechniques requiring only minute amounts of DNA andthe current demand for high-volume testing, a lessinvasive, simpler to perform, and cheaper method toobtain DNA is desirable. We developed a method toobtain high-quality genomic DNA from buccal cells that

has high acceptability and allows for a large number ofPCR assays from a single sample. Sixty subjectsvigorously swished 10 ml of undiluted commercialmouthwash in the mouth for 60 s and expelled the liquid

into a collection container. DNA was isolated from thebuccal cells with a rapid method using proteinase Kdigestion, phenol-chloroform extraction, and ethanolprecipitation. Electrophoretic analysis of the extractedDNA showed detectable levels of high molecular weightgenomic DNA in all samples. The DNA yields rangedfrom 0.2 to 134.0 ,zg, for an average of 49.7 �tg. Usingthese samples, all 60 subjects were successfully genotypedby PCR-based assays for polymorphisms in the CYPJAJ(MspI and exon 7), CYP2EJ (RsaI), GSTMJ, GSTTJ, andNQOJ genes, confirming that the quality of DNA isolatedfrom mouthwash samples was sufficient to reliablysupport PCR amplification. Storage of the (unprocessed)specimens at room temperature or at 37#{176}Cfor 1 week(temperature conditions that may be encountered whenmailing samples) or at -20#{176}C for at least 6 months didnot affect the DNA yield or ability to PCR amplify thesamples. The results suggest that this mouthwashprocedure may be suitable for large community-basedstudies of genetic susceptibility to disease in whichsamples can be collected by the participants themselves,mailed back to the study center, and stored for monthsprior to DNA analysis.

Introduction

Almost invariably, genomic DNA for PCR-based genetic anal-

yses has been derived from leukocytes prepared from whole

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

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

t This research was supported in part by NIH Grant CA60987.

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

968 I 3.

blood. Although this method yields large amounts of DNA

N 100 j.�g from a l0-ml blood sample), an alternative source ofDNA 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

cultural or religious reasons. As a consequence, it is not un-common in molecular epidemiological studies for a substantialproportion (20-40%) of the participants to refuse the blood

collection. Furthermore, obtaining blood samples becomes pro-hibitively expensive and logistically arduous when the studysize is large or when the participants reside across a largegeographical area, as is typical in family studies, in associationstudies examining gene-gene or gene-environment interactions,

and in prospective studies of rare diseases.DNA for genetic analysis has also been derived from

finger-stick blood, paraffin-embedded tissue, urine, hair roots,saliva, cheek scrapings. buccal brushes, buccal swabs, and oral

saline rinse samples (1-8). However, these methods are some-what invasive (finger stick, cheek scrapings or brushes, and

saline rinse) or do not yield an adequate amount (urine, hairroots, and saliva) or quality (paraffin blocks) of DNA. Also,some of these methods require the samples to be stored in apreservative solution that is toxic, which makes it problematicfor use by mail (buccal brushes and swabs). This work wasaimed at developing a method to obtain high-quality genomicDNA from buccal cells that would have high applicability andacceptability and allow for a large number of PCR assays from

a single sample. Our requirements were that, with this method,

samples could be collected by the participants themselves,mailed back to the study center, and stored for months prior toDNA extraction.

Materials and Methods

Subjects for this feasibility study were recruited among the

employees of the Cancer Research Center of Hawaii and theiracquaintances. A total of 64 individuals were given a 30-mi,wide-mouth, screw-capped jar that contained 10 ml of undi-

luted mouthwash (FreshBurst Listerine), along with writteninstructions for collecting the sample at home. About 1 h afterthey brushed their teeth, the subjects swished the mouthwashvigorously throughout the mouth for 1 mm and expelled it back

into the jar. On the same day or on the following day, the

participants returned the specimens to the laboratory. Themouthwash samples were either processed within 1 week or

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 m�i Tris (pH8.0), 10 mM EDTA (pH 8.0)]. The suspension was centrifugedat 2700 rpm for 15 mm, and the supernatant was discarded. Wemodified the protocol published by Walsh et a!. (9) to extractDNA. Briefly, the pellet was resuspended in 700 �l of lysis

buffer [10 mM Tris (pH 8.0), 10 m�i EDTA (pH 8.0), 0.1 M

NaCI, and 2% SDS} and transferred to a 2-mI microcentrifuge

on April 4, 2020. © 1998 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from

http://cebp.aacrjournals.org/

‘II II I I II� III I II

Fig. 1. Distribution of amounts of DNA in �.sg ob-

tamed by proteinase K digestion, phenol-chloroform

extraction, and ethanol precipitation from singlemouthwash samples collected by 60 subjects. The

DNA concentration of each sample was determined by

spectrophotometer (GeneQuant II: Pharmacia Bio-

tech) and ranged from 0.2 to 134 �sg.

t, � (� �

� � �cg � $ $

DNA �.Lg)

l�23 4 5 6 7 8 9 10 11 12 13 l4,,

720 Obtaining DNA with a Mouthwash Method

8

7

1n69-.

0w5

Cl)Is-03

0z2

I

0

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 molecularweight DNA.

tube containing 35 �l of 20 mg/mi proteinase K. The sampleswere mixed and digested at 58#{176}Cfor 2 h. The DNA was then

extracted from each sample with equal volumes of phenol-chloroform ( 1 : 1 ) and with an equal volume of chloroformalone, each time vortexing for 10 s and centrifuging at 14,000rpm for 2 mm. The DNA was removed from the supernatantwith 3 M NaOAc (pH 6.0; 1/10 volume of supernatant) and 2volumes of cold 100% ethanol and precipitated at -20#{176}Cfor

2 h. The DNA was pelleted at 10,000 rpm for 10 mm, washedwith 70% ethanol, and dried in a SpeedVac (Savant) for 15 mm.

The pellet was resuspended in 200 pA of TB, and the concem-

tratiom of DNA was calculated on a OeneQuant (PharmaciaBiotech). The integrity of the genomic DNA was determined byresolving 5 �l of the buccal DNA extract on a 1.5% agarose gelfollowed by visualization with ethidium bromide staining. The

DNA samples were then subjected to genotyping following thesame PCR-based protocols used in our laboratory for blood-

derived DNA samples.The first of the two CYPIA] polymorphisms studied was

a T-to-C transition 264 bp downstream from the poly(A) signal,which creates an MspI restriction site (m2 allele). Oenotypingof the CYPJA 1 alleles associated with the presence or absenceof this MspI site was carried out by PCR amplification usingprimers 5’-TAOGAGTC1TOTCTCATGCCT-3’ and 5’-CAO-

TGAAGAGGTGTAGCCGCT-3’ (10). Amplification was per-formed in a thermal cycler (Perkin-Elmer) using 300 ng of

DNA template with initial denaturation at 95#{176}Cfor 4 mm,

followed by 30 cycles with denaturation at 95#{176}Cfor 1 mm,annealing at 65#{176}Cfor 1 mm, and extension at 72#{176}Cfor 1 mm

and a final annealing and extension step at 65#{176}Cfor 1 mm and72#{176}Cfor 8 mm, respectively. The PCR product was thendigested with MspI and subjected to electrophoresis on anagarose gel.

The second CYPJA] polymorphism studied was an A-to-G transition, which results in the substitution of valine forisoleucine at residue 462 in the heme-binding region of theCYP1A1 protein. We assessed this polymorphism by the allele-specific PCR method described by Hirvonen et a!. (11). For this

purpose, each of the primers 5’-AAOACCTCCCAGCO-

GGCAAT-3’ (for the detection of the A allele) and 5’-AAOAC-CTCCCAOC000CAAC-3’ (for the detection of the G allele)were used in subsequent PCRs, together with the opposite

strand primer 5’-OAAAGGCT000TCCACCCTCT-3’, the 5’end of which is located 303 bp upstream of the AJG polymor-phic site. Three hundred ng of DNA were used in each reaction.The PCR conditions consisted of an initial denaturation step at

94#{176}Cfor 1 mm 30 s, followed by 25 cycles with denaturationat 94#{176}Cfor 1 mm and annealing and extension at 70#{176}Cfor 1mm 30 5. The PCR products were then subjected to electro-phoresis on an agarose gel.

We also genotyped subjects for a polymorphism in the 5’

flanking region of CYP2EJ, consisting of two distinct base



720bp

268bp215bp -

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

k- ‘ � . ..:� #{149}.�‘. __

340 bp -

200 bp -

140 bp -

Cancer Epidemiology, Biomarkers & Prevention 721

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�

.�:. �::�- � IFig. 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-F151 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 otherand create RsaI and PstI restriction sites (12). We used theprimers 5’-TFCATFCTGTCTFCTAACTGG-3’ and 5’-

CCAOTCOAOTCTACATFOTCA-3’ to amplify a region con-taming the two distinct base substitutions. Five hundred ng ofDNA were used for each PCR. Cycling conditions included aninitial denaturation at 94#{176}Cfor 4 mm, followed by 30 cycles of

denaturation at 94#{176}Cfor 1 mm, annealing at 55#{176}Cfor 1 mm,

and extension at 72#{176}Cfor 1 mm and a final annealing andextension step at 55#{176}Cfor 1 mm and 72#{176}Cfor 7 mm, respec-

tively. RsaI digestion of the PCR products followed by reso-lution on an agarose gel helped identify the genotypes.

To detect deletion of the GSTMJ and/or GS77’l gene loci,we used the multiplex PCR method described by Deakin et al.

(13), using primers 5’-OAACTCCCTGAAAAGCTAAAOC-3’and 5’-GTF0000TCAAATATACGGTGG-3’ for GSTMJ

and 5’-UCC’VfACTOOTCCTCACATCTC-3’ and 5’-TCAC-CGGATCATGGCCAGCA-3’ for GS7TJ. We coamplified a268-bp fragment of the f3-globin gene as an internal standard

using the primers 5’-CAAC’VfCATCCACOTTCACC-3’ and5’-GAAGAOCCAAOGACAGGTAC-3’. Three hundred ng ofDNA template were used for each reaction. PCR conditions

included an initial denaturation step at 94#{176}Cfor 4 mm, followed

by 30 cycles of denaturation at 94#{176}Cfor 1 mm, annealing at63#{176}Cfor 1 mm, and extension at 72#{176}Cfor 1 mm and a finalannealing and extension step at 70#{176}Cfor 10 mm.

Genotyping for the NQOJ polymorphism followed a PCRmethod modified from Traver et a!. (14), using primers 5’-TCCTCAGAGTOOCATFCTTGC-3’ and 5’-TCTCCTCATC-

CTOTACCTCT-3’. One hundred ng of DNA were used in eachreaction. The PCR conditions included an initial denaturation at

94#{176}Cfor 1 mm, followed by: 2 cycles of denaturation at 94#{176}C

for 15 5, annealing at 69#{176}Cfor 15 s, and extension at 72#{176}Cfor30 s; 2 cycles of denaturation at 94#{176}Cfor 15 s, annealing at67#{176}Cfor 15 s, and extension at 72#{176}Cfor 30 s; 31 cycles ofdenaturation at 94#{176}Cfor 30 s, annealing at 64#{176}Cfor 30 s, and

extension at 72#{176}Cfor 1 mm; and a final extension at 72#{176}CforS mm.

In a separate experiment, multiple mouthwash samples

were collected from four individuals I day apart and either

stored at room temperature or 37#{176}Cfor 1 week to mimicconditions that may be encountered when samples are mailed or

stored at -20#{176}Cfor 3 or 6 months to test the effect of long-termstorage. PCR assays were performed following the same pro-tocols on these stored samples.

Results

Sixty subjects returned a mouthwash sample, giving a partici-

pation rate of 94%. These included 23 males and 37 females,ages 26-68 years, and they were of various ethnic backgrounds

(19 Caucasian, 18 Japanese, 8 Chinese, 6 Hawaiian/part-Hawaiian, 3 Asian Indian, 2 Koreans, 2 Filipinos, and 2African-American subjects). Sixteen subjects were smokers.

Fig. 1 represents the distribution of the amounts of DNAextracted from the mouthwash samples. The DNA yieldsranged from 0.2 to 134.0 �g, for an average of 49.7 �g (SD3 1 .7 p�g). Electrophoretic analysis of the extracted buccal cell

DNA showed detectable levels of high molecular weightgenomic DNA in each sample (Fig. 2). The size of the generegions amplified with the PCR methods used here ranged from

215 bp (for GSTMJ) to 720 bp (for GST7’l). Ethidium bromidegels of PCR products after electrophoresis or after digestionwith restriction enzymes followed by electrophoresis are shown



1 2

AG AG

322bp- [�3 4 5 6

.. .. ..

A GAGAGAG

� -#{149}‘

i::. � : � .

7AG

8 9 10..

A G AG AG

,�Ja .�t

322 bp -

11..

12..

13-.

14..

15..

16..

17..

18..

19..

M AGAGAGAGAGAGAGAGAG

Fig. 7. CYPIAI exan 7 polymar-phism analyzed by PCR. The point

mutation at position 462 resulting in

a change from lIe to Val in CYP1AIwas studied by PCR (I I). Lane M,

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

18, positive control far Ile-Val het-

erozygote: Lane /9, negative can-

trals. Presence of the 322-bp frag-ment in Lane G indicates the

replacement mutation of lIe far Val

in exan 7.. � a� -- � - ‘� ..�.- .. � � . �

722 Obtaining DNA with a Mouthwash Method

4lObp -

360bp

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

-�-� �-� - � � -

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.

in Figs. 3-7 for the first 17 samples extracted. Using theextracted DNA samples, we were able to unequivocally geno-

type all 60 subjects for the six polymorphisms studied. ThePCR failure rates were 2% for CYPJA] MspI, 8% for CYPJAJ

J!e-Val, 5% for CYP2EJ RsaI, 12% for NQOJ, and 2% for

GSTMJ/GSTfl. All subjects were successfully genotyped forall genes as the result of a second attempt, except three subjects,for whom a third PCR was required for either CYP2EJ RsaI or

GSTMI/GSTTI. The null GSTMI and GSTJ’I genotypes werefound in 41 and 37% of subjects, respectively. The variantallele frequencies for the NQOJ, CYP2EI RsaI, CYPJA] MspI,and CYP/A1 I!e-Val polymorphisms were 0.22, 0.13, 0.28, and0. 17, respectively. These alleles frequencies are comparable to

past results for a predominantly Asian population (15). Withinthe constraints of our sample size, we did not detect anyobvious effect of age, sex, race, or smoking status on DNAyield or ability to PCR amplify the samples or on the intensityof the bands on the gels.

Table 1 represents the DNA yields obtained in the storage

experiment. There was no apparent change in DNA yield with

storage of the mouthwash samples from four individuals either

for I week at room temperature or 37#{176}Cor for 3 or 6 months

at - 20#{176}C.Fig. 8 compares the electrophoretic patterns of thePCR products obtained with these samples after amplificationof GSTMI . No difference could be detected in the intensity of

the bands that would have suggested a change in human DNAconcentration with storage. The genotyping of these samplesfor the other polymorphisms led to the same observation.

Discussion

With the availability of PCR techniques that require much less

DNA than traditional Southern blotting, the notion that bloodsamples are the specimens of choice for molecular genetic

Table I DNA yields (in jig) from mouthwash samples by storage condition

and duration before extraction

Storage.

duration

Storage

temperature ( C) A

Subj

B

ect

C IDMean

Extracted:

Immediately 80 24 31 41 44.0

After I week Room temperature 47 10 24 73 38.5

After I week 37 101 10 17 43 42.7

After 3 months -20 65 16 55 41 44.2

After 6 months -20 1 10 18 50 34 53.0

epidemiology may need reevaluation. Blood collection is inva-sive and expensive and is not always accepted or practical. Italso requires special handling and storage. By contrast, buccalcell collection is noninvasive and simple to perform and re-quires no special equipment or training. The use of such asource of genomic DNA in lieu of, or as an alternative to, blood

collection is likely to increase participation and reduce costs inmolecular epidemiological studies.

The validity of using DNA isolated from buccal cells has

been demonstrated in previous studies. Richards et a!. (16),collected buccal cells on cytology brushes or swabs from 533individuals for the multiplex amplification of five exons withinthe CFTR gene. The success rate of PCR multiplex amplifica-tion in this study was 99%. In a blind comparison of the

frequency of 12 mutations responsible for cystic fibrosis inproducts amplified with DNA from both blood and buccal cellsamples collected from 464 individuals, there was 100% agree-

ment in the results for the two types of DNA source (16).Buccal brushes or swabs may be perceived as invasive, espe-

cially outside a clinical setting. Additionally, if not analyzed



340 bp -

200 bp -l4Obp -

Cancer Epidemiology, Biomarkers & Prevention 723

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

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 placedin a preservative solution to optimize DNA yield and PCR

amplification (5, 8). The toxicity of this storage solution doesnot make the method appropriate for unsupervised collection

and transport by mail.In contrast to using brushes or swabs, several authors have

collected buccal cells by asking subjects to rinse their mouthwith isotonic saline. This simpler and noninvasive collectionmethod requires no supervision by trained personnel, no use oftoxic reagents, and the samples can be obtained through themail. The method has been validated in mass screening with

PCR amplifications for specific mutations with specificity andsensitivity of 100% (17, 18). In an investigation of the SF508

mutations of the cystic fibrosis gene in mouth rinse samples

collected in sputum containers from over 1 1 ,000 blood donors,the PCR failure rate was only 5.6% (17). These failures were

thought to be due to insufficient rinsing of the mouth, contain-

ers leaking during transportation, or residual food contamina-tion.

In another study testing the stability of the mouth rinse

specimens when exposed to a variety of temperature conditions(7 days at -20#{176}C,4#{176}C,25#{176}C,or 37#{176}C)possibly encounteredwhen samples are obtained by mail, it was noted that the

specimens stored at 25#{176}Cand 37#{176}Cwere more likely to yield

increased amount of high molecular weight DNA, possibly of

bacterial origin (19). Foreign DNA is unlikely to interfere withPCR amplification of specific alleles, and the subsequent visu-alization of the amplification products on the agarose gel.However, it was also noted in this study that “samples stored at

higher temperatures resulted in slightly less robust PCR reac-tions” (19). The method we propose here minimizes the chanceof substantial bacterial contamination by collecting samplesafter subjects brushed their teeth and by using an alcoholic

solution. The alcohol content of the mouthwash brand that weused was 21.6%. Indeed, we did not observe greater DNAyields in samples stored at room temperature or at 37#{176}Cfor 1week, and the PCRs worked well. Moreover, mint-flavoredmouthwash presented in a store-bought, sealed bottle of a

familiar brand is likely to be more acceptable to study subjects,as part of a sample collection kit, than the saline solution usedin previous studies. Possibility of leakage during transport can

be minimized with the use of an appropriate container.

Our studies thus far have also shown that the suitability for

PCR amplification of the DNA obtained by this method is notaffected by freezing the mouthwash samples at -20#{176}Cfor 6months before DNA isolation. Although we have not yet testedlonger storage durations, the samples are likely to remain stablefor a longer period of time.

In previous studies, samples were apparently collected fora single genetic test. Thus, these previous reports focused on thesuccessful amplification of a particular sequence and did not

include DNA yields. This is an issue in studies of disease

susceptibility genes in which a large number of PCR amplifi-

cations are typically conducted on the same samples. However,a few studies reported DNA yields that can be used for com-parison with this study. Meulenbelt et a!. (5) reported a DNAyield of 1.3 ± 0.05 � per buccal swab. Similarly, in the study

by Freeman et a!. (8), in which 10 buccal swabs were collectedper subject, the average total DNA yield was 32 �g, with arange of 3.2-1 10.8 �g. Thus, these results suggest that a singlemouthwash sample yields an amount of DNA that is compa-rable to that obtained from 16 to 38 buccal swabs. This amount

is sufficient to run several hundred PCR assays and can be

increased by collecting multiple samples.We believe that this noninvasive method of buccal cell

collection is likely to have a high acceptability, at least in

populations where mouthwash is commonly used in oral hy-giene, such as in the United States. The DNA extraction method

used is sufficiently short to be practical for the processing of alarge number of samples and yields human genomic DNA in aform that is easily amplified by PCR. Thus, this mouthwash

procedure appears suitable for large community-based studiesof genetic susceptibility to disease in which samples can be

collected by the participants themselves, kept at room temper-ature for several days during transportation back to the studycenter, and stored for months prior to DNA extraction.

Acknowledgements

We thank the subjects for their participation. Hangwei Chen far his work on the

NQOI protocol, and Ann Seifried far helpful comments.

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1998;7:719-724. Cancer Epidemiol Biomarkers Prev A Lum and L Le Marchand molecular epidemiological studies.A simple mouthwash method for obtaining genomic DNA in

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