Download A Simple Mouthwash Method for Obtaining Genomic DNA in

Vol.
7, 719-724,
August
1998
Cancer
Short
A Simple
Mouthwash
Annette
Etiology
Honolulu,
Lum
Program.
Hawaii
Research
Center
of Hawaii,
University
of Hawaii,
Abstract
Genomic
DNA for genetic
analyses
has traditionally
been
derived from blood samples.
With the availability
of PCR
techniques
requiring
only minute
amounts
of DNA and
the current
demand
for high-volume
testing,
a less
invasive,
simpler
to perform,
and cheaper
method to
obtain DNA is desirable.
We developed
a method
to
obtain high-quality
genomic
DNA from buccal
cells that
has high acceptability
and allows
for a large number
of
PCR assays
from a single
sample.
Sixty subjects
vigorously
swished
10 ml of undiluted
commercial
mouthwash
in the mouth
for 60 s and expelled
the liquid
into a collection
container.
DNA was isolated
from the
buccal cells with a rapid method
using proteinase
K
digestion,
phenol-chloroform
extraction,
and ethanol
precipitation.
Electrophoretic
analysis
of the extracted
DNA showed
detectable
levels
of high molecular
weight
genomic
DNA in all samples.
The DNA yields
ranged
from 0.2 to 134.0 ,zg, for an average
of 49.7 tg. Using
these samples,
all 60 subjects
were successfully
genotyped
by PCR-based
assays
for polymorphisms
in the CYPJAJ
(MspI and exon 7), CYP2EJ
(RsaI),
GSTMJ,
GSTTJ,
and
NQOJ
genes,
confirming
that the quality
of DNA isolated
from mouthwash
samples
was sufficient
to reliably
support
PCR amplification.
Storage
of the (unprocessed)
specimens
at room temperature
or at 37#{176}C
for 1 week
(temperature
conditions
that may be encountered
when
mailing
samples)
or at -20#{176}C for at least 6 months
did
not affect the DNA yield or ability to PCR amplify
the
samples.
The results
suggest
that this mouthwash
procedure
studies
samples
mailed
prior
may be suitable for large community-based
of genetic susceptibility
to disease in which
can be collected
back to the study
to DNA analysis.
by the
center,
participants
and stored
themselves,
for months
Introduction
Almost
invariably,
genomic
DNA for PCR-based
yses has been derived
from leukocytes
prepared
& Prevention
for
Obtaining
Genomic
DNA
in
Epidemiological
and LoIc Le Marchand2
Cancer
96813
Biomarkers
Communication
Method
Molecular
Epidemiology,
genetic
from
analwhole
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
in
accordance
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 of
DNA is desirable
because
venipuncture
is invasive
and uncomfortable
for the subject
and involves
risk of exposure
to bloodborne pathogens.
It is also unacceptable
in some individuals
for
cultural
or religious
reasons.
As a consequence,
it is not uncommon
in molecular
epidemiological
studies
for a substantial
proportion
(20-40%)
of the participants
to refuse
the blood
collection.
Furthermore,
obtaining
blood samples
becomes
prohibitively
expensive
and logistically
arduous
when
the study
size is large or when
the participants
reside
across
a large
geographical
area, as is typical
in family studies,
in association
studies 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 somewhat invasive
(finger
stick, cheek
scrapings
or brushes,
and
saline
rinse) or do not yield an adequate
amount
(urine,
hair
roots, and saliva)
or quality
(paraffin
blocks)
of DNA.
Also,
some of these methods
require
the samples
to be stored
in a
preservative
solution
that is toxic, which makes
it problematic
for use by mail (buccal
brushes
and swabs).
This work was
aimed
at developing
a method
to obtain
high-quality
genomic
DNA from buccal
cells that would
have high applicability
and
acceptability
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 to
DNA extraction.
Materials
and
Methods
Subjects
for this feasibility
study
were recruited
among
the
employees
of the Cancer
Research
Center
of Hawaii
and their
acquaintances.
A total of 64 individuals
were given a 30-mi,
wide-mouth,
screw-capped
jar that contained
10 ml of undiluted
mouthwash
(FreshBurst
Listerine),
along
with written
instructions
for collecting
the sample
at home.
About
1 h after
they brushed
their teeth, the subjects
swished
the mouthwash
vigorously
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.
The
mouthwash
samples
were either
processed
within
1 week or
stored
at - 20#{176}C
for later extraction.
Processing
consisted
of
transferring
the sample
to a 50-mi conical
tube for centrifugation 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
8.0), 10 mM EDTA (pH 8.0)]. The suspension
was centrifuged
at 2700 rpm for 15 mm, and the supernatant
was discarded.
We
modified
the protocol
published
by Walsh
et a!. (9) to extract
DNA.
Briefly,
the pellet was resuspended
in 700 l of lysis
buffer
[10 mM Tris (pH 8.0), 10 mi EDTA (pH 8.0), 0.1 M
NaCI, and 2% SDS} and transferred
to a 2-mI microcentrifuge
Downloaded from cebp.aacrjournals.org on August 9, 2017. © 1998 American Association for Cancer Research.
7/9
720
Obtaining
DNA
with
a Mouthwash
Method
8
7
1n6
9-.
0
w5
Fig. 1.
Distribution
tamed
by proteinase
Cl)
Is-
extraction,
mouthwash
03
0
z2
I
0
t,
‘II II I I II
I
of DNA
in .sg obphenol-chloroform
and
ethanol
precipitation
samples
collected
by 60
DNA concentration
spectrophotometer
tech) and ranged
III
of amounts
K digestion,
from
subjects.
of each sample
was determined
(GeneQuant
II: Pharmacia
from 0.2 to 134 sg.
single
The
by
Bio-
II
(
$
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 dcctrophoresis
in a I .8% agarose
gel and
visualized
with ethidium
bromide,
as
shown
here for the
extracted.
Arrow,
weight
DNA.
first
high
I 7 samples
molecular
tube containing
35 l of 20 mg/mi
proteinase
K. The samples
were mixed and digested
at 58#{176}C
for 2 h. The DNA was then
extracted
from each sample
with equal
volumes
of phenolchloroform
( 1: 1) and with an equal
volume
of chloroform
alone, each time vortexing
for 10 s and centrifuging
at 14,000
rpm for 2 mm. The DNA was removed
from the supernatant
with 3 M NaOAc
(pH 6.0; 1/10 volume
of supernatant)
and 2
volumes
of cold 100% ethanol
and precipitated
at -20#{176}Cfor
2 h. The DNA was pelleted
at 10,000 rpm for 10 mm, washed
with 70% ethanol,
and dried in a SpeedVac
(Savant)
for 15 mm.
The pellet was resuspended
in 200 pA of TB, and the concemtratiom
of DNA
was calculated
on a OeneQuant
(Pharmacia
Biotech).
The integrity
of the genomic
DNA was determined
by
resolving
5 l of the buccal DNA extract
on a 1.5% agarose
gel
followed
by visualization
with ethidium
bromide
staining.
The
DNA samples
were then subjected
to genotyping
following
the
same PCR-based
protocols
used in our laboratory
for bloodderived
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).
Oenotyping
of the CYPJA
1 alleles
associated
with the presence
or absence
of this MspI site was carried
out by PCR amplification
using
primers
5’-TAOGAGTC1TOTCTCATGCCT-3’
and 5’-CAOTGAAGAGGTGTAGCCGCT-3’
(10). Amplification
was performed
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}C
for 1 mm,
annealing
at 65#{176}C
for 1 mm, and extension
at 72#{176}C
for 1 mm
and a final annealing
and extension
step at 65#{176}C
for 1 mm and
72#{176}Cfor 8 mm, respectively.
The PCR
product
was then
digested
with MspI
and subjected
to electrophoresis
on an
agarose gel.
The second
CYPJA]
polymorphism
studied
was an Ato-G transition,
which
results
in the substitution
of valine
for
isoleucine
at residue
462 in the heme-binding
region
of the
CYP1A1
protein. We assessed
this polymorphism
by the allelespecific
PCR method described
by Hirvonen
et a!. (11). For this
purpose,
each
of the primers
5’-AAOACCTCCCAGCOGGCAAT-3’
(for the detection
of the A allele) and 5’-AAOACCTCCCAOC000CAAC-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 polymorphic site. Three hundred
ng of DNA were used in each reaction.
The PCR conditions
consisted
of an initial denaturation
step at
94#{176}C
for 1 mm 30 s, followed
by 25 cycles
with denaturation
at 94#{176}C
for 1 mm and annealing
and extension
at 70#{176}C
for 1
mm 30 5. The PCR products
were then subjected
to electrophoresis
on an agarose
gel.
We also genotyped
subjects
for a polymorphism
in the 5’
flanking
region
of CYP2EJ,
consisting
of two distinct
base
Downloaded from cebp.aacrjournals.org on August 9, 2017. © 1998 American Association for Cancer Research.
Cancer
M
1
2
3
4
5
6
7
8
9
10
11 12
Epidemiology,
13 14 15 16
Biomarkers
& Prevention
17 18 19
720bp
.
268bp
215bp
-
kFig. 3.
GSJ7’l
DNA molecular
at 268
bp: Lane
and GSTMI
polymorphisms
weight
marker:
Lanes
1-17.
/8.
negative
control.
The
analyzed
by PCR. The deletion
polymorphisms
PCR products
from the same 17 subjects
shown
720-
M
340 bp
200 bp
140 bp
and
1
2l5-bp
2
fragments
3
indicate
5
4
6
of GSTTI
and GSTMI
in Fig. 2. each showing
the presence
7
.:
.
‘
8
of the GS17’1
9
10
11
and
12
13
__
#{149}.
‘.
were studied
by PCR.
positive
amplification
GSTM/
genes.
14
15
Lane
ofthe
M. Hinfl-digested
internal
-glohin
respectively.
16
17
18
19
-
::-
.:.
Fig. 4.
CYPIAI
polymorphism
analyzed
by PCR.
The 3-end
polymorphism
Hinfl-digessed
4X174
DNA molecular
weight
marker:
Lanes
1-17,
same samples
200- and 140-bp
fragments
indicates
the variant
allele.
M
1
2
3
4
5
6
7
by MspI restriction
enzyme
control:
Lane /9. uncut PCR
was studied
by PCR.
followed
as in Fig. 2: Lane 18, negative
8
9
X I 74
control
10
11
12 13 14 15 16
The
point
mutation
17
18
I
digestion
product.
(6). Lotte M.
Presence
of the
19
195bp-F
151 bp
Fig.
5.
restriction
18, uncut
NAD(P)H:quinane
-
axidoreductase
(NQOJ)
gene
enzyme
digestion
(8). Lane M, Hinfl-digested
PCR product:
Lane 19. negative
control.
The
polymorphism
4Xl74
I51-bp
analyzed
DNA molecular
fragment
indicates
by PCR.
weight
marker:
Lanes
the variant
allele.
substitutions
that are in genetic
disequilibrium
with each other
and create
RsaI and PstI restriction
sites (12). We used the
primers
5’-TFCATFCTGTCTFCTAACTGG-3’
and
5’CCAOTCOAOTCTACATFOTCA-3’
to amplify
a region
contaming
the two distinct
base substitutions.
Five hundred
ng of
DNA were used for each PCR. Cycling
conditions
included
an
initial denaturation
at 94#{176}C
for 4 mm, followed
by 30 cycles of
denaturation
at 94#{176}C
for 1 mm, annealing
at 55#{176}C
for 1 mm,
and extension
at 72#{176}C
for 1 mm and a final annealing
and
extension
step at 55#{176}C
for 1 mm and 72#{176}C
for 7 mm, respectively.
RsaI digestion
of the PCR products
followed
by resolution 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’-TCACCGGATCATGGCCAGCA-3’
for GS7TJ.
We coamplified
a
268-bp
fragment
of the f3-globin
gene as an internal
standard
using the primers
5’-CAAC’VfCATCCACOTTCACC-3’
and
5’-GAAGAOCCAAOGACAGGTAC-3’.
Three hundred
ng of
DNA
template
were used for each reaction.
PCR conditions
included
an initial denaturation
step at 94#{176}C
for 4 mm, followed
by 30 cycles
of denaturation
at 94#{176}C
for 1 mm, annealing
at
63#{176}C
for 1 mm, and extension
at 72#{176}C
for 1 mm and a final
annealing
and extension
step at 70#{176}C
for 10 mm.
Genotyping
for the NQOJ
polymorphism
followed
a PCR
method
modified
from Traver
et a!. (14),
using
primers
5’TCCTCAGAGTOOCATFCTTGC-3’
and 5’-TCTCCTCATCCTOTACCTCT-3’.
One hundred
ng of DNA were used in each
reaction.
The PCR conditions
included
an initial denaturation
at
94#{176}C
for 1 mm, followed
by: 2 cycles
of denaturation
at 94#{176}C
at position
1-1 7. PCR
products
609
was
studied
for the same
by PCR.
I 7 subjects
kllowed
as in Fig.
by Hinfl
2: Lane
for 15 5, annealing
at 69#{176}C
for 15 s, and extension
at 72#{176}C
for
30 s; 2 cycles
of denaturation
at 94#{176}C
for 15 s, annealing
at
67#{176}C
for 15 s, and extension
at 72#{176}C
for 30 s; 31 cycles
of
denaturation
at 94#{176}C
for 30 s, annealing
at 64#{176}C
for 30 s, and
extension
at 72#{176}C
for 1 mm; and a final extension
at 72#{176}C
for
S 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 mimic
conditions
that may be encountered
when samples
are mailed or
stored at -20#{176}Cfor 3 or 6 months
to test the effect of long-term
storage.
PCR assays
were performed
following
the same protocols on these stored samples.
Results
Sixty subjects
returned
a mouthwash
sample,
giving
a participation
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/partHawaiian,
3 Asian
Indian,
2 Koreans,
2 Filipinos,
and 2
African-American
subjects).
Sixteen
subjects
were smokers.
Fig. 1 represents
the distribution
of the amounts
of DNA
extracted
from
the mouthwash
samples.
The
DNA
yields
ranged
from 0.2 to 134.0 g, for an average
of 49.7 g (SD
3 1 .7 pg). Electrophoretic
analysis
of the extracted
buccal
cell
DNA
showed
detectable
levels
of high
molecular
weight
genomic
DNA in each sample
(Fig. 2). The size of the gene
regions
amplified
with the PCR methods
used here ranged
from
215 bp (for GSTMJ)
to 720 bp (for GST7’l).
Ethidium
bromide
gels of PCR products
after electrophoresis
or after digestion
with restriction
enzymes
followed
by electrophoresis
are shown
Downloaded from cebp.aacrjournals.org on August 9, 2017. © 1998 American Association for Cancer Research.
721
722
Obtaining
DNA
with
a Mouthwash
Method
M
4lObp
1
2
3
4
5
6
-
Fig. 6.
CYP2EI
polymorphism
analyzed
by PCR.
Hinfl-digested
4X174
DNA molecular
weight
marker:
/9, negative
control.
Presence
of the 410-bp
fragment
1
4
..
AG
[
A
AG
9
-
-
5
..
7
6
i::.
11
12
..
8
AG
:
9
A
G
AG
AG
14
-.
a
15
..
--
.t
16
..
17
..
-
‘
...-
in Figs.
3-7 for the first
17 samples
extracted.
Using
the
extracted
DNA samples,
we were able to unequivocally
genotype all 60 subjects
for the six polymorphisms
studied.
The
PCR 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
for
all 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
were
found
in 41 and 37% of subjects,
respectively.
The variant
allele frequencies
for the NQOJ,
CYP2EI
RsaI, CYPJA]
MspI,
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
the constraints
of our sample
size, we did not detect
any
obvious
effect
of age, sex, race, or smoking
status
on DNA
yield or ability to PCR amplify
the samples
or on the intensity
of 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}C
or for 3 or 6 months
at - 20#{176}C.
Fig. 8 compares
the electrophoretic
patterns
of the
PCR products
obtained
with these samples
after amplification
of GSTMI
. No difference
could be detected
in the intensity
of
the bands that would
have suggested
a change
in human
DNA
concentration
with storage.
The genotyping
of these samples
for the other polymorphisms
led to the same observation.
Discussion
With the availability
of PCR techniques
DNA than traditional
Southern
blotting,
samples
are the specimens
of choice
(12). Lane
M,
(410 bp): Lane
Fig. 7.
CYPIAI
exan
7 polymarphism
analyzed
by PCR.
The point
mutation
at position
462 resulting
in
a change
from lIe to Val in CYP1AI
was studied
by PCR (I I). Lane M,
HaeIlI-digested
4X I 74 DNA malecular
weight
marker;
Lanes
1-17,
allele-specific
PCR paired
by sampIes as in Fig. 2; Lanes A, wild-type
specific
amplification;
Lanes
G, mutatian-specific
amplification:
Lane
18, positive
control
far Ile-Val
heterozygote:
Lane
/9, negative
cantrals.
Presence
of the 322-bp
fragment
in Lane
G
indicates
the
replacement
mutation
of lIe far Val
in exan 7.
18
..
19
..
..
AGAGAGAGAGAGAGAGAG
.
19
10
.
13
..
18
..
-#{149}‘
M
-
1617
-
..
GAGAGAG
,Ja
322 bp
10 11 12 13 1415
The 5-end
polymorphism
was studied
by PCR
followed
by Rsal restriction
enzyme
digestion
Lanes
1-17, samples
corresponding
to the same samples
as in Fig. 2: Lane 18, uncut PCR product
indicates
the variant
allele.
3
2
8
-
-
360bp
322bp-
7
that require
much less
the notion
that blood
for molecular
genetic
..
.
Table
I
DNA
Storage
.
duration
yields
(in jig) from
and duration
Storage
temperature
mouthwash
samples
before
extraction
by storage
condition
Subj ect
( C)
Mean
A
B
C
ID
Extracted:
Immediately
After
I week
After
I week
Room
temperature
37
80
24
31
41
47
10
24
73
38.5
101
10
17
43
42.7
44.0
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 invasive and expensive
and is not always
accepted
or practical.
It
also requires
special
handling
and storage.
By contrast,
buccal
cell collection
is noninvasive
and simple
to perform
and requires
no special
equipment
or training.
The use of such a
source of genomic
DNA in lieu of, or as an alternative
to, blood
collection
is likely to increase
participation
and reduce
costs in
molecular
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 533
individuals
for the multiplex
amplification
of five exons within
the CFTR
gene. The success
rate of PCR multiplex
amplification in this study
was 99%.
In a blind
comparison
of the
frequency
of 12 mutations
responsible
for cystic
fibrosis
in
products
amplified
with DNA from both blood and buccal
cell
samples
collected
from 464 individuals,
there was 100% agreement in the results
for the two types
of DNA
source
(16).
Buccal
brushes
or swabs
may be perceived
as invasive,
especially outside
a clinical
setting.
Additionally,
if not analyzed
Downloaded from cebp.aacrjournals.org on August 9, 2017. © 1998 American Association for Cancer Research.
Cancer
A
M
340 bp 200 bp -
l4Obp
-
1
2
3
B
4
5
;4
L.
1
2
C
3
Fig. 8.
GSTM 1 polymorphism
far samples
from four subjects
(A, B, C, and
temperature
(Lane 2) or at 37CC (Lane 3) far 1 week or at -20CC
for 3 months
marker:
Lane (-), negative
control.
Epidemiology,
4
5
1
2
3
Biomarkers
& Prevention
723
D
4
5
1
2
3
4
5
(-)
:i
D) using
freshly
collected
samples
(Lane
1) and samples
(Lane 4) or 6 months
(Lane 5). Lane M, Hinfl-digested
shortly
after collection,
the brushes
or swabs should
be placed
in a preservative
solution
to optimize
DNA
yield
and PCR
amplification
(5, 8). The toxicity
of this storage
solution
does
not 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 mouth
with isotonic
saline.
This simpler
and noninvasive
collection
method
requires
no supervision
by trained
personnel,
no use of
toxic reagents,
and the samples
can be obtained
through
the
mail. The method
has been validated
in mass screening
with
PCR amplifications
for specific
mutations
with specificity
and
sensitivity
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,
containers leaking
during
transportation,
or residual
food contamination.
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
encountered
when
samples
are obtained
by mail,
it was noted
that the
specimens
stored at 25#{176}C
and 37#{176}C
were more likely to yield
increased
amount
of high molecular
weight
DNA, possibly
of
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.
However,
it was also noted in this study that “samples
stored at
higher
temperatures
resulted
in slightly
less robust
PCR reactions” (19). The method
we propose
here minimizes
the chance
of substantial
bacterial
contamination
by collecting
samples
after subjects
brushed
their teeth and by using
an alcoholic
solution.
The alcohol
content
of the mouthwash
brand that we
used was 21.6%.
Indeed,
we did not observe
greater
DNA
yields in samples
stored
at room temperature
or at 37#{176}C
for 1
week,
and the PCRs
worked
well.
Moreover,
mint-flavored
mouthwash
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
used
in 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 not
affected
by freezing
the mouthwash
samples
at -20#{176}Cfor 6
months
before DNA isolation.
Although
we have not yet tested
longer storage
durations,
the samples
are likely to remain
stable
for a longer period
of time.
In previous
studies,
samples
were apparently
collected
for
a single genetic
test. Thus, these previous
reports
focused
on the
successful
amplification
of a particular
sequence
and did not
that have been stored
4X174
DNA molecular
at room
weight
include
DNA
yields.
This is an issue
in studies
of disease
susceptibility
genes in which a large number
of PCR amplifications
are typically
conducted
on the same samples.
However,
a few studies
reported
DNA yields
that can be used for comparison
with this study. Meulenbelt
et a!. (5) reported
a DNA
yield of 1.3 ± 0.05
per buccal
swab. Similarly,
in the study
by Freeman
et a!. (8), in which
10 buccal swabs were collected
per subject,
the average
total DNA yield was 32 g, with a
range of 3.2-1 10.8 g. Thus, these results
suggest
that a single
mouthwash
sample
yields
an amount
of DNA that is comparable 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 hygiene, such as in the United States. The DNA extraction
method
used is sufficiently
short to be practical
for the processing
of a
large number
of samples
and yields human
genomic
DNA in a
form that is easily
amplified
by PCR. Thus,
this mouthwash
procedure
appears
suitable
for large community-based
studies
of genetic
susceptibility
to disease
in which
samples
can be
collected
by the participants
themselves,
kept at room temperature for several
days during
transportation
back to the study
center,
and stored for months
prior to DNA extraction.
Acknowledgements
We thank the subjects
NQOI
protocol,
and
for their participation.
Ann Seifried
far helpful
Hangwei
Chen
comments.
far his work
on the
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A simple mouthwash method for obtaining genomic DNA in
molecular epidemiological studies.
A Lum and L Le Marchand
Cancer Epidemiol Biomarkers Prev 1998;7:719-724.
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