J. Biol. Macromol.2 (3) 91-94 (2002)

Detection of Aldehyde Dehydrogenase 2 Gene Fragment Using Human Nail Clippings and Saliva
Mami Toyasaki1) , Megumu Yano1) and Yukio Takii1,2)
1)

Department of Food Science and Nutrition, School of Human Environmental Sciences, and 2)Institute for Biosciences,

Mukogawa Women’s University, Nishinomiya, Hyogo 663-8558

Received August 17, 2002; accepted August 28, 2002

phenotypes relating to alcohol tolerance have beenclassified into three groups, consisting of normal

An individual genotype of aldehyde dehydrogenase 2

homozygotes (NN), heterozygotes (NM), and mutant(ALDH2) was observed when using nail clippings andhomozygotes (MM). A single G-to-A transition in thesaliva, respectively by the method consisting of DNA

3’-terminal sequences (size length:135 bp) of ALDH2

extraction, genomic DNA amplification by polymerase
chain reaction, and gel electrophoresis of DNAfragment. Gene analysis using fingernail DNA wasfound to be effective in that they were easily to collectfrom human samples, in that they were kept storable
at –80℃ until use, and in that DNA in the sample wasvery stable after repeated treatment of freezing andthawing.

exon 12 region led to substitute glutamic acid for lysine in
ALDH and resulted in the conversion of NN to NM orMM. The latter was intolerant to alcohol and highly
prevalent among Japanese4).

Therefore, a rapid andreliable survey should be conducted among young female
students that have not possessed alcohol-drinking habit,
since it is not always feasible to collect data from a varietyof female students.
The present study was undertaken to determine whether
it was feasible or not to collect biological materials and toprovide more information regarding the types of ALDH2

Key words: ALDH (aldehyde dehydrogenase), genotype,present in nails and in saliva.

phenotype, PCR (polymerase chain reaction), KOD (DNApolymerase derived from Thermococcus kodakaraensios
KOD1 strain), homozygote, heterozygote

Materials and methods
Collection of nails and saliva
After informed consents were obtained from 3 healthy

The oxidation of ingested ethanol to aldehyde is firstJapanese female students that have no problems in alcohol

catalyzed by alcohol dehydrogenase and then metabolized

ingestion, nails and saliva were collected, respectively.

to acetic acid by six classes of aldehyde dehydrogenase

Nail clippings were rinsed once with ethanol and kept for(ALDH; EC.1.1.1.1) 1,2). ALDH2 has a low Km value
and is regarded as the critical ALDH isoenzyme that
determines an individual’s intolerance to alcohol.
Genotype-phenotype analysis of ALDH2 is essential forclinical, or epidemiological studies of alcohol-relateddiseases3).

According to the genotyping studies, the10 min at room temperature. Each specimen was cut intopieces (1.0 x 2.0 mm) , weighed and stored at -80℃ until
use. Saliva (0.2 – 0.5 ml) was collected from subjects
after they had rinsed their mouth with deionized water andstored at -80℃. Alcohol patch tests and questionnaires

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M.Toyasaki et.al

about drinking habit to the subjects were conducted

for 20 sec, and 72℃ for 45 sec. After the cycle, thesimultaneously.

mixture was once incubated for 5 min at 72℃ and stored
at 4℃.

DNA extraction
Each specimen prepared from nail (1.0 – 20 mg) or saliva

Agarose gel electrophoresis(10 – 20μl ) was transferred into microcentrifuge tube andA 5-μl portion of the PCR product was analyzed in 3%

incubated for 20 min at 55℃ in 210μl of urea - SDS

agarose 21 gel6). Electrophoresis was carried out at 50 Vsolution by using a modification of the method of supplierfor 30 min. Molecular markers employed was HaeIII(Isohair kit, Nippon gene Co.) and further incubated for 15

digest ofφX174 DNA, consisting of 1353, 1078, 872, 603,5)

min at 55℃ after adding 5 μl of proteinase K solution .

310, 281, 271, 234, 194, 118 and 72 base pairs. DNA wasThe mixture was then converted gently for 5 min with 200

visualized after staining with ethidium bromide7).

μl phenol-chloroform-isoamylalcohol (25:24:1, v/v) atroom temperature. After centrifugation at 14,000 rpm for
15 min at 4 ℃, the upper layer was transferred to a new

Results and discussion

tube and mixed with 20μl of 3M sodium acetate and 2μl

Data from three healthy students were similar to the figuresethachinmate.

thedemonstrated in the typical patterns throughout the studies.

solution was kept at – 20℃ for 30 min. The supernatantFigure 1 and 2 clearly show that either 10μl and moreobtained by centrifugation was thoroughly decanted andamount of saliva preparation or 5 mg and more of nailthe resulting precipitate was washed once with 70%

segments is sufficient enough to amplify a 135 bp DNA

ethanol and centrifuged. After drying in vacuum for 5

fragment containing ALDH2 exon 12 region (lanes 4,6, 8,min, DNA was dissolved in 10μl of TE buffer (pH 8.0).

and 10 in figure 1: lanes 4, 6,8, 10 and 12 in figure 2).

After adding 400μl of ethanol,

On the contrary, negative control tests conducted (lanes
PCR amplification

2 and 3 in figures 1 and 2) indicated that the amplification

The allele-specific of ALDH2 gene was amplified withprocedures performed both in human saliva and in nails

primer set of FR1, consisting of forward primer (F)were not contaminated with other DNAs. Moreover, not(5’-CAAATTACAGGGTCAACTGCT-3’) and reverse

any of 135 bp bands was seen when R2 was employed as

primer R1 (5’-CCACACTCACAGTTTTCTCTTC-3’),

the reverse primer (lanes 5, 7 ,9 and 11 in figure 1 andor set FR2, consisting of the forward primer (F) andlanes 5, 7, 9, 11 and 13 in figure 2).

reverse primer R2 (5’-CCACACTCACAGTTTTCTCT

Faint bands of staining located at around 50 bp wereTT-3’). A total of 50 μl PCR solution, containing 1x

visible in lanes 3 thorough 10 indicated the proper

PCR buffer, a template DNA, 0.5 mM MgSO4, 0.2 mM

proceedings of the PCR (positive control).

dNTP, 0.4 mM forward primer, 0.4 mM reverse primer R1

As demonstrated in figures 3 and 4, 135 bp DNAor R2 and 1.25U KOD plus enzyme was pre-heated atfragment containing ALDH2 exon12 was not destructed

98 ℃ for 1 min ,and subjected to 35 cycles ofeven after 20 cycles of freezing and thawing.

amplification by PCR as follows: 98℃ for 20 sec, 60℃

Aldehyde Dehydrogenase 2 Gene

93

Fig. 1. Electrophoresis pattern of the PCR product of a

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

135 bp DNA fragment containing ALDH2 exon 12 fromhuman saliva.
Lane 1,φX174/ HaeIII; lanes 2 and 3, denoted the respective
135bp

template DNA was omitted (negative control). Either 1-μl, 5-μl,10-μl or 20-μl portion of saliva solution was employed in PCR mixture,and 5-μl of the product was applied to lanes 4 and 5, 6 and 7, 8 and 9,and 10 and 11, respectively. R2 was used for reverse primer in lanes 2, 4,6, 8 and 10., in figure 1, and lanes 3,5,7,9 and 11in figure 2.

Fig. 2.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Dependency of nails clippings extract

concentration on PCR production of a 135 bp DNA
fragment containing ALDH2 exon 12Lanes 4 and 5 representsDNA extract from 1 mg of nail clippings.
135bp

Lanes 6 and 7, 8 and 9, 10 and 11, and 12 and 13 demonstrate theDNA extracts from 2 mg, 5 mg, 10 mg and 20 mg nail clippings,
respectively. The other experimental procedures and denotes in the figure
were the same as described in figure 1.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Fig. 3. Effect of repeated freezing and thawing on PCR
production of a 135 bp DNA fragment containing ALDH2
exon 12 in saliva.
135bp

Lanes 4 and 5 represents 1 cycle of freezing and thawing
treatment of the template DNA. Lanes 6 and 7, 8 and 9, 10 and
11, and 12 and 13 demonstrated the repeated times of 5, 10, 15, and20,respectively offreezing and thawing. The other experimental

procedures and denotes in the figure were the same as described in figure 1.

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M.Toyasaki et al.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Fig. 4. Effect of repeated freezing and thawing on PCR
production of of a 135 bp DNA fragment containing
ALDH2 exon 12 in nail clippings.
135bp

Lanes 4 and 5 represent 1 cycle of freezing and thawing. Lanes 6and 7, 8 and 9, 10 and 11, and 12 and 13 demonstrate the repeated cyclesof 5, 10, 15, and 20,respectively. The other experimental procedures anddenotes in the figure were the same as described in figure 1.

From these results together with the alcohol patch tests andReferences

questionnaires, the genotype of subject was normal

1) Dyck, L.E.

homozygotes (NN), and not heterozygotes (NM) nor(1995)

mutant homozygotes (MM).
Nail clippings are relatively easy to obtain from humanvolunteers especially when they are restricted to female
students. Plucking hair roots with attached bulbs fromthe people’s scalp was not successful, since it caused
sometimes pains on the volunteers and template DNA was

Alcohol Clin. Exp. Res., 19: 420-426

2) Edenberg, H.J. Prog. Nucleic Acid Res. Mol. Biol., 64:
295-341 (2000)
3) Harada, S, Agrwal, D.P. and Goedde, H.W. HumanGenet. 44: 181-185 (1978)
4) Hus, L.C., Bendel, R.E. and Yoshida, Y. Genomics 2:57-65 (1988)

extremely low in hair sample preparations (data not

5) Chomczynski, P.and Sacchi, N. Anal. Biochem. 162:shown).

156-159, (1987)

Genetic and epidemiological studies on ALDH2 using
nails from more than 150 students are now in progress.

6) Duley, J.A., Harris, O. and Holmes, R.S. Alcohol Clin.
Exp. Res., 9: 263-271 (1985)
7) Sambrook,J., Fritsch,E.F. and Maniatis,T. Molecularcloning: A laboratory manual (2nd ed.) (1989)

Acknowledgement
The research was supported in part by the Grants from theIijima Memorial Foundation for the Promotion of Food
Science and Technology.

Communicated by Ohnishi Masatake