Protocol for Advanced laboratory skills in field biology
Shimei Shirasawa
Kyoto University WRC M 1

Abstract

In this study, our aim is to understand the procedure of analysing DNA extracted fromanimals and humans. We have extracted DNA from buccal swab and survey individual
difference of androgen receptor gene which is reported to be related with personality evaluation
by questionnaire survey. This gene encodes poly-glutamine and poly-glycine amino acidsequences in the exon region and repeat numbers are polymorphic. The papers reported therepeat length of poly-glutamine was related with aggressiveness in humans and dogs. For theprocedure, we have extracted DNA we have taken from buccal swab and then measured theconcentration. Then we amplified the targeted sequence by PCR. Once we finished amplifying
DNA, we have measured the number it was repeated in our genes, and the result differed inparticipants. Comparing the survey results with the PCR amplification, we found thatparticipants with fewer repeats exhibited lower levels of Dominance, Neuroticism, and Harm,
while participants with numerous repeats exhibited higher levels of these categories respectively.
On the second day, we have used same DNA to extract genotype ALDH2 gene. This is todetermine weather the participants carry genes that indicates tolerance to alcohol or not. Before
we begin the PCR amplification, we have conducted a small experiment by using 70% ethanoland distilled water. Two cloths, one moistened with ethanol and the other with distilled water,
were applied to the arm and left in place for several minutes. By conducting this small
experiment at the beginning, we could assume our body’s tolerance to ethanol. While the PCR
amplification was in progress, we prepared an agarose gel for electrophoresis. After creating thegel, we loaded the PCR products into wells in the gel and ran it at 100V for 20minutes until thedye migrated to the centre of the gel. We observed that participants who exhibited ethanol
tolerance in the patch test had the ALDH R gene as homozygous, while those who did not were aheterozygous combination of ALDH R and ALDH R2 or were homozygous of ALDH R2. It is
intriguing we can predict alcohol tolerance only through PCR amplification but also via the patch
test, and the results are remarkably consistent.

Introduction
Personality analysis and comparison of poly-Q, poly- G
In contemporary studies, there is a growing emphasis on examination of personality
through DNA analysis, signifying its increasing relevance in current research discussions(Sanchez-Roige et al., 2018; Weiss et al., 2009). Personality describes behaviours, emotions andcognitive styles that are stable throughout life (McCrae & Costa, 2003). Five factors have been
identified as essential in the study of personality – Neuroticism, Extraversion, Openness toExperience, Agreeableness, and conscientiousness, which are collectively referred to as FiveFactor Model (FFM) [Digman, 1990]. Later research suggests that FFM is human universal andare heritable (Bouchard & Loehlin, 2001). Early laboratory primatologists recognised thesignificance of personality in chimpanzees, although subsequent efforts to draw parallel between

nonhuman species and humans were criticised as anthropomorphic (Povinelli, 1997).
Nonetheless, recent evidence suggests that analogues of the five human personality factors arepresent in various nonhuman species, exhibiting strong inter-rater reliability, construct validity
and internal consistencies (Gosling, 2021; Gosling & Vazire, 2002). In 1997, King andFigueredo conducted a study on chimpanzee personality by utilizing a questionnaire derived
from the FFM (Goldberg, 1990). They have identified five chimpanzee personality factors
resembling the FFM and a chimpanzee specific factor called Dominance (Goldberg, 1990). To
explore cross – cultural differences in the perception of chimpanzee personality, a study was
conducted in Japan with the translated questionnaires used in the US. The study aimed to addressquestions regarding interrater reliabilities, culture- specific trait intercorrelations, and the impactof culture on the perception of sex, age, and subjective well-being in relation to chimpanzee
personality (King & Figueredo, 1997). To evaluate FFM numerically, genetic regions linked to
specific behaviours were extracted and the extent to which these regions were repeated wasverified. This experiment centred around studying aggression and involved comparing the
subject’s responses to FFM questionnaire with their DNA results. Aggression was the primary
focus, and it was investigated in the context of intraspecific polymorphism. This was related to
the length variability on the poly- glutamine (poly- Q) and poly-glycine (poly- G) regions within
the androgen receptor. The association between androgens and various traits has been
documented in several animal species such as Old-world primates, as well as in various
mammals, for instance dogs (Konno et al., 2011). Several studies indicate shorter polyglutamine
repeats were linked to reports of increased aggression (Konno et al., 2011; Aluja et al., 2011).
In this study, the results obtained from the FFM were compared to the DNA analysis withshorter poly- glutamine repeats in the participants DNA would exhibit higher scores on the FFM
aggression item.

Material and Methods

Materials
Tools
Micro pipettor
Heat block
Vortex mixer
Tube stand
Timer
Cooling tube stand for 1.5ml
Cooling tube stand for PCR tubes
Centrifuge for 1.5ml
Centrifuge for 8 PCR tubes
Thermal Cycler for PCR
Reagents
DNeasy Blood & Tissue Kit (QIAGEN)
0.9% NaCl
H2O (deionised)
Ethanol 99.5%
LA Taq with GC buffer (-20 ℃)
Primers (-20 ℃)

Control DNA samples (4℃)
Size standard (4 ℃)
HDFA (-20 ℃)
Others
Cotton buds
Paper towel
Marker pen
Filter tip (1000µl, 200µl, 10µl)
Tube (1.5ml)
94 well PCR plate and cap
94 well plate for the sequencer
Methods
DNA extraction
Buccal swab
1.Add 1000µl saline (0.9% NaCl) to a new 1.5ml tube.
2.Rub buccal mucous membrane with a cotton bud.
3.Wash the cotton bud in Step 1 tube.
4.Centrifuge at 14000rpm for 1 min.
5.Discard supernatant. Around 100µl remains.
Extraction of DNA
6.Mix 100µl saline (0.9% NaCl) and 20µl protease per sample. Add 120µl of mixture to Step5
and mix thoroughly.
7.Add 200µl of Buffer AL and mix thoroughly.
8.Incubate at 56℃ for 10 min. Mix thoroughly every 3 min.
9.Add 200µl of ethanol and mix thoroughly.
10.Pipet solution in Step 9 to spin column tube and centrifuge at 14000rpm for 1 min.
11.Place spin column a new collection tube, add 500µl of Buffer AW1, and centrifuge at
8000rpm for 1 min.
12. Place spin column a new collection tube, add 500µl of Buffer AW2, and centrifuge at
14000rpm for 4 min.
13.Place spin column in a new 1.5ml tube, add 50µl of incubated H2O, and incubate at 56℃ for
5 min.
14.Centrifuge at 8000rpm for 1 min.
15. Discard column and store DNA at 4℃.
Measuring DNA concentration
1µl of step 15 is used for Nano drop
PCR amplification
For glutamine (Q) repeat
ARhFF:
5’-TCCAGAATCTGTTCCAGAGCGTGC-3’ (FAM labelled)
ARhR:
5’-GCTGTGAGGGTTGCTGTTCCTCAT-3’ around 280bp

For glycine (G) repeat
ARGFH:
5’-CAGTGCCGGCTATGGGGACCTGGCGA-3’ (HEX labelled)
ARGR:
5’-GGACTGGGATAGGGCACTCTGCTCACC-3’ around 320bp
PCR mixture
2x GCI buffer
H2O
sNTP
Primer1(20µM)
Primer2(20µM)
LATaq
Total
Template DNA

5.0
1.8
1.6
0.25
0.25
0.1
9.0µl
1.0µl

Mix reagents (x samples +1) except DNA in 1.5ml tube, and distribute 9µl into each PCR tube,
then add template DNA. PCR reaction (LA60C30):95℃ 2min (95℃ 30s, 60/65℃ 30s, 74℃
1min)x 30/35, 74℃ 10min, 10℃∞
Genotyping
1.Diluting PCR product: 100µl water and 1µl PCR product in a new 96 well plate.
2.Mix the following reagents in a tube
Mixture of Hi-Di and size standard (for 1 run: 16 sample):
The amount should be changed for No. of samples
Hi-Di Formamide 160µl
Size Standard
1µl
3. Add 10µl of the mixture above to 96 well plate.
4.Add 1µl of Step1 into the mixture in 96 well plate.
5.Heat the sample for 5 min, at 95℃.
6.Immediately chill on ice box for 5min.
7.Set the plate in a sequencer and run.
Genotype analyses
Check and select one or two peaks for each sample, create and export table.
Change the value to the integer.

Results

Personality analysis
The self-assessment results exhibited significant variations among the participants (Table1). To measure these variations, specific behavioural traits, such as aggression, were quantified.
next, the lengths of poly-Q and poly- G regions were determined from the extracted DNA. Since
androgen receptors are encoded on the X chromosome, the results exhibited gender-based
differences. In males, a single peak was observed in the results, whereas females displayed two
distinct peaks (Figure 1 and 2).
Subsequently, the frequency of repeats for both poly-Q and poly-Q regions wasdetermined, resulting in two parameters: ARQ and ARG. ARQ values ranged from 24 to 26, and
ARG values ranged from 21.5 to 23. For female participants, ARQ and ARG values werecalculated as averages, allowing the of the length of the glutamine and glycine regions within the
androgen receptor gene for each individual (Table 2 and Table 3). Participants were categorised
into two groups based on the length of their ARQ repeat regions, specifically those with short(24.00) and long (25.00 or more) repeats. A comparison was conducted by assessing the meanscores on the FMM items within these groups. Notably, individuals with shorter repeat regions in
Dominance exhibited lower scores compared to those with linger repeat regions. Additionally,
participants with shorter genes scored slightly higher on the Neuroticism item (Table 2).

Discussion

The results of the study yielded diverse outcomes, and no clear patterns or tendency
could be discovered. Even when the number of repetitions of the ARQ domains was the same,
there were significant individual differences reflected in the values of the FFM items. These
differences did not align with the expected outcomes based on the prior research mentioned in
the introduction. One possible reason for this discrepancy could be the limited sample size of theparticipants in the current experiment.
The primary aim of this study was to explore the degree to which an individual’s DNA
influences their personality by combining personality assessments with DNA diagnostics. It is
worth nothing that as the accuracy of DNA diagnostics advances in the future, it may become
feasible to quantitatively express aspects of one’s personality and unnoticed habits that cannot beeffectively assessed through traditional personality tests. However, the enhanced precision inpersonality diagnosis through DNA analysis also raises certain ethical concerns. There is a riskthat using another person’s DNA could inadvertently reveal their personality traits without theirconsent, potentially resulting in the inadvertent disclosure of personal information. Therefore, it
is imperative to handle such information, along with other biometric data like fingerprints andvoiceprints, with great care and consideration for privacy and consent.

Tables and figures
Table 1. The quantified results of FMM questionnaire survey
Dominance Neuroticism Harm
S 2,3,6

1.84-0.11 39.20

L 1,4,52.22-0.22 60.00

S, L represents participants with Short and Long ARG repeat, respectively.
Table 2. The result of DNA amplification of participants

ARG_1 ARG_2 AR AR
Har_Repea _Repea Q
Gm
S AR AR AR ARtt
ave ave Domiavoi
I e Q_ Q_ G_ G_ ARQ_1_Rep ARQ_2_Rep Numbe Numbe rag rag nanc Neuro denc
D x 1
2
1
2 eat_Number eat_Number
r
r
e
e
e
ticism e279 285 323 332
1 F .00 .00 .00 .00

25.00

27.00

20.00

23.00

26.0 21.5
0
0

2.00

-0.33 80.00

276
2 M .00

24.00

－

23.00

－

24.0 23.0
0
0

2.18

-0.33

24.0 23.0
23.00
0
0

1.35

-0.44 49.00

332
－ .00

－

276 276 332 332
3 F .00 .00 .00 .00

24.00

24.00

23.00

279 282 332 332
4 F .00 .00 .00 .00

25.00

26.00

23.00

23.00

25.5 23.0
0
0

2.53

-0.11 40.00

276 288 332 332
5 F .00 .00 .00 .00

24.00

28.00

23.00

23.00

26.0 23.0
0
0

2.12

-0.22 40.00

276
6 M .00

24.00

ー

23.00

－

24.0 23.0
0
0

2.00

-0.44 68.60

332
－ .00

－

Table 3. Repeat number of ARQ/ARG relative to the number of codons in the gene.
ARQ

ARQ Repeat Number
273

ARG

23.00
ARG Repeat Number

314

17.00

Figure 1 and 2. The result of the PCR amplification.Wave coloured in Blue and Green represents
the amount of Glutamine and Glycine in the DNA fragment, respectively.

Reference
•

•
•
•
•
•
•

Aluja A, Garcia LF, Blanch A, Fibla J. Association of androgen receptor gene, CAG and
GGN repeat length polymorphism and impulsive-disinhibited personalitu traits in inmate:
the role of short-long haplotype. Psychiatric Genetics. 2011; 21:229-239
Bouchard TJ, Loehlin JC. Genes, evolution, and personality. Behavior Genetics 2001;
31(3): 243-273
Digman JM. Personality structure: Emergence of the five-factor model. Annual Review
of Psychology. 1990; 41:417-440
Goldberg LR. An alternative “description of personality”: the Big-Five factor structure.
Journal of Personality and Social Psychology. 1990;59(2):1216-1229
Gosling SD. & Vazire S. Are we barking up the right tree? Evaluating a comparative
approach to personalitu. Journal of Research in Personality 2001; 36(3):607-614
King JE, Figueredo AJ. The five-factor model plus dominance in chimpanzee personality.
Journal of Research in Personality. 1997;31(2):257-271
Konno A, Inoue-Murayama M, Hasegawa T: Androgen receptor gene polymorphism inzebra species. Meta Gene 2015; 5:120-123

•
•
•
•
•

McCrae RR, Costa PT, Personality in adulthood: A five-factor theory perspective.
Guilford press
Povinelli DJ, Gallup GG, Eddy TJ, & Bierschwake DT. Chimpanzees recognize
themselves in mirrors. Animal behaviour 1997; 53(5): 1083-1088
Rentfrow PJ, Gosling SD. Putting personality in its place: A geographical perspective on
personality traits. The Guilford press. Theory and research. 2021; 824-836
Sanchez- Roige S, Gray JC, MacKillop J, Chen CH, Palmer A. The genetics of humanpersonality. Genes Brain Behav. 2008; 17: e12439
Weiss A, Inoue-Murayama M, Hong K-W, Inoue E, Udono T, Ochiai T, Matsuzawa T,
Hirata S, and King JE. Assessing Chimpanzee Personality and Subjective Well-Being inJapan American Journal of Primatology 2009; 71: 283-292

Introduction

In the human body, there are 19 aldehyde dehydrogenases (ALDHs) that serve various
roles in preserving biological functions (Jackson et al., 2011; Marchitti et al., 2008). One of these
ALDH2, plays a critical role in metabolizing aldehydes, which are by-product of alcohol(Marchitti et al., 2008). Various ALDH enzymes, apart from ALDH2, are known to be
associated with impaired biological function when they are deficient (Marchitti et al., 2008).
Interestingly, it is not able to distinguish from a person’s phenotype whether they possess
ALDH2 defects, as these defects lack distinctive outward manifestations and are generally
considered to have minimal impact on the preservation of biological functions (Matsumoto et al.,2014).
While there are multiple genetic polymorphisms of ALDH2, only rs671 is notably
distinct (Matsumoto et al., 2014). The wild type allele is termed to as ALDH2*1, and the mutant
type allele is known as ALDH2*2. This results in three genotypes: homozygous wild type,
heterozygous, and homozygous mutant type. ALDH2 mutations are primarily observed in
Mongoloid populations, and in Japan, they are relatively less common in regions such as
Okinawa, Kyushu, Hokkaido, and Tohoku (Goedde et al., 1992). In this experiment, we haveused the DNA samples collected from participants in the previous study.

Material and Methods

Materials
Patch test
1.Prepare two 1.5cm square cut paper towel
2.Dip one towel in 70% ethanol, another one to H2O
3.Put them on inside of arm for 5 min
4.After reaction, store at 10℃
Glu (GAA) strong↓
Lys (AAA) weak
Detection of SNP (single nucleotide polymorphism)
F primer……………….GAA……………
CTT remirp R
F primer……………….AAA……………
TTT remirp 2R

F primer (ALDH F): CAAATTACAGGGTCAACTGCT
R primer (ALDH R): CCACACTCACAGTTTTCACTTC
R2 primer (ALDH R2): CCACACTCACAGTTTTCACTTT
Two reactions (F+R, F+R2) per sample
PCR Mixture
AmpliTaq Gold 360 Master Mix
H2O
Primer aet A or B
F primer
R/R2 primer
Total
Template DNA

5.0
2.5
0.25
0.25
8.0µl per person
2.0µl per person

PCR reaction
Agarose gel electrophoresis
Preparation of 1.5% agarose gel
1. Measure 60ml of 1 x TBE and 0.9g agarose
2. Melt in a microwave, add H2O to restore to original volume, cool down to 60℃
3. Wait more than 30 min until the gel hardens, add a small volume of 1 x TBE andremove the comb
Electrophoresis and taking a picture
1. Fill a tank with 400ml of 1 x TBE
2. Set the gel
3. Add 1µl of dye to parafilm, add 3µl of size marker, mix by pipetting and load all to
the well within the gel
4. Add 1 µl of dye to a parafilm, add 3µl of size marker, mix by pipetting and load all to
the well in the gel
5. Run at 100V for 20 min until the dye moves to the middle of the gel
6. Put the gel on the UV Trans illuminator and take a photo

Results

Patch test
Among the five participants in the patch test, one displayed a reddish discoloration in thepatched area as a reaction to alcohol (Table 4). This reaction indicated that the participant likely
to carry a genotype of heterozygous of ALDH2*1 and ALDH2*2 or homozygous of ALDH2*2.
The other four participants, excluding the one who displayed discoloration in the patch test, did
not show any such redness and were predicted to carry a genotype of homozygous of ALDH2*1.
Electrophoresis
Electrophoresis results indicated that all six participants carried the gene of ALDH2*1,
while among the participants, only one participant carried the gene of ALDH2*2 (Figure 3). This

implies that five of the participants were homozygous for ALDH2*1, and one participant washeterozygous for ALDH2*1 and ALDH2*2.
Comparing the results of the two experiments, we can predict that participants who exhibited
reddish discoloration in the patch test carries the genotype of heterozygous of ALDH2*1 and
ALDH2*2 genotype or the genotype of homozygous of ALDH2*2. In contrast, those who did
not show reddish coloration in the patch test were found to have the genotype of homozygous of
ALDH2*1 at the patch test stage.

Discussion

As mentioned in the introduction, it is believed the ALDH2 variant is predominantly
found in Mongoloids. The proportion of individuals possessing this variant varies from country
to country and region to region, but it is generally estimated that over 40% of the population in
Japan possesses the variant. In Japan, the prevalence of the wild type of ALDH2 is reported to behigher in the Tohoku, Okinawa, and Kyushu regions in order. In the current experiment, five outof the six participants have Asian, Extreme Orient roots, which, in terms of expected values,
suggests two of them carry the mutation. However, only one of these five participants possessed
the heterozygous genotype. Interestingly, the participant with the heterozygous genotype wasfrom the Tohoku region.
This variant of ALDH2 is believed to have originated in neo-Mongoloids and is widespread in
specific areas of the Extreme Orient, likely spreading through invasions and ethnic migrations.
Even within the Extreme Orient, this mutation is almost absent in Siberia, Oceania, North andSouth America, suggesting its expression may have originated in southern to central China. How
this mutation entered the Japanese archipelago remains uncertain and may be a factor to consider
when examining rhe migration of ancient Japanese to the archipelago.
However, if this gene was introduced, there should be much more variants in the Kyushu,
and Okinawa islands, which re geographically close to the continent. Nevertheless, the variant isstill abundant from Honshu to the south part of Tohoku region. This persistence might be due tothe fact that alcohol-containing food/drink was not essential for survival, and individuals with thevariant gene were not subject to the strong natural selection. Consequently, it accumulated in the
population as a neutral gene, a phenomenon known as genetic drift.
Furthermore, the removal of restrictions on freedom of migration within Japan, startingfrom the Meiji era with the abolition of feudal domains, contributed to the disappearance ofregional differences. These differences are believed to have persisted only in areas with limited
human interaction and migration, such as remote islands.

Tables and figures
Table 4. The result of the patch test and PCR amplification of all participants (1-6). Black cell
represents person who did not take the patch test (or control).
participant patch test genotype A genotype B

result1－＋－strong2－＋－strong3－＋－strong4＋－strong5－＋－

storong

6

＋

＋

＋

medium

C1＋－strongC2＋＋

mediumFigure 3. The result of Electrophoresis.

Reference••••

Goedde HW, Agarwal DP, Fritze G, Meier- Tackmann D, Singh S, Beckmann G, et al.
Distribution of ADH2 and ALDH2 genotypes in different populations. Hum Genet 1992;
88:344-346.
Jackson B, Brocker C, Thompson DC, Black W, Vasiliou K, Nebert DW, et al. Update on
the aldehyde dehydrogenase gene (ALDH) superfamily. Hum Genomics 2011; 5: 283303.
Marchitti SA, Broker C, Stagos D, Vasiliou V. Non-P450 aldehyde oxidizing enzymes:
the aldehyde dehydrogenase superfamily. Expert Opin Drug Metab Toxicol 2008;4: 697720.
Matsumoto A, アルデヒド脱水酵素２(ALDH2)遺伝子多型の予防医学的重要性.
Jpn. J. Hyg 2018; 73:9-20.

Acknowledgement

This experiment was carried out smoothly under the guidance of Professor Murayama
and Assistant Professor Sato. As I mainly do fieldwork, it has been a long time since I have done
experiments in the lab. I also felt that it takes a lot of efforts to do both fieldwork and laboratory
work. If there are any opportunity, I would be grateful to receive advice on analysing the samples
gained in the fieldwork. I would like to appreciate Professor Murayama and Assistant Professor
Sato again.