Lab B: Identification of human ALDH2 genotype by PCR-RFLP method

Abstract
ALDH2 encodes aldehyde dehydrogenase, which is an essential component of human ethanol
metabolism. The SNP (Single Nucleotide Polymorphism) of the human ALDH2 gene inactivates
aldehyde dehydrogenase due to the replacement of base 37,030 in the genomic sequence, and the
frequency of the mutant ALDH2 gene is particularly high in the East Asian population. Aimed at the
difference of this single base, this experiment applies PCR-Restriction fragment length polymorphism
(RFLP) to distinguish the three ALDH2 genotypes after extraction, purification, and enzymatic
digestion of the genomic DNA from human oral epithelium.

Introduction
Aldehyde dehydrogenase (ALDH) is a protein that catalyzes the oxidation of aldehydes, including
acetaldehyde [1]. The human genome has identified 19 functional ALDH genes, categorized into four
classes: ALDH1-4. Among them, the ALDH2 gene is unique due to its high expression level and
genetic polymorphism compared to the other three classes [2].

The ALDH2 gene exhibits sequence polymorphisms, with the most common mutation being
g.37030G>A. This mutation replaces the glutamate residue at position 487 of the ALDH2 amino acid
sequence with lysine, resulting in a significant loss of catalytic activity in the synthesized aldehyde
dehydrogenase. The wild-type gene encoding the normal enzyme is referred to as ALDH2*1 (G), while
the mutated gene encoding the inactivated enzyme is referred to as ALDH2*2 (A). Biochemical studies
have shown that the absence or structural alteration of any subunit in the ALDH2 tetramer is sufficient
to decrease or even abolish the enzymatic activity. Therefore, enzymes encoded by the heterozygous
Glu487/Lys487 (ALDH2*1/2) genotype do not exhibit high catalytic activity, with only approximately
10-20% of the activity observed in the homozygous wild-type (ALDH2*1/1) genotype. The lysine487
homozygous mutation (ALDH2*2/2) results in a 96% loss of enzymatic activity, rendering it virtually
incapable of ethanol oxidation metabolism. In the primary hepatocytes utilizing the ethanol metabolic
pathway II, a decrease in aldehyde dehydrogenase catalytic activity leads to the inefficient metabolism
of acetaldehyde produced after alcohol ingestion. Consequently, a large number of acetaldehyde
molecules enter the bloodstream and participate in systemic circulation. Accumulation of acetaldehyde
in facial capillaries, due to its vasodilatory effect, leads to the phenomenon commonly known as "Asian
Glow," characterized by facial flushing when consuming alcohol [3, 4].

The frequency of ALDH2 gene polymorphisms varies among different ethnicities. Compared to
European and African populations, the ALDH2*2 gene is more widely distributed in East Asian
populations, resulting in a larger proportion of individuals with impaired acetaldehyde metabolism and
a higher susceptibility to alcohol-related complications such as facial flushing. In the Chinese Han
population, the frequency of ALDH22 allele is 17-29%, while the genotype frequency of ALDH2*1/*2
heterozygotes is 36-44%, and ALDH2*2/*2 homozygotes frequency is 7-8% [5, 6], which are
relatively high levels worldwide.

PCR is a technique used to amplify specific DNA sequences in a sample. It involves repeated cycles
of DNA denaturation, primer annealing, and DNA synthesis using a heat-stable DNA polymerase. In
the case of ALDH2 mutation detection, specific primers are designed to target the region of interest in
the ALDH2 gene. If a mutation is present in the targeted region, the PCR amplification will result in a
different-sized DNA fragment compared to the wild-type allele. By analyzing the sizes of the PCR
products, we can identify the presence of ALDH2 mutations.

Fig.1 Schematic presentation of the PCR principle

Enzyme restriction analysis, also known as restriction fragment length polymorphism (RFLP), utilizes
specific restriction enzymes that recognize and cut DNA at specific DNA sequences. These enzymes
are sensitive to sequence variations, including point mutations or single nucleotide polymorphisms
(SNPs). In the case of ALDH2 mutations, specific restriction enzymes, such as AcuI, are selected to
cut the DNA at or near the mutation site. If the mutation alters or creates a recognition site for the
restriction enzyme, the digestion pattern will be different between the wild-type and mutant alleles.
This difference can be visualized by gel electrophoresis, allowing for the identification of ALDH2
mutations.
 Fig.2 Schematic presentation of the enzyme restriction principle

Fig.3 Fragments of different lengths from a PCR reaction are run on a gel.

2. Experimental
2.1 Materials
1) The saliva DNA extraction kit
2) The PCR reagents (2* Rapid Taq Master Mix)
3) TrackIt™ 100 bp DNA Ladder
4) Primers
5) The restriction endonuclease AcuI
6) PCR tube, 1.5mL tube, PCR rack
7) Agarose, TAE buffer, DNA gel plate
8) Disposable paper cups

2.2 Instrument
1) benchtop centrifuge
2) microplate reader
3) vortex mixer
 4) PCR machine
5) metal bath
6) water bath
7) electronic balance
8) microwave oven
9) electrophoresis system
10) gel imaging system, etc.

3. Sample Preparation and PCR analysis

3.1 Extraction of human oral epithelial cell DNA
Genomic DNA from oral epithelial cells was extracted following the instructions provided in the saliva
DNA extraction kit. The column-based method for genomic DNA extraction from oral swabs utilizes
Buffer ACL and CL to lyse the cheek epithelial cells, releasing genomic DNA along with protein and
cellular debris. Impurities are then precipitated through centrifugation. The supernatant is transferred
to a new centrifuge tube, where genomic DNA is selectively adsorbed onto a specialized adsorption
membrane using a binding solution. A washing solution is used to remove the small amount of
impurities adsorbed on the membrane. Finally, genomic DNA is eluted from the membrane using an
elution buffer, resulting in high-quality genomic DNA.
Before the experiment, participants rinsed their mouths with drinking water to remove food residues.
They then held approximately 5 mL of distilled water in their mouths and vigorously performed
chewing and gargling movements. After 3 minutes, they spat the water into a paper cup.
• Sampling: Use an oral swab to swipe the inner wall of the mouth 6-10 times.
• Cut the cotton tip of the swab from its handle using scissors and place it in a 2 ml centrifuge tube.
Add 400 μl of Buffer PBS to the centrifuge tube.
• Add 400 μl of Buffer ACL, 200 μl of Buffer CL, and 20 μl of Proteinase K to the centrifuge tube.
Shake to mix well, incubate at 65°C in a water bath for 10 minutes, occasionally mixing.
• Add 400 μl of ethanol (100%) to the centrifuge tube, mix thoroughly, and transfer the entire
solution and precipitate to a silica membrane adsorption column (place the column in a collection
tube). Centrifuge at 10,000 rpm for 1 minute, discard the solution in the collection tube, and return
the adsorption column to the collection tube.
• Add 500 μl of Wash Solution to the adsorption column, centrifuge at 10,000 rpm for 1 minute,
discard the solution in the collection tube, and return the adsorption column to the collection tube.
• Repeat step 5 once.
• Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm for 2
minutes.
• Open the lid and let it stand for 5 minutes until the Wash Solution evaporates.
• Remove the adsorption column and place it in a new 1.5 ml centrifuge tube. Add 50 μl of sterile
deionized water preheated to 60°C to the center of the adsorption membrane, let it stand for 5
minutes, and centrifuge at 12,000 rpm for 2 minutes.
• The concentration and relative purity of the extracted DNA were determined using the microplate
reader.

3.2 PCR amplification of sequences containing mutation sites

For the design of primers near the ALDH2 gene mutation site, the researcher considered factors such
as primer sequence specificity, length, and distribution of base types. Primers, ALDH2-F and ALDH2-
R, were designed upstream and downstream of the AcuI restriction enzyme site, respectively, at
positions 134 bp and 253 bp. The primer sequences are as follows:
• ALDH2-F: 5'-TCAAATTACAGGGTCAACTGCTA-3' (23 nt)
• ALDH2-R: 5'-GGGAAATTAGTAGGAAACACTGATG-3' (25 nt)
Under appropriate PCR conditions, this primer pair can specifically amplify a 387 bp fragment.

The PCR system and amplification conditions are as follows:

PCR reaction mixture composition:
• ALDH2-F: 0.5ul
• ALDH2-R: 0.5ul
• 2* Rapid Taq Master Mix:25ul
• DNA template:100ng
• ddH2O:to 50ul

PCR program:
1) Initial denaturation: 95.0°C for 3 min
2) Denaturation: 95.0°C for 15 s
3) Annealing: 58°C for 15 s
4) Extension: 72.0°C for 10 s
5) Additional extension: 72.0°C for 5 min
6) The cycle is repeated 35 times.

3.3 Purification of PCR amplification products

According to the purification instructions for PCR products, the PCR amplification products were
purified. Kits contain a silica membrane assembly for binding DNA in high-salt buffer and elution
with low-salt buffer or water. The purification procedure removes primers, nucleotides, enzymes,
mineral oil, salts, agarose, ethidium bromide, and other impurities from DNA samples
• Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix.
• Place a QIAquick column in a provided 2 ml collection tube or into a vacuum manifold. For details
on how to set up a vacuum manifold, refer to the QIAquick Spin Handbook.
• To bind DNA, apply the sample to the QIAquick column and centrifuge for 60s. Discard flow-
through and place the QIA quick column back in the same tube.
• To wash, add 750μl Buffer PE to the QIAquick column centrifuge for 60s or apply vacuum.
Discard flow-through and place the QIAquick column back into the same tube.
• Centrifuge the QIAquick column once more for 2 min to remove residual wash buffer.
• Place each QIAquick column in a clean 1.5 ml microcentrifuge tube.
• To elute DNA, add 40 μL of sterile deionized water preheated to 60°C to the center of the
QIAquick membrane and centrifuge the column for 5 min.
• The concentration and relative purity of the purified PCR products were determined using a
microplate reader.

3.4 Enzyme digestion of PCR amplification products`

1) In a total system of 20 μL, add 500ng of PCR product, 0.4μL of AcuI, and 2.0 μL of 10×
 CutSmart buffer.
2) After thorough mixing, incubate at 37°C for 60 minutes.
3) Subsequently, replace the enzyme with an equal volume of sterile deionized water as a negative
control for the enzyme digestion reaction.

3.6 Electrophoresis detection of enzyme digestion products

Take the enzyme-digested products and the negative control group, add 1xloading buffer, and load
them onto a 1% agarose gel.
Perform gel electrophoresis at a constant voltage of 130 V for 30 minutes in a standard electrophoresis
tank.
Use a gel imaging system to detect the band distribution to determine the genotypes.

4. Questions
1) Discuss the electrophoresis results of enzyme digestion products
2) Briefly explain the mechanism of enzyme digestion
3) Discuss viable methods to increase the purification concentration of PCR amplification
products
4) Explain the mechanism of PCR amplification and application

Reference
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