Practice in Medical Genetics PhD.

Le Minh Thong

VIETNAM NATIONAL UNIVERSITY HO CHI MINH CITY

INTERNATIONAL UNIVERSITY

SCHOOL OF BIOTECHNOLOGY

LAB REPORT

PRACTICE IN MEDICAL GENETICS

Instructor: Ph.D. Le Minh Thong

Class: Practice in Medical Genetics (Saturday Morning)
Group 02

Group members:

Phạm Khánh Duy– BTBTIU19011

Lê Thị Phương Thùy – BTBTIU19125
Lê Văn Phong – BTBTIU19102
Lý Nguyễn Hoàng Trinh – BTBTIU19134

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Practice in Medical Genetics PhD. Le Minh Thong

TABLE OF CONTENT

LAB 2: DNA EXTRACTION AND DNA QUANTIFICATION

I. INTRODUCTION ...............................................................................................3

II. MATERIALS AND METHOD ........................................................................3

III. RESULT ............................................................................................................6

IV. DISCUSSION.....................................................................................................7

LAB 3: DETECTION OF 𝛽-GLOBIN GENE MUTATION (CD26-AG) AMONG

𝛽-THALASSEMIA CARRIERS BY USING ARMS-PCR

I. INTRODUCTION ...............................................................................................8

II. MATERIALS AND METHOD ........................................................................8

III. RESULT ............................................................................................................10

IV. DISCUSSION.....................................................................................................11

LAB 4: DETECTION OF 𝛽-GLOBIN GENE MUTATION (CD26-AG) AMONG

𝛽-THALASSEMIA CARRIERS BY USING TETRA- PRIMER ARMS-PCR

I. INTRODUCTION ...............................................................................................12

II. MATERIALS AND METHOD ........................................................................12

III. RESULT ............................................................................................................14

IV. DISCUSSION.....................................................................................................15

LAB 5: DETECTION OF 𝛽-GLOBIN GENE MUTATION (CD26-AG) AMONG

𝛽-THALASSEMIA CARRIERS BY USING REAL - TIME PCR

I. INTRODUCTION ...............................................................................................16

II. MATERIALS AND METHOD ........................................................................16

III. RESULT ............................................................................................................17

IV. DISCUSSION.....................................................................................................18

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Practice in Medical Genetics PhD. Le Minh Thong

LAB 2: DNA EXTRACTION AND DNA QUANTIFICATION

I. Introduction

a. DNA Extraction

DNA extraction is the process that involves separating DNA from proteins, membranes, and other
biological components and three necessary processes in the procedure are lysate, precipitation, and
purification. DNA extraction is frequently a preliminary step in many diagnostic procedures used to
identify environmental viruses and bacteria, as well as to diagnose illnesses and hereditary diseases.
The number and types of steps vary between different methodologies; however, there are various
alternative commercial methods for DNA extraction that can get over these challenges. Commercial
kits also provide consistency in extraction across labs and from various samples.
The process of DNA extraction is essential to biotechnology. It serves as the basis for a wide range
of applications, from basic study to regular diagnostic and therapeutic decision-making. Determining
the distinctive properties of DNA, such as its size, shape, and function, also requires extraction and
purification.

b. DNA Quantification

DNA quantitation of purified DNA samples is a standard nucleic acid research workflow, providing
confidence for downstream applications such as Next Gen Sequence, PCR, Cloning, and DNA
transfections. There are some recommended techniques that are used for identifying the DNA
concentration such as agarose gel electrophoresis, spectrophotometry and fluorometry.
● Gel electrophoresis is a technique used to separate molecules like DNA, RNA or proteins
based on their size and charge.
● Spectrophotometry measures the proportion of light of a certain wavelength that is absorbed
by a sample. The absorbance values allow both qualitative and quantitative analysis of the
sample
● Fluorometry is a technique used to identify and quantify analytes in a sample by adding
fluorophores that bind to the analyte of interest to the sample, exciting them with a beam of
UV light and detecting and measuring the emitted wavelength

In this experiment, we will learn and discuss the required technique in DNA extraction and
quantification.

II. Materials And Method:

DNA Extraction:
Materials
• CH3COONa 3M
• Chloroform
• Ice-cold isopropanol

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Practice in Medical Genetics PhD. Le Minh Thong

• Ethanol 70-80%
• Proteinase K (20mg/mL)
• Lysis buffer:
o Tris-HCL 50mM
o NaCl 50mM
o EDTA 25mM
o SDS 0.5% (stored at room temperature)
*Lysis buffer preparation for 1 sample (70µl)
Procedure:

DNA Quantification:
Part A: DNA quality assessment using absorbance method:
• Micropipette 10µl and tips
• Thermos ScientificTM NanoDrop 2000
• Sample
• Distilled water
• Extracted DNA
Method:
• Set the mode of Nanodrop for dsDNA measurement.
• Clean the drop mount using One paper and distilled water.
• Add 2 µl distilled water and BLANK.
• Add 2 µl of extracted DNA and measure the DNA amount.
• Record all information: DNA concentration. 260/230, 260/280 ratio.

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Practice in Medical Genetics PhD. Le Minh Thong

Part B: DNA quality assessment using electrophoresis method:

Equipment and Materials:
• DNA solution
• Agarose gel system
• Agarose powder
• Gel-red staining dye.
• 1kbp DNA ladder
• 0.5X TAE buffer
• UV illuminator with camera
Method:
Set-up:
• Weigh 0.8g of agarose powder.
• Add 100mL of distilled water and dissolve them in the microwave.
• Prepare the gel tray and balance it.
• Insert the 8-well comb into the tray.
• Cool down the gel solution for a bit and pour the gel carefully into the tray.
• Wait until the gel cools completely minutes.
Gel electrophoresis:

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III. Result
DNA Extraction and Quantification.

Friday AM

Friday PM

Saturday (our class)

Table 1. Gel electrophoresis results of three classes in comparison.

Class Group ng/ul A260/A280 A260/A230

1 -0.3 0.20 0.20
2 0.3 -1.77 0.27
Friday AM 3 112.8 1.80 2.21
4 317.3 1.85 1.89
5 1.7 -6.39 0.82
Friday PM 1 41.0 1.85 1.83

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Practice in Medical Genetics PhD. Le Minh Thong

2 192.0 1.31 0.30

3 317.5 1.65 1.10
4 213.2 1.83 2.22
5 17.4 1.99 1.59
6 -0.1 0.09 0.22
1 83.1 1.82 1.79
2 168.2 1.85 2.22
Saturday
3 1352.2 1.8 1.63
4 283.0 1.81 1.81

Table 2. Nanodrop results of DNA concentrations and purification.

IV. Discussion
Speculation from the results of our class.
As far as we know, the expected ratio of A260/A280 is between 1.8-2.0 and its ratio of A260/A230
is between 2.0-2.2. Regarding absorbance result, the ratio of A260/A280 almost belonged to perfect
ratio when it was around 1.8. On the other hand, the ratio of A260/A230 was opposite, only group 2
had the acceptable ration approximately 2.22 while the others was under 2.0, it means that the DNA
sample of the others were not completely purification when it may contaminate the unknown
substance, such as salt, specifically, here is SDS and EDTA, which one is a strong anionic detergent,
if present in high concentrations can interfere with A260/A230 measurement, and the other one is
chelating agent that can form complexes with metal ions, respectively. Furthermore, chloroform is
strongly believed to interfere with the ratio in the extraction step as the technique of performers is
not sufficient to handle the withdrawal of top layers which contain DNA.
In gel electrophoresis, as our forecast, the band of group 3 was very bright, but accompanied by the
smear also as bright as its band, it could be explained by the highest concentration (1352.2 ng/ul) is
not only contain DNA but salt and might be protein as well. It was generally accepted, when the
other band of another group had shiny but slightly blurry than group 3’s as the lower concentration
of absorbance and the smears belonged to each group as the purification of DNA was quite not
successful. One more detail is that since it was the whole genome gel electrophoresis, the band ran
not far from the gel because it was huge, and our result which band on the top of the ladder was
reasonable.
Speculation of the other classes.
In summary, in Friday PM class, despite the acceptable ratio of both A260/A230 and A260/A280,
the band of gel electrophoresis of group 4 was likely to stuck, it might be the amount of loading dye
they added too much, hence the DNA cannot ran out as fast as the others. Whilst, in the Friday AM
class, the absorbance result of group 1 had band, in spite of a negative figure of concentration and
out range of ratio A260/A280 and A260/A230. It can be explained that the band was unspecific, and
it needed to perform electrophoresis again. According to group 2 and 5, as the concentration of
DNA, certainly no band was observed.

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Practice in Medical Genetics PhD. Le Minh Thong

LAB 3: DETECTION OF 𝛽-GLOBIN GENE MUTATION (CD26-AG) AMONG

𝛽-THALASSEMIA CARRIERS BY USING ARMS-PCR

I. Introduction
Overview about 𝛽-thalassemia disease:
Beta thalassemia is an inherited blood disease marked by low levels of functional hemoglobin. Red
blood cells contain hemoglobin, which is the oxygen-carrying, iron-rich component of the blood.
There are three primary kinds of beta thalassemia—minor, intermedial, and major—distinguish the
severity of the condition.
The hemoglobin beta (HBB) gene undergoes modifications (variants or mutations), which result in
beta thalassemia. While both HBB genes are mutated in the intermediate and major types of beta
thalassemia, only one HBB gene is mutated in the minor form.

Overview about ARMS-PCR

The Amplification Refractory Mutation System PCR (ARMS-PCR) is now one of the most accurate
methods for diagnosing genetic diseases. Refractory primers are used in ARMS-PCR to genotype
SNPs (single nucleotide polymorphisms). Selective amplification is made possible by creating
primers for the mutant (with SNP) and normal (without SNP) alleles, allowing for simple analysis
following electrophoresis.
The aim of this experiment is studying about how to design a diagnosis protocol for identifying 𝛽-
globin gene mutation by ARMS-PCR

II. Materials and Method

Materials
• PCR machine
• Electrophoresis system
• Gel tray and comb
• UV light and camera
• Micropipettes
• PCR tube 0.2 ml
• Eppendorf tube 1.5 ml
• Agarose powder
• DNA ladder 100bp
• 0.5X TAE buffer
• Gel-red staining dye
• DNA solution
• Top Taq DNA polymerase master mix
• 3 sets of primers
Methods:
Three pairs of primer were used:
• Internal control:

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Long-hGCG-F(control A): CAGGAATAACATTGCCAAACGTCACGATG

Long-hGCG-R(control B):CTCTCGCCTTCCTCGGCCTTTCAC
• Disease detection primers:
Common E: TGAAGTCCACTCCTAAGCCAGTG
Cd26: TAACCTTGATACCAACCTGCCCAGGGC
• Disease- detection primers(CD41-42)
CD4142-TTCT-F: CTGTGTTCACTAGCAACCTCAAA
CD4142-TTCT-R: GAAAACATCAAGCGTCCCATA
Components for running PCR:
Master mix 10 µl
Forward primer 1 µl
Reverse primer 1 µl
DNA 2 µl
Distilled water 6 µl
Total 20 µl

Add components to the tube in descending order of volume and add primers on top of tube
with different direction

Figure 3.1: Positions for PCR components.

Set up temperature program in PCR machine

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Practice in Medical Genetics PhD. Le Minh Thong

Prepare 2% agarose gel.

• Measure lg agarose powder
• Prepare a bottle with 50ml 0.5X TAE buffer.
• Mix well lg agarose with 50m1 0.5X TAE buffer.
• Boil the mixture in microwave for 1-2 minutes.
• Leave gel to cool for few minutes.
• Prepare the tank.
• Pour gel into gel tank.
• Insert gel comb.
• Leave the gel cool at room temperature for 10 min.
Analysis of PCR products

III. Result

Friday AM

Friday PM

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Practice in Medical Genetics PhD. Le Minh Thong

Saturday

Table 3. The result of ARMS PCR (hGCG, CD26, CD4142) from three classes.
IV. Discussion

In Saturday class, all groups had hGCG’s band as internal control at first line of each group
indicating that our sample was valid as it was the band from human sample, despite group 2, the IAC
was quite blur. In comparison with the other classes, the IAC and sample wells should have two
bands indicating that the IAC or the whole process work well, but in our class, IAC and sample wells
just only had one band (it should belong to IAC control, because if IAC did not appear, the whole
process was invalid). The only considerable point is that the band of mutation well of group 1 of our
class. had the band too low, nearly the same band size as IAC. It can be explained that they added
wrong pair of primers, long-hGCG instead of Common E. In case of group 2, there was no
appearance of mutation band indicating that the donator have had not contain mutation at this point
proven by the illustration of band on whole genome gel electrophoresis.

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Practice in Medical Genetics PhD. Le Minh Thong

LAB 4: DETECTION OF 𝛽-GLOBIN GENE MUTATION (CD26-AG) AMONG

𝛽-THALASSEMIA CARRIERS BY USING TETRA-PRIMER ARMS-PCR

I. Introduction
The ARMS-PCR reaction (tetra-primer amplification refractory mutation system-polymerase chain)
is a simple and cost-effective method for genotyping single-nucleotide polymorphisms (SNPs).
Tetra- PCR is conducted with four primers, and gel electrophoresis is the only step after that.
The difference between the tetra-primer and the ARMS techniques is the mismatch location. For the
tetra-primer, the mismatch is located in the middle of the allele-specific primers and four primers are
utilized in the reaction. However, in the ARMS system, the mismatches are located in the 3’terminus
of the allele-specific primer and five primers are used. Another difference is that the tetra-primer
genotyping is done in a single reaction, with two different annealing temperatures (TA), one higher
in the first cycles and lower in the remaining cycles. However, only one TA is used in the ARMS,
but the genotyping is made on two different reactions, amplifying on each reaction one of the allele.
In the tetra-primer ARMS–PCR, the high specificity of the reaction does not rely only on the 30
terminus mismatch, but also on a deliberate mismatch at position-2 (second to the terminal) from the
3' terminus of the same allele-specific primer. This extra mismatch destabilizes the base pairing
between the primers and their corresponding non-targets templates and have been found to increase
the specificity of the reaction by eliminating false-positives results

II. Materials & Methods

Materials & Instruments
PCR machine
Electrophoresis system
Gel tray and comb
UV light and camera
Micropipettes
PCR tube of 0.2 ml
Eppendorf tube of 1.5 ml
Agarose powder
DNA ladder 100bp
0.5X TAE buffer
Gel-red staining dye
Our DNA sample.
Top Taq DNA polymerase master mix
4 sets of primers

Methods:
There are 2 pairs of primers:
• CD26-GA-inner:
o Forward: GAACGTGGATGAAGTTGGTGTTG
o Reverse: CAACCTGCCCAGGGCATT
• CD26-GA-outer
o Forward: GCCAATCTACTCCCAGGAGCA
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o Reverse: CATCACTAAAGGCACCGAGCA
• CD4142-TTCT-inner:
o Forward: CCTTGGACCCAGAGGTTC
o Reverse: GATCCCCAAAGGACTCAACCT
• CD4142-TTCT-outer
o Forward: CTGTGTTCACTAGCAACCTCAAA
o Reverse: GAAAACATCAAGCGTCCCATA
Components for running PCR:
Master mix 10 µl
Inner forward primer 1 µl
Inner reverse primer 1 µl
Outer forward primer 1 µl
Outer reverse primer 1 µl
DNA 1 µl
Distilled water 5 µl
Total 20 µl

DNA sample is added right above the water droplet on the edges of PCR tube, so that water will
create a slippery path for DNA sample to slide down easily. The 2 sets of primers droplets must not
overlap with each other, otherwise the primers will bind with each other and not bind with the DNA
sample, leading to false results. After finishing adding the components, vortex the PCR tube.

Setting up the PCR machine

● 94oC in 5 minutes
● 35 cycles of
○ 95oC for 30 seconds
○ For CD26: 60oC for 30 seconds – For CD4142: 52oC for 30 seconds
○ 72oC for 30 seconds
● 72oC for 7 minutes
● Hold at 4oC

Running the PCR assay

- Prepare 2% agarose gel with 0.5X TAE buffer.
- Mix 1.5 μl of loading dye with 5 μl of our PCR product.
- Load the mixture into the gel we have prepared before.
- Run the gel with 100 voltages of 100bp DNA ladder.
- Visualize the gel under UV light.

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III. Result

Picture 1. The result of ARMS PCR (CD26 inner-outer, CD4142 inner-outer), 100bp ladder
from Saturday am classes.

Picture 2. The result of ARMS PCR (CD26 inner-outer, CD4142 inner-outer), 100bp ladder from Friday pm

Picture 3. The result of ARMS PCR (CD26 inner-outer, CD4142 inner-outer), 100bp ladder from Friday am.

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IV. Discussion
β- thalassemia is caused by the SNP from G to A transition. The normal person will have G/G, the
carrier possesses G/A, and the infected person is A/A. Because the SNP does not lie right in the
middle of our DNA section, therefore we can observe the difference in sizes between the PCR
product containing nucleotide G and the one containing nucleotide A. The PCR product containing
G is larger in size compared with the one containing A, therefore travels further in gel
electrophoresis. In theory, the carrier (G/A) lane should have 3 bands (1 control, 1 G, and 1 A), the
normal (G/G) and the infected person (A/A) should have 2 bands (1 control and the other one is
either G at a higher position in gel electrophoresis, or A at lower position).
First, when looking at this result, we can see that the chemical and electrophoresis were working
well, and the ladder showed up clearly, in addition, in the positive control, it showed that there is a
band and all 4 groups have a band effect, only group 3 was showing a brighter band because this
group used a different master mix, so the band of group 3 was clearer than the other groups. Looking
at the band we saw in our class, all 4 groups 1, 2, and 4 had no main band in group 3 we saw a band
appearing and very clear, but it still deviated a little from control, it could be the piece of DNA we
need to find, but to be sure we need to do another test. Moreover, if we assumed that in the group of
three bands that appear to be pathogenic DNA, it might be in a heterozygote state so it did not cause
disease in the donor because if it was homozygote, the donor would not live until now. Regarding
the remaining groups there was no band and talking about group 2, our group, well with our sample,
the band didn't show up which means that the first reason might be in our sample there was no
pathogenic sample or the DNA in our sample was lost in the process or not enough for expression in
the gel.
Looking at the sample of the Friday afternoon class, and looking at the results, we saw that the
groups had no bands on their sample, although the control and ladder clearly showed that there was
no problem with electrophoresis. This could be in samples of groups that did not have pathogenic
DNA, so it did not show up, or during DNA loss. But looking closely, we could see that in group 4,
the control band was there but it was quite fuzzy, this could be guessed that while adding gel, this
group might be added out, or while the technique of this group was not very good or maybe lost so
makes the dye not bright enough and could not be visible on the gel.
About Friday morning class, we could see that the results of the first 2 groups did not give us any
conclusions because the band was in good control and the sample did not appear so these 2 groups
during work could be a problem technically, although we could see the control of the last 3 groups
and the ladder goes up. Group 3,4,5 did not band in the well containing the sample, this may be
because, in the sample of this group, there is no disease-causing DNA like the groups above.

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Practice in Medical Genetics PhD. Le Minh Thong

LAB 5: DETECTION OF 𝛽-GLOBIN GENE MUTATION (CD26-AG) AMONG

𝛽-THALASSEMIA CARRIERS BY USING REAL - TIME PCR

I. Introduction
Real-time polymerase chain reaction (real-time PCR) is commonly used to measure gene expression.
In real-time PCR, the accumulation of amplification products is measured as the reaction progresses,
in real time, with product quantification after each cycle. Real-time detection of PCR products is
enabled by the inclusion of a fluorescent reporter molecule in each reaction well that yields
increased fluorescence with an increasing amount of product DNA. The main advantage of real-time
PCR over PCR is that real-time PCR allows you to determine the initial number of copies of
template DNA (the amplification target sequence) with accuracy and high sensitivity over a wide
dynamic range. Real-time PCR results can either be qualitative (the presence or absence of a
sequence) or quantitative (copy number).

II. Materials and Methods

Loading sample into qPCR tubes
• Transfer 10𝜇L of PCR products to new qPCR tubes
• Add 1 𝜇L of Eva Green dye to each tube

Set up temperature program in qPCR machine

• Denature step: 95℃ for 15 seconds
• Annealing step: 60℃ for 15 seconds
• Plate reading step: the temperature is increasing from 60-95℃ slowly and the camera in
qPCR machine will record the flurorescene signal of Eva Green in this time
• Stop reading step: 95℃ for 15 seconds

Running qPCR machine – melting curve assay

• Place qPCR tubes into the qPCR machine in the right position (remember that position for
setting up on the computer in”Plate display” option)
• Recheck to sensure that all tubes are in the right positions with the caps surface cleaned.
• Close the lid of the machine tighly
• Run the program
• Get the result

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III. Result

Figure 1: The results of fluorescence profile during a time of run qPCR of our class.

Figure 2: The results of melting curve SYBR of our class.

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Figure 3: The results of fluorescence profile during a time of run qPCR of Friday am class.

IV. Discussion
Initially, the gathered sequences in a sample were double-stranded, therefore the fluorescence signals
were strong at ambient temperature. When the temperature exceeded 90oC, the signals began to
decline gradually. The curved fluorescence profile was obtained as a result of this. In general, the
fluorescence signal is large at the start of qPCR and declines with time. This is due to the
measurement of variations in fluorescence with increasing temperature. The two strands of the
amplicon split to create single-stranded DNA as the temperature rises, causing the fluorescent
intercalating dye to disassociate from the DNA and stop fluorescing.
Our group sample is in position C3 in the qPCR machine.
- Threshold with 33%:
+ If peaks are higher than the threshold→ point have a relevant amplification signal.
+ If peaks are lower than the threshold→ point does not have a relevant amplification signal.
- The control sample is in position B3 which has no peaks higher than the threshold
- Our group sample is in position C3 and has no peaks higher than the threshold → no separate
amplicons.
Looking at the melting curve, we saw that groups 1,2,3,4 correspond to curves C2, C3, C4, and C5.
Based on the results after running real-time PCR, we could see that in group 3 there was a high peak,
which means that the gel would show 1 band but look closely, the curve of group 3 would have 2
peaks if the middle segment was pulled down but in fact only saw a peak inferred showing only 1
band on the gel. The remaining groups also had peaked but the peak of group 1,2 was not very
visible, and it was quite low, lower threshold. In addition, group 4 also had a higher peak than the
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threshold and corresponds to a band. But in fact, compared with the results of electrophoresis, our
group only saw the band in group 3 and the rest of the groups did not saw the band it could be
because when we did the electrophoresis of our sample. Not enough to attach to the dye or in the
process lost DNA or our DNA degradation could also be the issue.

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