FUNDAMENTAL MEDICAL SCIENCE I

FINAL REPORT (GENOMIC)

ANGELINE TANCHERLA
01071170034
GROUP A2-2

Universitas Pelita Harapan

Mochtar Riady Institute for Nanotechnology
Faculty of Medicine
2017
 ABSTRACT

Genes are found in living organisms and are passed on to their offspring.
They are in charge of producing proteins. Proteins are needed by the body in order
to work normally. Genes are also responsible for various traits in our body. Genes
have been studied by researchers to identify similarities or differences that may be
associated with diseases. Application of technology to analyze genes has been
beneficial for human health and medical knowledge.

And in this experiment, we aim to apply genetic technology to obtain and

identify human gene. The experiment process includes blood separation to obtain
DNA, DNA isolation, quantitation of DNA concentration and purity using absorbance
as measured by spectrophotometry, DNA gel electrophoresis to observe the size of
DNA, Polymerase Chain Reaction to amplify specific DNA, DNA sequencing (Sanger
method) and finally using BLAST to identify the gene by comparing the sequence
with the NCBI database. Through these experiments, we can know more about
genetics, especially human genes. We can also gain skills in genetic technology.

After doing the experiments, we achieved the aim wherein we successfully

obtained and identified the gene. Although there were some mistakes made
throughout the experiments, the gene could still be used for identification. The
conclusion of this experiment is that there is 99% similarity of our DNA sequence
with the Homo sapiens transforming growth factor beta receptor 2 (TGFBR2) in the
NCBI database.

I. INTRODUCTION

Genetic analysis involves the process of studying DNA sample to observe

differences that may be associated with diseases or drug responses [1]. In the
following experiments, genetic analysis is done to study and identify the type of
human gene. And the gene (DNA) will be obtained from the blood.

Blood is a constantly circulating fluid in blood vessels [2]. It delivers nutrition

and oxygen required for the body. Blood consists of specialized cells and proteins
suspended in liquid matrix. The liquid matrix (plasma) constitutes about 55% of
blood. Plasma contains proteins that help in blood clotting and transporting
substances, while serum is blood plasma without clotting factors (fibrinogen). The
remaining 45% blood content is made up of red blood cells (RBCs), white blood cells
(WBCs) and platelets. RBCs contain hemoglobin protein, which are responsible for
oxygen transport throughout the body. WBCs consist of leukocytes, macrophages
and monocytes that help fight infections. And platelets are in charge in blood clotting.

DNA, or deoxyribonucleic acid, is a long double helix molecule that contains

genetic information and blueprint for protein production. The building blocks of DNA
are nucleotides, which are made up of phosphate group, a sugar group
(deoxyribose) and a nitrogen base [3]. The four types of bases form base pairs with
each another: adenine (A) with thymine (T); guanine (G) with cytosine (C). DNA is
found mostly in the nucleus and less often in the mitochondria [4]. DNA can be
obtained in WBCs, but not in RBCs. This is because RBCs need to carry oxygen
efficiently so they lack nucleus and mitochondria. Therefore, to obtain DNA from
whole blood, we need to isolate WBCs from other blood components by conducting
DNA isolation.

After DNA is isolated, it is important to find out the concentration and purity of
DNA, which are important factors for a good genetic analysis. DNA concentration
and purity can be quantified using their absorbance. The relationship between
absorbance and concentration of DNA is shown in Beer-Lambert Law [5]. It shows
that: Absorbance (A) = absorption coefficient (ε) x concentration (c) x length of light
path (l). Therefore, concentration can also be expressed as C = (A ÷ ε) x dilution
factor. The purity of DNA can obtained through A260 ÷ A280 or through A260 ÷ A230.
DNA can be considered pure if the purity ranges from 1.80-2.00 for A260/A280 or 2.00-
2.20 for A260/A230.

To compare the size of the DNA samples, electrophoresis is conducted.

Electrophoresis is a way of distinguishing substances according to the rate of
movement under the influence of electric field [6]. During electrophoresis, the
agarose gel is placed in the chamber containing a buffer solution and is connected to
electricity. The electrical current will drive the DNA sample through pores of the gel,
from negative (black) to positive electrode (red). Factors that influence the speed of
DNA movements are the strength of the electrical field, the concentration of agarose
and the size of DNA molecules. Smaller DNA molecules will move faster than larger
molecules. DNA is not visible within an agarose gel, thus it will be visualized by
adding ethidium bromide (dye). A set of DNA molecules of known length is used,
which is known as DNA ladder, to help determine the size of DNA fragments in an
electrophoresis gel.

In order to gain enough desired DNA but also to minimize the wasting of
excess DNA samples, PCR is conducted. PCR (Polymerase Chain Reaction) is an
easy way to amplify a small amount of DNA. Firstly, the DNA sample is denaturated
to form single stranded DNA (template DNA). Next, the short and specific DNA
sequences (oligonucleotide primers) are annealed with DNA template. New
nucleotide bases are added to end of primer with the help of Taq DNA polymerase
enzyme (thermostable), which produces new double stranded DNA. This process is
repeated for numerous cycles to obtain desired amount of DNA strands copies [7].

To identify the type of gene, we need to know the DNA sequence. Sanger or
dideoxy sequencing is a sequencing method in which target DNA undergoes
denaturation, annealing to an oligonucleotide primer, and extension by DNA
polymerase [8]. The process will be stopped using dideoxynucleotide triphosphates
(ddNTPs), which does not have 3' OH group. Each of the four ddNTPs shows
different colors. ddATP is green, ddGTP is yellow, ddCTP is blue and ddTTP is red.
The computer will read the sequence (from smallest to largest fragment) and
interprets the colors. Cromas Lite software will be used to edit the sequence for
BLAST.

Bioinformatics is a branch that integrates computer technology, statistics and

applied maths to study biological data [9]. An example of bioinformatics tool is
BLAST (Basic Local Alignment Search Tool). Using BLAST, we can search gene
sequences in the whole genomic libraries. The National Center for Biotechnology
Information (NCBI) website (http://www.ncbi.nlm.nih.gov) has a complete database
of DNA and protein sequences. Therefore, we can use the website identify the gene
we obtained.
II. MATERIALS AND METHODS

To separate blood components, blood samples were drawn from 2 voluntary

students (Anastasia and Christine) into 5 mL EDTA vacutainer and empty
vacutainer. One mL of whole blood was then transferred into a separated vial (each
were labeled properly). The remaining blood samples were centrifuged at 3000 g,
20oC, for 10 minutes using Allegra X 15 centrifuge. Next, the serum (upper layer)
was transferred into a new vial and is stored at -80oC.

The stored blood samples were then used for DNA isolation. Firstly, 0.8 ml 1X
SSC buffer were mixed in the samples. The samples were centrifuged for 1 minute,
at 12,000 rpm in a microcentrifuge. One mL of supernatant was removed and 1 ml of
1X SSC buffer was added into samples. The samples were then vortexed and
centrifuged at 12,000 rpm in a microcentrifuge for 1 minute. All of the supernatant
were removed. Next, 375 μl of 0.2M NaOAc were added to each pellet, which is then
vortexed. Then, 25 μl of 10% SDS and 5 μl of proteinase K were added and the
samples were vortexed and incubated at 55oC for 30 minutes. Phenol/chloroform/
isoamyl alcohol (120 μl) were added and vortexed for 30 seconds. The samples
were centrifuged at 12,000 rpm in a microcentrifuge for 2 minutes. The aqueous
layer was removed carefully to a 1.5 mL microcentrifuge tube and 1 ml of cold 100%
ethanol were mixed. They were centrifuged at 12,000 rpm in a microcentrifuge for 2
minutes. The aqueous layer was moved to a new 1.5 ml microcentrifuge tube and 1
ml of 100% ethanol was mixed. The supernatant was decanted and 180 μl 1X TE
buffer was mixed. Then, 20 μl of 2 M sodium acetate and 500 μl of cold 100%
ethanol were mixed in the samples and they were centrifuged at 12,000 rpm in a
microcentrifuge for 1 minute. The supernatant was decanted and the pellet was
rinsed with 1 ml of 70% ethanol. The pellet was centrifuged at 12,000 rpm in a
microcentrifuge for 1 minute. The supernatant was decanted and the pellet was
dried. The pellet was resuspended by addition of 200 μl of 1X TE buffer. Lastly, the
samples were incubated at 55oC for 30 minutes and were stored at -20oC.

To calculate DNA concentration, DNA sample was diluted 1:5 (but C2 sample
was then changed to 1:2 as instructed). Next, 50 μl 1X TE buffer was added to the
cuvette, which was then set on the holder of spectrophotometer. The type of nucleic
acid was set as dsDNA and the conversion factor was modified. The spectrophoto-
meter was then blanked with 1x TE buffer. The cuvette was inserted to read the
absorbance (50 μl final volume). The absorbance at 230, 260 and 280nm were read
and recorded to calculate purity and concentration.

To detect DNA using electrophoresis, agarose 1% gel is needed. The agarose

1% gel was previously prepared and loaded in the electrophoresis chamber. Then, 5
μl of DNA was mixed by pipetting with 1 μl of loading dye and TAE buffer on parafilm
sheet. Then, the mixture and 6 μl DNA marker were loaded orderly into the agarose
gel well. The lid was closed and electrode was connected to the power supply. The
electrophoresis was run at 100V, 6 Watts, 0.6 A. After 30 minutes, the gel was taken
out and washed under tap water. Versa Doc instrument was used to save the picture
and record the data.

To conduct PCR, a reaction mixture is required. It should contain 11.85 μl of

dH2O, 5 μl of 5X Buffer PCR, 0.5 μl of 2.5mM dNTP mix, 0.15 μl of Taq DNA
Polymerase, 2 μl of 25mM MgCl2, 0.75 μl of 10 μM Forward and Reverse Primer,
and 4 μl of DNA template. To efficiently make reaction mixtures for 4 DNA samples,
a master mix was made with a quantity of 4 times from the original reaction mix. It
was then divided into 4 PCR tube. The tubes were placed in the PCR machine and
were set in the program: step 1 in 95oC for 30 seconds; step 2 (set for 40 cycles) for
denaturation at 95oC for 30 seconds, annealing at 56oC for 30 seconds, and
extension at 72oC for 7 minutes; and step 3 for final extension at 72oC for 7 minutes.
Lastly, the PCR product was stored at 4oC. Electrophoresis was done again to
observe the DNA.

To determine DNA sequence, a laptop is needed to operate the Basic Local

Alignment Search Tool (BLAST). Firstly, Cromas Lite (a Bioinformatics Software
tool), which was installed in the laptop, was used to edit DNA sequence. Any N base
had to be changed into the base according to the appropriate color. After editing, the
sequence was copied in Fasta format. In the http://www.ncbi.nlm.nih.gov/ website,
the nucleotide blast menu was opened and the DNA sequence was copied into the
'Enter Query Sequence Area'. Human genomic database was chosen as search set
and the DNA sequence was blast. Lastly, the result of blast was analyzed.
III. RESULTS

A. Blood Separation

Figure 1: Blood Separation Result in EDTA Vacutainer

Figure 2: Blood Separation Result in Empty Vacutainer

B. DNA Isolation and DNA Electrophoresis

DM A2 C2

>10000 bp

DNA Ladder

Figure 3: DNA Electrophoresis Result Description

Lane DM : DNA Marker
Lane A2 : A2 DNA Sample
Lane C2 : C2 DNA Sample
C. Quantitation of DNA Concentration

Absorbance of DNA Samples (Tables 1 and 2)

A2 Sample
First measurement Second Measurement Mean
(1:5)*
A230 0.032 0.03 0.031
A260 0.178 0.183 0.1805
A280 0.112 0.116 0.114

Table 1: Absorbance of A2 Sample

C2 Sample
First measurement Second Measurement Mean
(1:2)**
A230 -0.030 -0.032 0
A260 0.154 0.151 0.1525
A280 0.106 0.107 0.1065

Table 2: Absorbance of C2 Sample

* Dilution
** Dilution of C2 was changed from 1:5 to 1:2 as instructed

Concentration Quantitation of DNA Samples

𝐴260 × Dilution Factor 0.1805 × 5

DNA Concentration = =
ε 20
of A2 Sample
= 𝟎. 𝟎𝟒𝟓𝟏𝟐𝟓 𝐦𝐠/𝐦𝐥

DNA Concentration 𝐴260 × Dilution Factor 0.1525 × 2

of C2 Sample = = = 𝟎. 𝟎𝟏𝟓𝟐𝟓 𝐦𝐠/𝐦𝐥
ε 20

(ε for double stranded (ds) DNA = 20 g-1cm-1L)

Purity Index of DNA Samples (Tables 3 and 4)

A2 Sample
Quantitation Result***
Purity Index

A260 0.1805
P1 = = 𝟓. 𝟖𝟐𝟑 > 2.2 RNA Contamination
A230 0.031

A260 0.1805
P2 = = 𝟏. 𝟓𝟖𝟑 < 1.8 Protein Contamination
A280 0.114

Table 3: Purity Index of A2 Sample

C2 Sample
Quantitation Result***
Purity Index

A260 0.1525
P1 = =∞ > 2.2 RNA Contamination
A230 0

A260 0.1525
P2 = = 𝟏. 𝟒𝟑𝟐 < 1.8 Protein Contamination
A280 0.1065

Table 4: Purity Index of C2 Sample

*** Purity Indicator

Purity Pure Contamination (cont.)
A260
2.00-2.20 < 2.00 = Phenol cont. > 2.20 = RNA cont.
A230
A260
1.80-2.00 < 1.80 = Protein cont. > 2.00 = RNA cont.
A280

Table 5: Purity Indicator

D. PCR (Polymerase Chain Reaction)

DM A2 C2 (+) (-)

DNA Ladder

Figure 4: Polymerase Chain Reaction Result

Description
Lane A2 : A2 DNA Sample Lane (+) : Positive Control
Lane C2 : C2 DNA Sample Lane (- ) : Negative Control
Lane DM : DNA Marker
E. DNA Sequencing

Figure 5: Editing DNA Sequence using Cromas Lite (Forward Direction)

Figure 6: Blast Result

Figure 7: Blast Result-Alignment Scores

 Figure 8: Blast Result-Type of Gene

Figure 9: Blast Result-Alignments

IV. DISCUSSION

The result of the blood separation experiment shows that there are different
layers formed after centrifugation. The layers are developed due to the different
densities of blood contents. After centrifugation, heavier blood components will sink
to the bottom of the tube, while lighter components will appear on the upper layer.

For blood sample in EDTA vacutainer, three layers are formed. They are
plasma (yellowish and transparent), buffy coat (thin and white) and RBC layer. Buffy
coat is made up of WBCs and platelets. The appearance of buffy coat is due to the
anticoagulant in EDTA tube that prevents WBCs and RBCs from clotting together.
EDTA vacutainer contains anticoagulant that maintains blood in the fluid state. Since
the empty vacutainer has no anticoagulant, blood components (such as RBCs,
WBCs and platelets) will clot together and forms only two layers, which are serum
(yellowish transparent layer without coagulating factor) and blood clots. From the
results above, we can conclude that our experiment results match with the
references. Blood is composed of various components with different sizes and
functions.

To conduct the next experiment, we need to obtain DNA. DNA isolation

requires some substances to help isolate DNA from whole blood. Firstly, SSC buffer
is added to lyse red blood cells that do not contain DNA. Then, NaOAc (salt) is
added to break down nucleus wall. SDS detergent is used to lyse the cell membrane
and form complexes with lipid and proteins. Proteinase K will then degrade the
unwanted protein. Phenol/chloroform/isoamyl alcohol is added to dissolve non-polar
substances (lipids). And ethanol is added to isolate DNA from water. The final
product of this experiment is precipitated DNA, which appears as white pellet and will
be used for concentration quantitation and electrophoresis.

The DNA absorbance is measured to count the concentration and purity. A2

sample has an approximate concentration of 0.045 mg/ml, while C2 sample is lesser,
which is 0.015 mg/ml. The difference in concentration may be due to error in
pipetting during DNA isolation. A2 sample has a purity index of 5.823 for A260/A230
and 1.583 for A260/A280. C2 sample's purity index is ∞ for A260/A230 and 1.432 for
A260/A280. According to the purity indicator, both samples have RNA and protein
contamination. From the results of DNA purity quantitation, we can conclude that the
samples are not pure. They are all contaminated because of the mistakes done
during DNA isolation. We were not careful enough to keep the pipets and DNA
samples sterile.

Through DNA electrophoresis, we can analyze the size of our DNA samples.
By comparing the samples with the DNA marker (see Figure 3), we can conclude
that our DNA samples have a size of more than 10.000 bp. However, the band
shown in the picture is very thin and unclear. The thickness of the band is associated
with DNA concentration. Our DNA samples have low concentration, which results to
the appearance of thinner bands. In the previous experiment, we found out that A2
sample has greater concentration than C2 sample. In electrophoresis, this
relationship is shown, wherein A2 sample has a band, which is slightly more visible
than C2 sample.

DNA amplification is done through PCR. Electrophoresis is also done again to

check our PCR result. From Figure 4, we cannot see any band formed. The absence
of the bands is due to the error in inserting the DNA samples and controls into the
agarose gel wells. Contaminated DNA samples may also result to invisible bands.
However, the absence of band in the negative control lane is not an error. This is
because negative control has no DNA and there should not be any band formed.
Normally, the bands of DNA samples will appear thick and clear if the DNA is pure
and well-amplified.

In the last experiment, we blast our DNA sequence in the NCBI website. The
blast result shows that our sample is the most similar with the Homo sapiens
transforming growth factor beta receptor 2 (TGFBR2). The graph in the alignment
scores section (Figure 7) shows alignments of our sample (purple colored lines) that
matches to the database (red lines). From the graph, we can know that our sample is
an amplified DNA fragment, because the purple line (our sample) is quite short,
compared to the long red line (sequence in database). The alignment score is
approximately 80 and is labeled as purple color. The more the length of a sequence
matches the database, the greater the alignment score is. In Figure 9, we see that
our sequence has identities of 80/81 matches, which is 99% similar. This means that
our DNA sequence is very similar to TGFBR2 gene in the database. Meanwhile, the
single different identity may be due to error in editing the sequence using Cromas
Lite.

Therefore, from the experiment results above, we can conclude that we

successfully achieved the aims. Despite that there were some mistakes, we still
managed to identify the gene (DNA) that we obtained from human blood. And it is
99% similar with the Homo sapiens TGFBR2 gene in the NCBI database.
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