FUNDAMENTAL MEDICAL SCIENCE 1

FINAL REPORT (GENOMIC)

JEREMIAH SUWANDI

01071170045

GROUP C4-2

UNIVERSITAS PELITA HARAPAN

Mochtar Riady Institute for Nanotechnology

Faculty of Medicine

2017
ABSTRACT

Genomes are the entire hereditary information or complete set of DNA. Genome
organized all of human body function. In this experiment our goal to isolated Human
Transforming Growth Factor Beta Receptor 2 from human blood sample.

Two student volunteer for this experiment (James and Keshia). After blood was
taken and the DNA was isolated, DNA electrophoresis was done to measure the length
of DNA. And to quantified the DNA sequence with spectrophotometry. Then PCR was
conducted to multiply the target DNA sequence and gel electrophoresis for the DNA
length. Chromas lite in FASTA format was used for sequence and editing. BLAST for
compare the DNA with the database from ncbi.com. The experiment result for Human
Transforming Growth Factor Beta Receptor 2 is 98% match.
I. INTRODUCTION

This experiment use blood from two subject, James and Kezia. Blood is a highly
specialized tissue with more 4000 with main function to transport oxygen from the lungs
to all of the living tissues of the body and carry away carbon dioxide back to lung for
expiration. It also use as body immune system against foreign material. Blood consist of
erythrocytes, leucocytes, platelet, and plasma. Plasma is a salt and protein liquid that
made up to 55% of our blood component. When blood is extract from our body we need
to add anticoagulant to prevent platelet to form clot before we do blood separation 1.
Since erythrocyte doesn’t have nucleus, we must take the leucocyte for our experiment.
DNA is a polymer made up of nucleotide and nucleotide contain deoxyribose that bind a
phosphate in 5’ carbon and 4 base (ATGC) nitrogen in 1’carbon 2.

The purity of DNA that have been isolated can be calculated by the ratio of
absorbance. Absorbance value 260nm and 280nm needed to be obtained by using
spectrophotometer. A DNA is considered to be pure if the ratio is around 1.8 - 2,0
(A260/A280), For contamination we is 230nm (A260/A230) and considered pure if the ratio is
between 2,0 - 2,2. DNA concentration is calculated by using the general formula,
(Optical Density / ε) x dilution factor3.

To analyze further, the DNA will undergo Polymerase chain reaction (PCR), a
common laboratory technique used to make many copies (millions or billions) of a
particular region of DNA. PCR use DNA polymerase enzyme to make new strand DNA
using existing strand as templates. The PCR have three major steps, denaturation,
annealing, and elongation. The new target DNA copy region can be used for analyzing,
sequencing, and electrophoresis4. PCR requires a DNA polymerase enzyme that
makes new strands of DNA, using existing strands as templates. The DNA polymerase
typically used in PCR is called Taq polymerase, after the heat-tolerant bacterium from
which it was isolated (Thermus aquaticus). T. aquaticus lives in hot springs and
hydrothermal vents. Its DNA polymerase is very heat-stable and is most active

1
https://histo.life.illinois.edu/histo/457.pdf
2
https://ghr.nlm.nih.gov/primer/basics/dna
3
NanoDrop Technologies; 2007
4
https://www.ndsu.edu/pubweb/~mcclean/plsc431/cloning/clone9.htm
around 70°C (a temperature at which a human or E. coli DNA polymerase would be
nonfunctional). This heat-stability makes Taq polymerase ideal for PCR. high
temperature is used repeatedly in PCR to denature the template DNA, or separate its
strands.
Electrophoresis using Gel as template is a technique that separates pieces of
DNA by size so researcher can further analyzed them. Gels are made of agarose, a
seaweed extract similar to gelatin. The finished gel has a consistency similar to very
firm jello. This consistency offers resistance to the pieces of DNA as they try to move
through the gel. The gel is prepared with wells at one end so that DNA samples can be
loaded into the gel. DNA in the gel is invisible. To visualize DNA, gels are stained with
ethidium bromide. Once the DNA samples are loaded onto the gel, an electric current is
applied to the gel. DNA is negatively charged due to all the phosphate groups in the
backbone of DNA. Thus, DNA will move towards the positive electrode. Larger pieces of
DNA will have more difficulty moving through the gel than smaller fragments. The
electrophoresis UV light is then used to visualize the EtBr. Ethidium bromide
intercolates between the bases. Shining UV light on ethidium bromide will cause it to
fluoresce at visible wavelengths5.

To know exact sequence of DNA after calculating the purity and the size of DNA.
DNA sequencing is needed to determining all part of the nucleotide sequence of a
specific DNA molecule. One of the DNA sequencing methods is Sanger Method or
chain termination method. This sequencing process are just like the PCR reactions but
dideoxyribonucleotide (ddNTP) was added to halt the process. At the end of incubation,
the fragments are seperate from shorter to the longer fragments. Each of ddNTP will
also have the different color when illuminated by laser beam. BLAST is a program used
to compare our DNA sequence with the NCBI database, BLAST will also confrim the
gene that we isolate is AFP gene or not6.

II. MATERIALS AND METHODS

5
https://www.colorado.edu/Outreach/BSI/pdfs/Electrophoresis%20Notes.pdf
6
Luke Alphey1997
 All these steps were taken from the laboratory protocol for Fundamental Medical
Science 1. First, Blood separation. Blood were drawn from 2 students (James and
Keshia) and put into vials then labeled them. Next was transferring blood into separated
vacutainers, with and without anti-coagulants. Then centrifuged them at 3o00 g,20°C,
for 7 minutes. We transfered the serum (upper layer transparent liquid) that we got from
the vacutainer without anti-coagulant into a new vial then stored them at -80°C.

Second, for DNA isolation we used the sample from the previous experiment
which stored at -80°C. we added 0,8 ml 1X SSC buffer to the stored vacutainer tubes in
EDTA and then mixed. Centrifuge for 1 minute at 12000rpm in a microcentrifuge then
remove the supernatant. Next was adding 1 ml of 1X SSC buffer, mix it then centrifuge
for 1 minute. After that remove all the supernatant. Add 375ul 0,2M NaOAc to each
tubes and vortex briefly, then add 25 µl of 10%SDS and 5µl proteinase K. vortex and
incubate 30 minute at 55°C. Add 120 µl phenol/chloroform/isoamyl alcohol and vortex
for 30 second then centrifudge for 2 minute at microcentrifudge at 12000rpm. Aqueous
layer were carefully removed to a new 1.5 ml microcentrifuge tube, add 1,5ml 100%
alcohol, vortex, and incubated for 15 minutes at -20C. Samples were microcentrifuged
for 2 minutes at 12000 rpm. Supernatant was decanted and drained 180 ul of 1X TE
buffer was added, vortex and incubated at 55C for 10 minutes. Add 20 ul of 2 M sodium
acetate, 500 ul of cold 100% ethanol and mixed. Samples was microcentrifuged for 1
minute at 12000 rpm. Decant the supernatant and rinse the pellet with 1 ml 0% ethanol.
Microcentrifuged for 1 minute at 12000 rpm. Decant the supernatant, and the pellet was
air dried for 10 minutes. The pellet was resuspended by adding 200 ul of 1X TE Buffer.
Samples were incubated at 55C for 30 minutes, periodically vortexed to dissolve the
genomic DNA. Samples were stored at -20C.

Third, in quantitation of DNA concentration experiment, DNA samples were

diluted (1:5). The cuvette was filled with 50 ul of 1X TE Buffer. The cuvette was set on
the cuvette holder of spectrophotometer. The type of nucleic acid was selected (dsDNA)
and the conversion factor was determined. The spectrophotometer was blanked with 1X
TE buffer. The cuvette was inserted with DNA Sample to read the absorbance (50 µl
final), and make sure absorbance fall between 0.1 to 1.0 for accurate result. Then the
concentration results were recorded.

Fourth, DNA detection using electrophoresis. First to prepared agarose 1% gel,

0.5 gr agarose was mixed in 50 ml tAE buffer in an Erlenmeyer flask, boiled in the
microwave, and let temperature reach 60C then 1 ul ethidium bromide was added. The
agarose was poured off in the tray. The comb set to 30 minute to make allow gel
become hard. The electrophoresis chamber was prepared and the agarose gel was
loaded. 5 ul of DNA was placed on to 5 mm parafilm sheet on the 9 well plate, and 1 ul
of loading dye added and mixed by pipetting. The mixture was loaded to the agarose
gel well according to the sample number. 6 ul DNA marker was loaded. TAE buffer was
filled until it reached about 1 mm above gel surface. The lid was closed, electrode was
connected to the power supply, and the electrophoresis was run at 100 V, 6 watts, 0.6 A
for +- 30 min. The gel was took out and washed under tap water. Results picture data
were recorded using Versa Doc instruments.

Fifth, for PCR experiment a reaction mixture must be made by 43.5 ul dH2O, 20 ul 5X
PCR buffer, 8 ul 2.5 mM dNTP mix, 5 ul 25 mM MgCl2, 0.5 ul taq DNA polymerase, 3
ul10 uM forward primer, 3 ul 10 uM reverse primer, 8 DNA template. The PCR tubes
wer put in the PCR machine, and the program was set as following step 1 was 95C for
10 minutes. Step 2 was consisted of 40 cycles of denaturation (94C for 30 seconds),
annealing (56C for 30 seconds), and extension (72C for 1 minutes). Step 3 was
consisted of final extension (72C for 10 minutes) and storage (4C for indefinite time).
The machine was run, and the PCR product was kept at 4C until further study.

Sixth, DNA sequencing using computer Bioinformatics software tools, Cromas

Lite and DNA sequence were saved in pasta format. Cromas Lite installed by the
laboratory assistant. Cromas lite was operated and the DNA sequence file was opened
in fasta format. Switch the direction from reverse to forward direction. Then the DNA
sequence was analyzed. Changed or edit the N base according to right color.
(Adenine=green, thymine =red, guanine=black, cytosine =blue). After editing the
sequence, it was saved and was copied in FASTA format. Open
http://www.ncbi.nlm.nih.gov/ Blast menu and Nucleotide blast menu. Edited DNA
sequence was pasted into Enter Query Sequence Area. Search set was chosen due to
DNA sequence from human genome then Human genomic database was clicked.
Lastly, BLAST was clicked and our DNA was compared with NCBI database.

III. RESULT

A. BLOOD SEPARATION

Figure 1 – Blood separation result

Left: tube with anti-coagulant. Right: tube without anti-coagulant.

 B. DNA Isolation and Electrophoresis

A B C D E F G

Description

A,D,G = DNA marker

B,E = Keshia Sample’s

C,F = James Sample’s

>10.000 bp

Figure 2 - Agarose 1% gel electrophoresis of DNA isolated from whole blood sample
and 1 kb DNA ladder

C. Quantitation of DNA Concentration

We used this following formula to determine the DNA concentration:

DNA concentration (mg/mL) = OD : e x dilution factor
Optical Density (OD) = absorbance, when the spectrophotometer has a path
length of 1 cm.
E = extinction coefficient (dsDNA = 20 g-1 cm-1 L)
Dillution factor = concentration of H20 that we use to reduce the chemical
concentration (in this experiment we use 1:5 for Keshia and 1:1 for James)
 Absorbance Results Mean of the
Wavelength Absorbance
I II
Results
A 230 0,064 0,065 0.064
A 260 0,446 0,442 0.444
A 280 0,231 0,232 0.231
Table 1. Absorbance results on James Sample 1’s DNA
Sample 1 = A 260 = 0,444
DNA concentration = 0.444/20 x 1 = 22.2 µg/mL
A 260
Purity index = = = 0.444/0.064 = 6.9375  RNA contamination
A 280

A 260
= = 0.444/0.231 = 1.922  pure
A 230

Absorbance Results Mean of the

Wavelength Absorbance
I II
Results
A 230 0,013 0,011 0,012
A 260 0,257 0,256 0,2565
A 280 0,138 0,136 0,137
Table 2. Absorbance results on Keshia Sample 2’s DNA
Sample 2 = A 260 = 0,2565
DNA concentration = 0.2565/20 x 5 = 64 ul/mL
Purity index =
A 260
= 0.2565/0.137 = 1.872  pure
A 280
A 260
= 0.2565/0.012 = 21.375  RNA contamination
A 230

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

1,6,11= DNA Marker

2,7 = Negative control
3,8 = Keshia
4,9 = Positive control
5,10 = James

400-500 bp

Figure 3 - Agarose gel electrophoresis of PCR experiment

E. DNA sequencing
Figure 4 - BLAST result of DNA sequencing experiment
 Figure 5 – DNA sequence

IV. DISCUSSION

In blood experiment, after the centrifugation process the blood in vacutainer with
anticoagulant (EDTA) was separated into three layers. The centrifugation split the blood
into three layer according to their weigh. The yellowish upper layer was the plasma that
contains, the white layer or the buffy coat in the middle, and the dark red layer in the
bottom was erythrocytes. The blood in empty vacutainer there were only two layers. The
first upper layer was serum and in the lower layer was the clotting blood that contains
RBC, WBC, platelets, and other coagulant factors. It’s because there wasn’t any
anticoagulant (EDTA) so the blood platelet was coagulated. This result is perfect.

In DNA isolation, the DNA were isolated from the white blood cells in the buffy
coat layer because in red blood cell there is no nucleolus. To get the DNA we wanted,
we must remove several components in the whole blood like protein, RNA, and lipids.
Then the isolated DNA was tested with electrophoresis. First, to lyse the erythrocytes
hypertonic SSC was added. Then to precipitate DNA NAOAc was added. To lyse
leukocytes SDS detergent was added. PCL alcohol to remove protein from nucleid acid,
will TE buffer was used to solute and protect the DNA. After this process DNA will form
small white precipitate at the bottom of solution.

To measure DNA length electrophoresis was conducted and bu comparting DNA

with marker in agarose gel for measurement. Human have 30000 base pairs, so the
result will be greater than 10000 base pairs. The fluorescence or the thickness of the
band in electrophoresis was from the amount of DNA present when isolated. In the
result we can see that Keshia’s sample have more DNA from the DNA isolation process
because it had a thicker band than James’s sample. In this experiment there was an
error because one of james’s sample was thrown away during electrophoresis. This
make one of James’s sample is blur.

In quantitation of DNA concentration experiment, DNA is considered pure if the

purity index is 1.80 -2.00. If the index <1.8, there were protein contamination and if the
index >2.0 there were RNA contamination. With the DNA contamination index, if the is
<2.0, there is a phenol, urea, or other organic compounds contamination. If the DNA
contamination index >2.2, there is a RNA contamination. From the references, we could
say that both of our DNA’s sample were not pure with purity index >2.0 so, both
samples was contaminated with RNA.

The PCR experiment was ran, after 40 cycles to amplify TGFBR2’s gene and
with the electrophoresis shown that the thickness was good with + 400-500 base pairs
and nearly same with the control sample. TGFBR2 human gene work by encodes a
member of the Ser/Thr protein kinase family and the TGFB receptor subfamily. The
encoded protein is a transmembrane protein that has a protein kinase domain, forms a
heterodimeric complex with another receptor protein, and binds TGF-beta. This
receptor/ligand complex phosphorylates proteins, which then enter the nucleus and
regulate the transcription of a subset of genes related to cell proliferation. Mutations in
this gene have been associated with Marfan Syndrome, Loeys-Deitz Aortic Aneurysm
Syndrome, and the development of various types of tumors.  The DNA samples from
James are 4477 base pairs and Keshia are 4704 base pairs. There wasn’t any
contamination, because there weren’t any band in the negative control area.
 BLAST result shown that the amplified DNA was the human transforming growth
factor beta receptor 2 gene. Editing and sequencing using Chromas lite in FASTA
format. The result was 98% similar with the TGFBR2 gene from NCBI database. The
2% may cause by RNA contamination

In conclusion that from all the experiment mainly goal to obtain the TGFBR2
gene was a success. Even though some mistake still happen in experiment

V. REFERENCES

 histo.life.illinois.edu. (2017). Formed Elements of Blood. URL:

https://histo.life.illinois.edu/histo/457.pdf [cited 2017 november 7]
 Makałowski W. (2017). The human genome structure and organization. URL:
https://www.ncbi.nlm.nih.gov/pubmed/11833767 [cited 2017 november 7]
 ghr.nlm.nih.gov. (2017). What is the DNA? URL:
https://ghr.nlm.nih.gov/primer/basics/dna [cited 2017 november 7]
 khanacademy.org. (2017). Polymerase Chain Reaction (PCR). URL:
https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-
sequencing-pcr-electrophoresis/a/polymerase-chain-reaction-pcr [cited
november 8]
 khanacademy.org. (2017). Gel Electrophoresis. URL:
https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-
sequencing-pcr-electrophoresis/a/gel-electrophoresis [cited 2017 november 8]
 colorado.edu. (2017). DNA Gel Electrophoresis. URL:
https://www.colorado.edu/Outreach/BSI/pdfs/Electrophoresis%20Notes.pdf
[cited 2017 november 9]
 amp.pharm.mssm.edu (2017). Transforming growth factor, beta receptor II
(70/80kDa). URL: https://amp.pharm.mssm.edu/Harmonizome/gene/TGFBR2
[cited 2017 november 9[