FUNDAMENTAL MEDICAL SCIENCE 1

FINAL REPORT (GENOMIC)

JESSICA ADHYKA MARGARETH

00000018861
Group C5-2

Universitas Pelita Harapan

Mochtar Riady Institute for Nanotechnology
Faculty of Medicine
2015
 ABSTRACT

Every living creature in this world has special codes in its body. Those codes
determine its traits. That codes called Gene, which is in human, it regulates what is
seen (the phenotype) and what is happening inside a human’s body (the genotype).
Gene is located in the DNA, which is located in all cells on human’s body.

The objectives of this genomic experiment is to extract, obtain, identify,

and amplify the P53 gene from the blood sample. The experiment was arranged by
extracting blood from appointed students and started with separating the blood
components from the blood sample with centrifugation method. After the blood is
successfully separated into three layers, the process continued with DNA isolating,
determining the DNA concentration with spectrophotometer instrument, confirming the
DNA size from electrophoresis using Versa Doc Machine to compare it with DNA
ladder, amplifying the targeted gene in DNA through PCR technique, and sequencing it
using the dideoxy method. The sequence then was edited and analyzed using a
software to determining the nucleotides order in the sequence called Chromas Lite. The
edited sequence then was compared and confirmed with the sequence that listed in
database in NCBI site using BLAST and our amplified target gene was finally known as
P53 gene.

Eventhough there were some errors in the results but it was still good enough to
be used for other experiments. It was shown as the sample had 100% identities
compared to the database, which meant that our sample was indeed homo sapiens
tumor protein P53 (TP53).
I. INTRODUCTION

Blood consists of four constituent parts: red blood cells, white blood cells,
plasma, and platelets. Blood consists of 45% cells components (packed cell volume)
and 55% of blood plasma. Plasma is mainly water, but also contains many important
substances such as proteins (albumin, clotting factors, antibodies, enzymes, and
hormones), sugars (glucose), and fat particles, it serves as transporter for materials
carried in the blood. The composition is made up of protein plasma that includes
albumin, globulin, and fibrinogen. If a test tube of blood is left to stand for half an hour,
the blood separates into three layers as the denser components sink to the bottom of
the tube and fluid remains at the top. The fluid that forms the top layer is called plasma
and forms about 60% of blood. The middle layer (buffy coat) is composed of white blood
cells and platelets, the bottom layer is the red blood cells. These bottom two layers of
cells form about 40% of the blood (http://www.ncbi.nlm.nih.gov/books/NBK2263/).

DNA (deoxyribonucleic acid) is the hereditary material in humans and almost all
other organisms. Most DNA is located in the cell nucleus (it’s called nuclear DNA), but a
small amount of DNA can also be found in the mitochondria (it’s called mitochondrial
DNA or mtDNA). The information in DNA is stored as a code made up of four chemical
bases: adenine (A), guanine (G), cytosine (C), and thymine (T). Human DNA consists of
about 3 billion bases, and more than 99 percent of those bases are the same in all
people (http://ghr.nlm.nih.gov/handbook/basics/dna). Several genes made up the
segment of DNA and its hereditary information passes from generation to generation.
(Tortora, 2014) Therefore, only WBCs which are a nucleated blood cells, which could
provide the human DNA. RBCs do not capable of providing any genetic materials due to
the absence of nucleus.

Polymerase chain reaction (PCR) is a revolutionary method developed by Kary

Mullis in the 1980s. PCR is based on using the ability of DNA polymerase to synthesize
new strand of DNA complementary to the offered template strand
(http://www.ncbi.nlm.nih.gov/probe/docs/techpcr/). This process allows scientist to
produce DNA copies in a larger amount within a shorter period of time. The technique
involves 3 main processes which are DNA template denaturation, primers annealing
and elongation or extension of the DNA strand. (Campbell, 1996)

To confirm the DNA isolation result, concentration and purity of the isolated DNA
need to be analyzed. This is done using spectrophotometer, a device which measures
the absorbance of light with certain wavelengths. It works according to Lambert-Beer’s
law, which states that the amount of light absorbed in a medium is proportional to the
medium’s concentration (Fankhauser, 2007). The fact that nucleic acid could absorb UV
light potentially in the length of 260 nm, an absorbance at 260 nm is used when
calculation the concentration of DNA. The purity index of a DNA was measured through
a ratio between absorbance 260 nm against 280 nm. The value of the ratio needs to
stay in between 1.8 – 2.0 for maximum purity. (Bazzi, 2009)

Electrophoresis is a technique used in a laboratory to identify, quantify, and purify

nucleic acid fragments that result in the separation of charged molecules (this technique
uses agarose gel). Samples are loaded into wells of an agarose or acrylamide gel and
subjected to an electric field, causing the negatively charged nucleic acids to move
toward the positive electrode. Shorter DNA fragments will travel more rapidly, whereas
the longest fragments will remain closest to the origin of the gel, resulting in separation
based on size. (http://www.lif.illnois.edu/molbio/geldigest/electro.html;
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/DNA-
RNA-Purification-Analysis/Nucleic-Acid-Gel-Electrophoresis/DNA-Electrophoresis.html)

Sequencing DNA means determining the order of the four chemical building
blocks - called "bases" - that make up the DNA molecule. The sequence tells scientists
the kind of genetic information that is carried in a particular DNA segment
(https://www.genome.gov/10001177). There two types of sequencing method. The first
one is Maxam-Gilbert’s method which involves chemical substances and radiation to
break down DNA fragments, and the second one is Sanger’s method, which involves
dideoxyribonucleic acid mixtures. Sanger’s method is considered to be more practical
and does not cost more DNA samples. BLAST (Basic Local Alignment Search Tool) is
an algorithm which helps comparing primary biological information. It calculates the
statistics on how similar the DNA fragments sequence compare to the database present
on the internet. (http://blast.ncbi.nlm.nih.gov/Blast.cgi)

II. MATERIALS AND METHODS

Procedures are conducted based on the Laboratory Protocol for Fundamental

Medical Science 1, Faculty of medicine, Universitas Pelita Harapan with several minor
changes upon it.

Blood Separation

In blood separations, blood samples were drawn from two appointed students
(Joseph and Josephine) and collected using a vacutainer coated with K 3EDTA (anti-
coagulant) and additional empty vacutainer. From each sample, one mL of whole blood
were transferred into a separated vial and labeled properly. The remaining blood
samples were then centrifuged at 3000 gravitation or 3824 RPM using Alegra X15
centrifuge, for 10 minutes, and in 20oC. The plasma (upper transparent layer) was
transferred into separated vials and stored at -80˚C.

DNA Isolation

To isolate the DNA, 0.5 ml of whole blood from the EDTA vacutainer was
transferred into separated pellets and mixed with 0.8 ml 1X SSC buffer. The samples
then where centrifuged at 12,000 rpm for 1 minute in a microcentrifuge and 1 ml of
supernatant was removed. 1 ml of 1X SSC buffer then added to the samples, vortexed
and was centrifuged for another 1 minute at 12,000 rpm in a microcentrifuge. After that,
all of the supernatant were removed and each sample was added with 375 µl of 0.2M
NaOAc to each pellet. The samples then vortexed, then 25 µl of 10% SDS and 5 µl of
proteinase K (10mg/ml H2O) were added. The samples were again vortexed and
incubated for 1 hour at 55°C. 120 µl of phenol/chloroform/isoamyl alcohol was added
and the samples were vortexed for 30 seconds, centrifuged at 12,000 rpm for 2 minutes
and the aqueous layer was transferred into separated 1.5 ml microcentrifuge tube. 1 mL
of cold 100% ethanol was added, mixed, and centrifuged using the previous setting.
The supernatant was removed. Addition of 180 µl 1X TE buffer was given before
incubation at 55˚C for another 10 minutes. 20 µl of 2M Sodium Acetate (NaOAc) and
500 µl of cold 100% ethanol were and mixed. The samples were centrifuged at 12,000
rpm for 1 minute and supernatant was decanted. The pellets were rinsed with 1 mL of
70% ethanol and centrifuged again for a minute at 12,000 rpm. The supernatant was
decanted and the pellets were dried for 10 minutes. The pellets were re-suspended by
adding 200 µl of 1X TE buffer and incubated for 30 minutes at 55˚C. Lastly, the DNA
samples were stored at -20˚C.

Quantitation of DNA Concentration

In quantitation of DNA concentration, all of the DNA samples were diluted diluted
for the ratio of 1:5 with 10 µl of used DNA and 40 µl water. The cuvette was then filled
with 1X TE buffer and set onto the spectrophotometer. The nucleic acid type was
selected as dsDNA and conversion factor was determined. The spectrophotometer was
blanked with 1X TE buffer and cuvettes with DNA samples were set in the
spectrophotometer and the concentration was recorded.

DNA Detection using Electrophoresis

For the electrophoresis process, Agarose 1% gel and the tray were prepared. The
agarose mixture was then poured on the tray and comb was set for the formation of well
and let 30 minutes in room temperature for the gel to get hard. Furthermore, the gel was
loaded onto the electrophoresis chamber. 1 µl of DNA was placed in tube or using
parafilm sheet on the 96 well plate that was mixed by pipetting technique with 1 µL of
loading dye. The mixture of DNA and 2 µl of DNA marker were loaded into the agarose
gel. Next, TAE buffer was filled into the chamber until 1 mm above the gel. The lid was
closed and the electrode was connected the power supply. It was occurred at 100 V, 6
Watts, 0.6 A for 30 minutes. Then, the data was recorded and the picture was saved by
using Versa Doc.

Polymerase Chain Reaction

In Polymerase Chain Reaction (PCR), a reaction mixture (for 25 µl) was set up in
a PCR tube which consists of 9,875 µl of dH2O, 5 µl 5X Buffer PCR, 2 µl 2,5 mM dNTP
mix, 1,5 µl 25 mM MgCl2, 0.125 Taq DNA Polymerase, 0.75 µl 10 µM Forward primer,
0.75 µl 10 µM Reverse primer, and 5 µl of DNA template. The mixture was prepared for
4 tubes (positive, negative, and 2 DNA Samples) and was put in the PCR machine. The
program was set for several steps. For the first step, the program was set at 95 oC for 30
seconds. The second step consisted 3 parts: denaturating at 94 oC for 30 seconds,
annealing at 58,6oC for 30 seconds, and extension at 72oC for 1 minute. The third step
consisted 2 parts: final extension at 72oC for 10 minutes and stored at 4oC for indefinite
time.

DNA Sequencing

Finally on DNA sequencing, a bioinformatics software tools called Chromas Lite

was installed and operated to open DNA sequence file in FASTA format. If DNA
sequence has reverse direction, it has to be switched to forward direction and properly
analyze the sequence. The file was saved and copied in FASTA format. Next, BLAST
menu was opened in http://www.ncbi.nlm.nih.gov/ and nucleotide BLAST was chosen.
The edited DNA sequence was pasted onto the Entry Query Sequence area, a human
genome category was chosen before clicking onto the BLAST button. After that, the
results was compared and recorded.
RESULTS

A. Blood Separation

Figure1. Image of Blood in Figure2. Image of Blood in

K3 EDTA (anti-coagulant) blanked vacutainer
vacutainer

B. DNA Isolation and DNA Electrophoresis

A B C D E F G H I
Well C = Joseph’s 1st Sample (C5-2)

Well D = Josephine’s 1st Sample (C5-2)

Well G = Josephine’s 2nd Sample (C5-2)

Well H = Joseph’s 2nd Sample (C5-2)

Well I = DNA Marker

Figure3. Agarose Gel Electrophoresis

after DNA Isolation
C. Quantitation of DNA Concentration

After these experiment, it’s possible to calculate the DNA purity and quantitation of
DNA concentration using calculation below:

Purity = A260/A280
= A260/A230

DNA concentration = (OD : ε) x dilution factor

ε = extinction coeficient
OD = optical dilution
= concentration x ε
This is the calculation that has been done to our DNA sample

Josephine’s Samples
Mean
First Second
A230 0,288 0,203 0,2455
A260 0,761 0,629 0,695
A280 0,428 0,325 0,3765
Joseph’s Samples
Mean
First Second
A230 0,093 0,093 0,093
A260 0,088 0,087 0,0875
A280 0,020 0,017 0,0185

Purity and contamination of samples:

Josephine

- Purity Index = A260/A280 = 0,540

- Contamination Index = A260/A230 = 1,534
- Concentration = 0,0875/20 x 5 = 21,875 µg/mL

Joseph
 - Purity Index = A260/A280 = 0,211
- Contamination Index = A260/A230 = 0,200
- Concentration = 0,695/20 x 5 = 173,75 µg/mL

D. Polymerase Chain Reaction (PCR)

Well A = Josephine’s Samples (C5-1)

Well B = Joseph’s Sampels (C5-1)

ABCDEFGHI
Well C = Negative Control

Well D = Positive Control

Well E = DNA Marker

Well F = Josephine’s Samples (C5-2)

Well G = Joseph’s Sampels (C5-2)

Well H = Negative Control

Well I = Positive Control

Figure4. Agarose Gel Electrophoresis

after PCR
E. DNA Sequencing
 Figure5. BLAST Result of DNA Sequencing, P53 Tumor Suppressor gene sample (forward)

Result : Showing high percentage of match to homo sapiens Tumor Supressor protein
(P53) gene.
 Figure6. BLAST Result of DNA Sequencing, P53 Tumor Suppressor gene sample (reverse)

Result : Showing high percentage of match to homo sapiens Tumor Supressor protein
(P53) gene.

III. DISCUSSION

From the result of blood separation, three layers of blood components formed in
the K3EDTA-coated vacutainer consisted of plasma (top), buffy coat (middle), and
erythrocytes (bottom), while the uncoated vacutainer only contained two layers, serum
(top) and clotted erythrocytes (bottom). According to theory, anticoagulated blood has
its clotting system inactivated and three layers are formed after contrifugation (plasma,
buffy coat, and erythrocytes), while blood without anticoagulant has its clotting factors
and thrombocytes clot the erythrocytes so only two layers are formed (serum and
clotted erythrocytes). The results were consistent with the theories stated in the
Introduction section, thus the experiment was successful (Schottenfeld and Fraumeni,
2006).

The concept of DNA isolation is to separate the DNA from other substances
present inside the cell and the cell itself. This was conducted through adding several
buffer and enzymes and removed the unwanted materials in order to obtain a purified
DNA. (Bruce, 1996) SSC buffer was used to lyse RBCs, NaOAc facilitates SDS
detergent (Sodium Dodecyl Sulphate) to lyse WBCs, Proteinase K to degrade proteins,
Phenol/Chloroform/Isoamyl alcholo (PCI) to remove non-nucleic acid molecule, and
lastly TE buffer to maintain pH environment and re-suspend the isolated DNA. There
are several errors might be caused by contamination and error when the experiment
was run. In the first electrophoresis, samples didn’t appear as thick band which means
the concentration of the samples low (might be caused by contamination, because DNA
is very sensitive with contamination).

The contamination is proved by the calculation. Calculation of DNA purity for

A260/A280 is below 1.8 for each sample, it means DNA sample has been contamined
with protein. The calculation for A260/A230 is below 2, it means DNA sample has been
contamined with organic compound
(http://www.phenogenomics.ca/transgenics/docs/NanoDrop%20Nucleic-Acid-Purity-
Ratios.pdf). Therefore, we have you have to be careful to do the DNA Isolation
experiment when taking the sample from the polar phase and the next steps. From the
DNA Electrophoresis, we also know that the samples we used is from the human DNA.
It’s because we found the size more than 10,000 base pairs
(http://www.oml.gov/sci/techresources/Human_Genome/project/info.shtml). It’s shown
that the samples are above the DNA marker.

PCR Experiment shown that the samples have thick bands, it means the samples
have high concentration and purity (no contamination in this experiment). Within the
PCR process, all the DNA samples were mixed with several substances in order to
amplify specific part of the gene, specifically the P53 gene. The result of DNA
sequencing shown that the sample are truly human genome.

In conclusion, some of the experiment ended successfully although there were

some mistakes during DNA Isolation experiment. However, all problems were resolved
after PCR is done. The objective to identify the P53 gene was met and confirmed with
satisfactory BLAST results of the sample sequences.
IV. REFERENCES

Cseke LJ, Peter B. Kaufman, Gopi K. Podila, Chung-Jui Tsai. Molecular and Cellular
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Tortora GJ, Bryan Derrickson. Principles of Anatomy & Physiology. 14 th ed. John Wiley
& Sons, Inc: Hoboken; 2014

Bruce A. Roe, Judy S. Crabtree. DNA Isolation and Sequencing. John Wiley & Sons,
Inc; 1996

Campbell M et al. Polymerase Chain Reaction 4 th ed. Benjamin/Cummings Publishing

Co.: New York: 1996.

Westermeier R. Electrophoresis in Practice. 4th ed. Wiley-VCH Verlag GmbH & Co.,
Weinheim; 2005

Sherwood L. Human Physiology From Cells to System. 7 th ed. Brook/Cole, Belmont,

USA; 2010

Schottenfeld D, Fraumeni JF. Cancer Epidemiology and Prevention. New York : Oxford
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Zdanowics MM. Concepts in Pharmacogenomics. Bethesda : America Society of Health

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Bazzi. DNA Quantification; Website 2009 (Accessed 2015, November 16th) ; Available
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Website 2014 (accessed 2015, November 16th). Available from: URL:
http://ghr.nlm.nih.gov/handbook/genomicresearch/snp.

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http://www.ncbi.nlm.nih.gov/books/NBK2263/

http://www.ncbi.nlm.nih.gov/probe/docs/techpcr/

http://www.lif.illnois.edu/molbio/geldigest/electro.html

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/DNA-
RNA-Purification-Analysis/Nucleic-Acid-Gel-Electrophoresis/DNA-Electrophoresis.html

https://www.genome.gov/10001177

http://blast.ncbi.nlm.nih.gov/Blast.cgi

http://www.phenogenomics.ca/transgenics/docs/NanoDrop%20Nucleic-Acid-Purity-
Ratios.pdf

http://www.oml.gov/sci/techresources/Human_Genome/project/info.shtml