FUNDAMENTAL MEDICAL SCIENCE

FINAL REPORT (GENOMIC)

YORDAN KUSUMA

00000015703

Group E-6

Universitas Pelita Harapan

Mochtar Riady Institute for Nanotechnology

Faculty of Medicine

2015
ABSTRACT

The genetic information lies within the cell nucleus of each living cell in the
body. The information can be considered to be retained in a book for example. Each
gene is a piece of genetic information. All the DNA in the cell makes up for the
human genome. DNA was determined to be the genetic material between 1920s and
mid-1950s. By understanding genetic function, it will help genetic disease treatment,
specific drugs, and many more beneficial things.
This experiment is done to obtain DNA sample from human blood and to
determine human gene, tumor protein gene p53, in the DNA by comparing in
database of National Center of Biotechnology Information (NCBI). DNA can be
amplified using a procedure called the Polymerase Chain Reaction (PCR). Obtained
the blood sample and then undergoes lysis of its component by using chemicals until
only remain the DNA. The absorbance of the DNA is measured next using
spectrophotometer, followed by its purity and concentration. The DNA confirmed
using Electrophoresis method. After that, tumor protein gene p53 of the DNA is
amplified during PCR Experiment. The DNA undergoes one more electrophoresis.
Finally, we had to access gene database in NCBI website and submited our DNA
sequence. The website will search for the DNA on their database for a match. It also
show us how identical it is by percentage.
Although we had problem in the process of experiment, we still managed to
acquire the DNA sequence and compare it with NCBI database. After we submited
the database of DNA sequence from sample, the result showed there were DNA
sequence in the NCBI database that are similiar with the samples we obtained from
our friend, Yordan and Wening.
INTODUCTION

Blood is the red fluid that circulates in our blood vessels to the most remote
part of the body. It mainly functions as the body’s transport system, but is also
responsible for immune responses. (America’s Blood Center, 2012)

Whole blood has two components, the first is called blood plasma, a watery
liquidextracellular matrix that contains dissolved substances, and the second are
formed elements, which are cells and cell fragments. If a sample of blood is
centrifuged in a anticoagulant glass tube, the cells which are more dense will sink to
the bottom of the tube while the plasma which is less dense, forms a layer on top.
There will be a 3 different layer in the tube. The first upper layer is the blood plasma.
Plasma contains fibrinogen which is essential for blood clotting. In the middle area, a
pale or white layer called buffy coat that contains white blood cells (WBCs) and
platelets, and the last layer in the bottom is erythrocytes or red blood cells. (Tortora
& Derrickson, 2011)

The greatest quantity of DNA is located in the white blood cell within the buffy
coat layer. The DNA of these cells must be separated or extracted from the other cell
components with some methods of DNA extraction. After DNA extraction, we can
amplify tumor protein gene p53 in the DNA using PCR (polymerase chain reaction)
technique.There are 3 major steps in PCR, the first step is the denaturation, which is
a heating of the DNA strands that results in the separation of double stranded DNA
into single-stranded DNA. The second step is called the annealing step, in which the
tube is cooled for the PCR primers bind tothe DNA strands. And the last step is
extension where DNA polymerase duplicates the DNA. During the denaturation step,
the temperature is quite high to degrade the DNA, that is the reason to use Taq DNA
Polymerase which is resistant to high temperature / thermal resistance. These all
steps are repeated to amplifywith exponential amplificationfor the DNA strands.
(Stephen M. Roth, 2007)

In electrophoresis, DNA fragments will migrate with different distance

according to its sizes, and length. The fragments will move to positive site of the gell.
Larger fragments will move slower than smaller fragments. This method can
separate DNA fragments in the range of 0.1 to 5 kbps (Ryan, 2010). A
polyacrylamide sequencing gel separates strands that differ in length from one to
several hundred of sequence databases s and reveals DNA sequences of varying
lengths (Brooks, 2010).

Since the goal of this experiment is to confirm the specific gene in our body, a
big quantity of the specific nucleotide sequence where the gene located is needed
before it can be studied properly. Polymerase Chain Reaction (PCR) is a technique
to amplify specific nucleotide sequence (the P53 gene in this case) to be copied
exponentialy. PCR is based on repeated cycles of replication using specially
designed primers, DNA polymerase, dATP, dGTP, dCTP, dTTP. The steps consist of
denaturation of DNA fragments at high temperature, anealing the primers to the
complementary regions in the DNA tamplate strands, and primers extention by DNA
polymerase (H.A Erlich, 1989).

After amplification, DNA sequencing is used to determine the nucleotide order

of a DNA sequence. One of the most popular methods is Sanger Method or chain
termination method by using dideoxyribonucleotide (nucleotides without 3’ hidroxyl
group) to stop DNA synthesis proses (Cantor CR & Smith CL, 1999). The sequence
could be read after after incubation from the shorter and the longer strand. After that,
the sequence is edited by Chromas Lite software. Basic Local Alignment Search
Tool (BLAST) is used to compare our DNA sequence with the database and as the
final confirmation if our DNA sample has P53 gene or not.
MATERIALS AND METHODS

The materials and the methods in all experiments are taken from the
Laboratory Protocol for Fundamental Medican Science 1 with some revisions in the
procedures.

The experiment was started by taking 5mL of blood sample from two students
(Yordan and Wening) and stored in EDTA vacutainer tubes. And then, we prepared
and labeled each vial properly. The blood were transferred into a separated vial.
Then centrifuged the vacutainer at 3.300 g on 20o C temperature, for 10 minutes. We
transferred blood’s plasma into a new vial and stored it at -80o C.

For DNA isolation, blood samples needed is 0.5mL of blood stored in EDTA
vacutainer tubes which was frozen in later at -80o C temperature. Then 0.8mL of 1X
SSC buffer had been added into EDTA vacutainer tubes and we mixed it. The
samples were centrifuged for 1 minute at 12,000 rpm in microcentrifuge. We
removed the 1 mL of the supernatant and discarded it into disinfectant. We added 1
mL of 1X SSC buffer and vortexed it. The blood samples were centrifuged for
second time. All of the supernatant were removed. Then we added 375 uL of 0.2M
NaOAc to each pellet and we vortexed briefly. After that, 25 uL of 10% SDS and 5 uL
of proteinase K (10 mg/mL H2O) and vortexed it. The blood samples incubated for
30 minutes at 55 oC. Then we added 10 uL of isoamyl alcohol and vortexed for 30
seconds. We were centrifuged the samples for 2 minutes at 12,000 rpm in a
microcentrufuge tube. We removed the aqueous layer to a new 1.5 mL
microcentrifuge tube and added 1 mL of cold 100% ethanol and then mixed it. Next,
we centrifuged the samples for 2 minutes at 12,000 rpm in a microcentrifuge. We
decanted the supernatant and drained it. We added 180 uL 1X TE buffer, and
vortexed it and incubated it at 55 oC for 10 minutes. Then we added 20 uL 2 M
sodium acetate and mixed it. 500 uL of cold ethanol we had been added and mixed.
Then we centrifuged for 1 minute at 12,000 rpm in a microcentrifuge. The
supernatant we had decanted and we rinsed the pellet with 1 mL of 70% ethanol. We
centrifuged the samples for 1 minute at 12,000 rpm in a microcentrifuge and
decanted the supernatant then air dried the pellet ‘till dry. Then the pellet
resuspended by adding 1X TE buffer 200 uL and incubated it at 55 oC for 30
minutes. We vortexed periodically to dissolve the genomic DNA. The samples stored
in -20 oC.
 We diluted the DNA sample (1:5) and filled the cuvette with 50 uL of 1X TE
buffer. The cuvette we were set on the cuvette holder of spectrophotometer. In this
experiment, we used dsDNA as the type of nucleic acid. To use this
spectrophotometer, we set blank the spectrophotometer with 1X TE buffer. After that,
we inserted the cuvette with DNA sample to read the absorbance (50 uL final
volume). Then we recorded the concentration.
RESULTS

DISCUSSIONS

REFERENCE
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Brooks GF, Carroll KC, Butel JS, Morse SA, Mietzner TA. Jawetz, Melnick, &
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H.A Erlich. PCR technology: principles and applications for DNA amplifications.
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