FUNDAMENTAL MEDICAL SCIENCE 1

FINAL REPORT (GENOMIC)

SHEREN ALEXIS
01071180186
GROUP E1-1

UNIVERSITAS PELITA HARAPAN

MOCHTAR RIADY INSTITUTE OF NANOTECHNOLOGY
FACULTY OF MEDICINE
2018
ABSTRACT
A genome is an organism’s complete set of DNA, including all of its genes.
The genomics is the study of all DNA set in organism. There are more than 3
billion DNA base pairs in humans which has each nucleus. Nowadays many
diseases are found in human such as inheritance disease and mutation that
cause disease. So the genomic technology help us to understand about DNA.
The purpose of doing this lab is to identify the specific gen in our blood
sample called TGFBR2 gene (Transforming Growth Factor Beta Receptor 2) . At
first, our blood sample were taken out by injection and then the blood is
centrifuged for separation of blood. DNA is isolated from the buffy coat of the
sample. The concentration and purity of DNA was determined using
spectrophotometer. And we used electrophoresis to detect DNA size whether the
experiment of isolate DNA had successful. The isolated DNA sample is amplified
using PCR (Polymerase Chain Reaction) to multiply the DNA. And then, we did
DNA sequencing by Sanger method. After that, the sequence of DNA was edited
in computer using Chromas Lite. Then, the nucleotide sequence was copied to
compare with the nucleotide sequence from NCBI using Basic Local Alignment
Search Tool (BLAST).
I. INTRODUCTION
In this genomic experiment, blood as the main material that we used to.
Blood is made up of liquid and solids. Red blood cells do not have DNA, as they
lose their nuclei ( the compartment in a cell that contains DNA). So the DNA in
your blood is in your white blood cells. Therefore, we have to separate the cells
from the blood fluid. After blood fluid was separated using centrifuge, it contains
3 different layers in tube. The upper layer is the blood plasma. In the middle
area, a buffy coat that contains white blood cells and platelets. And the last
layer, in the bottom is red blood cells.
Most DNA is available in white blood cells within the buffy coat layer.
DNA must be separated from the others by using DNA extraction. After that, we
can amplify the TGFBR2 gene by using PCR (polymerase chain reaction). There
are 3 steps in PCR which is denaturation (heat the reaction strongly to separate,
or denature the DNA strands, this provides single-stranded template for the next
step), Annealing (cool the reaction so the primers can bind to their
complementary sequences on the single-stranded template DNA), Extension
(raise the reaction temperatures so Taq Polymerase extends the primers,
synthesizing new strands of DNA. These all steps is to amplify the DNA strands.
The most common technique to determine DNA yield and purity is
measurement of absorbance. All the needed for the absorbance method is a
spectrophotometer and a solution of purified DNA. Absorbance readings are
performed at 260 nm (A260) where DNA absorbs light most strongly. Good-
quality DNA will have an A260/A280 ratio of 1.8 to 2.0.
Then there is also Agarose Gel Electrophoresis which is another way
to quickly estimate DNA concentration. This technique is the most effective way
of separating DNA fragments of varying sizes.
After we know the size of our DNA, we also need DNA sequencing
which is the process of determining the sequence of nucleotides in a piece of
DNA. One of DNA sequencing is Sanger Sequencing which is the target DNA is
copied many times, making fragments of different lengths. Fluorescent “chain
terminator” nucleotides mark the ends of the fragments and allow the sequence
to be determined. The end of incubation, this fragments are separate shorter to
longer fragments. Each of ddNTP have a particular nucleotide which is labeled
with different coloured dye.Is a unique analysis of illuminated area as a function
of inrensity in a plane of the laser beam. BLAST (basic local alignment search
tool) find regions of similarity between biological sequences. The program
compares nucleotide to sequence databases with the NCBI database. BLAST is
also confirm if we isolate TGFBR2 gene or not.
II. MATERIALS AND METHODS
In this experiment, first we must take 8-9 ml blood samples from
2 students in each group (E-1 group : Sharon Chen and Ryan Shidqi) and
will be placed on each vacutainer which has been labeled with its name.
Then take 1 ml of blood and transferred it into a separated vial. Then
centrifuge vacutainer at 3000 g at 20 degrees celcius for 10 minutes.
Later transferred serum to the new vial. And stored it at -80 degrees
celcius.
Further 0,5 ml of whole blood stored in EDTA vacutainer tubes,
and added 0,8 ml 1X SSC Buffer and mixed it, then centrifuged for 1
minute at 12.000 rpm in a microcentrifuge. Supernatant was removed by
1 ml. Afterwards, added 1 ml 1X SSC buffer, vortex, centrifuged at the
same level and removed all of the supernatant.375 µl of 0,2 M NaOAc
was added to each pallet and will be vortexed later. Furthermore, added
25 µl of 10% SDS and 5 µl of proteinase K, vortexed and incubated for 30
minutes at 55 degrees celcius. Added phenol/alcohol after the incubation.
The aqueous layer was taken to a new 1,5 ml microcentrifuge tube and
added 1 ml of cold 100% ethanol, mixed, and incubated for 15 minutes at
-20 degrees celcius. The supernatant was decanted and drained.
Thereafter, added 180 µl 1X TE buffer, vortexed, and incubated at 55
degrees celcius for 10 minutes. Next, 20 µl 2 M sodium acetate was
added and mixed it. Subsequently, 500 µl of cold 100% ethanol was
added and mixed it. The sample was centrifuged. The supernatant was
decanted, then centrifuged again, and was airdried for 10 minutes.
Finally, the pellet was resuspended with 200 µl of 1X TE Buffer, incubated
again.
The concentration was recorded after the absorbance was
made sure between 0,1 to 1,0.
 After that, to do the electrophoresis, we must prepare 1%
agarose gel by mixing 0,5gr agarose in 50ml TAE buffer in an Erlenmeyer
Flask, boil in the microwave, by letting the temperature reach 60 degrees
celcius, added 1 µl Ethidium Bromide, pouring off into the gell tray. To
amplify the TGFBR2 gene by using PCR technique,
We have to install Bioinformatic software tools (Cromas Lite). If
the DNA sequence had reverse direction, it was switched to forward
direction. Next, DNA sequence was analysed by changing N base with
base according to appropriate color. After the DNA sequence was edited,
saved it and the data was copied in fasta format. NCBI site was opened
(http://www.ncbi.nlm.nlh.gov/). Blast menu and nucleotide blast menu was
clicked, the DNA sequence was pasted into Enter Query Sequence Area.
After search set was chosen, the Human Genomic database was clicked.
And the last the BLAST was clicked.
III. RESULTS
A. Blood Separation
B. DNA Isolation and DNA Electrophoresis
DNA Isolation ->

DNA Electrophoresis ->

C. Quantitation of DNA Concentration

This is the formula of DNA concentration :
DNA concentration = (OD : ε) x dilution factor
1. 1:5 I n 50 µl
 10 µl DNA sample and 40 µl solvent (TE buffer)
2. R 230 = 0,693/0,697 = 0,695
260 = 0,17/0,19 = 0,18
280 = 0/0,006 = 0
S 230 = 0,856/0,860 = 0,858
260 = 0,60/0,063 = 0,0615
280 = 0,040/0,049 = 0,0445

Concentration
 R = (0,18/20) x 1/5 = 0,18 x 5/20 = 0,045 mg/ml
 S = (0,0615/20) x 1/5 = 0,0615 x 5/20 = 0,015375 mg/ml
Ratio
 R = 260 : 230 = 0,18 : 0,695 + 0,26
260 : 280 = 0,18 : 0 = -
 S = 260 : 230 = 0,0615 : 0,858 = 0,072
260 : 280 = 0,0615 : 0,0445 = 1,38
3. DNA is considered pure if the index is between 1,80-2,00.
However both DNA shows result that it is both contaminated.
Because both is below 1,80, this shows that our DNA is
contaminated with protein.
D. PCR

1. PCR

technique can amplify the specific segmen of DNA because we use

primer in the process which limits the target region in the DNA. It also
amplifier the DNA exponentally. Because it uses the product from the
previous cycle and duplicates it again, resulting in 2^n product at the
end.
2. Because Taq polymerase is extracted from Thermus aquatious which
is heat stable and we need heat stable polymerase because PCR
requires high temperature (50°C-80°C) and Taq polymerase is
optimum at 72°C.
E. DNA Sequencing
IV. Discussion
The specific gravity of blood components is plasma, platelets
and leucocytes(buffy coat), packed red blood cells (RBC). The
centrifugation split the blood into three layer first we called this with anti-
coagulant, the upper layer was plasma 55%(pale yellow sticky liquid)
which contained albumin (protein), fibrinogen (responsible for clotting of
blood), and globulins (antibodies). The second layer was the buffy coat,
and the third layer was red blood cell 45% which contained erythrocytes.
This happened because the anti-coagulant prevented clotting factors to
coagulate the blood .While without anti-coagulant, there are only 2 layers
which is first layer is serum (transparent orange) , this is plasma but it
doesn’t contained clotting factors because it lacks of fibrinogen(fibrinogen
and platelet as clotting factor coagulated red blood cell and white blood
cell together as clot blood at bottom layer and would stand still even the
vial was shaken). The second layer was the clotting red blood cell (dark
red). This experiment was successed and the result was matched with
the theory.
In DNA isolation, the aim to do this is to isolate DNA from
human blood and to understand the basic principle of DNA isolation
technique and its application. And for doing this, we needed component
of DNA and only leukocytes in buffy coat has it. The isolated DNA was
tested with electrophoresis. Fluorescence in electrophoresis was present
when isolated. There are also found in human error if band have different
thickness when DNA was placed to to the agarose gel. Where blood was
taken, unneeded material was immediately discarded. Specifically,
because red blood cells was lacked of nucleus, so in here we used it from
buffy coat while buffy coat do not contain pure DNA so we have to extract
DNA. First, the protein would be degraded by proteinase K. In next,
NaOAc was added and SDS detergent was used to lysis the membrane.
PCL function was to remove the unneeded component and DNA have to
be precipitated by ethanol and TE buffer was used to resuspend it. Which
 the point was the DNA was extracted also shown in our result in white
dot. Ours have were very small than intended.
In gel electrophoresis, this result was shown by compare to DNA
ladder that both our DNA’s samples are above the 10.000 base pairs. (the
DNA ladder is 30,000 base pairs).The thin bands functions as DNA ladder
from 250-10.000 base pairs, while thick bands, show that the lane F was
thinner. This was an error during the process. We can conclude that,
there was error during the process causing the DNA be undetected, but
there was still some sample that can be used for further experiment.
PCR technique amplify the gene of DNA and Taq DNA
polymerase was used in order if the enzyme was not denaturated in high
temperature while we must needed heat to cut DNA double strand into a
single strand. Electrophoresis was determine the DNA sequence was
amplified or not. Our experiment was succeed because detect the size of
DNA sample and the result is more than 400 nm.
Latter, the aim of DNA sequencing is to know that TGFBR2
gene was amplified and compared to database (NCBI). This result was
told us that TGFBR2 gene was amplified. And we have similarity with the
gene ERCC2. And finally our genomic experiment was finished, although
some errors occurred.

V. REFERENCES
Websites :
U.S National Library of Medicine. Genetics Home Reference.
( published 2018 November 13; cited 2018 Nov 13). Available from :
https://ghr.nlm.nih.gov/primer/hgp/genome

U.S National Library of Medicine. MedlinePLus. (Exported from 2018

November 13; cited 2018 Nov 13). Available form :
https://medlineplus.gov/blood.html

Promega.
https://worldwide.promega.com/resources/pubhub/enotes/how-do-i-
determine-the-concentration-yield-and-purity-of-a-dna-sample/

OpenStax College, Biology (CC BY 4.0).Khan Academy. Lewis, T

(2013, April 14). National Human Genome Research Institute (2016,
January 15). Retrieved from : http://www.livescience.com/28708-
human-genome-project-anniversary.html.
https://www.genome.gov/sequencingcosts/.

The University Of Queensland. Diamantina Institute. (updated: 27 Apr 2017).

Available from : https://di.uq.edu.au/community-and-alumni/sparq-
ed/sparq-ed-services/sequencing-dna-using-dye-terminators

E.D. Hirleman and W.H. Stevenson. OSA Publishing. Available from :

https://www.osapublishing.org/ao/abstract.cfm?uri=ao-17-21-3496