FUNDAMENTAL MEDICAL SCIENCE I

FINAL REPORT (GENOMIC)

ANDRE FARNANDES
07120120011
GROUP A1-3

UNIVERSITAS PELITA HARAPAN

MOCTHAR RIADY INSTITUTE FOR NANOTECHNOLOGY
FACULTY OF MEDICINE
2012
ABSTRACT

Today, genetics are very important information in this life. It could tell a lot
of things from what kind of genetic mutation(s) which could be responsible for
one or more disease , who are your siblings, and many more.

Our main goal for this experiment was to find a presence of HGNC:317
gene that is responsible for the expression of alpha fetoprotein. Started from
isolating the human blood using syringe, then we centrifuged the blood that was
mixed before with K3EDTA, and then we transferred the serum (the transparent
liquid, upper layer) into a new vial, and then we isolated the DNA from serum,
later we dilute the DNA sample and transferred it into the cuvette then we
measured the quantation of DNA concentration, and then we put the isolated
DNA into the agragose gel so we could detect the number of base pairs of DNA
using electrophoresis, after the number of base pairs was detected we amplified
the HGNC:317 through PCR, and then we sequenced the DNA using a
bioinformatics software tools named “Chromas Lite” and matched it using Basic
Local Aligment Sequence Tool (BLAST) to confirm the DNA which was amplified
was HGNC:317 gene.

Although there were many errors occurred in some experiment, we

succeed our main goal which is finding the HGNC:317 gene. We were satisfied
with our result because indeed our gene was perfectly match with the BLAST
database.
I. Introduction

Genetics itu apa. Alpha fetoprotein itu apa gen isinya tentang apa dan
kalo tinggi kenapa kalo rendah kenapa. DNA itu apa dimana dan cara
ngambilnya, lalu apa aja yang diperhatikan, lalu menterjemahkannya.

DNA is a very long, threadlike macromolecule made up of a large number

of deoxyribonucleotids, each composed of a base, a sugar, and a phosphate
group. The bases of DNA molecules carry genetics information, whereas their
sugar and phosphate groups perform a structural role. The structure of the DNA
itself is double helix. And when this structure was discovered, the complementary
of the structure suggest that each is a template for the other in DNA replication.
DNA polymerase is an enzymes that replicate the DNA by taking instructions
from DNA template. (Stryer, 1999)
II. MATERIALS AND METHODS

There are procedures with specific materials and methods on every step
of finding the gene itself. There were some revisions for the materials itself due
the limited stocks of our materials.

The first experiment would be extract the blood from voulenteers and
separate the blood. For this experiment, we need blood, vacuntainer, syringe,
vials, centrifuge tubes. The first step, we drawn the blood from the voulenteers.
And then we transferred one ml blood into two different vials and labeled it. One
of the vial contains EDTA and the other one doesn’t. And then we centrifuged the
vacutainer at 3700 rpm using Alegra X15 at 20°C for ten minutes. After that we
transfer the serum, the transparent liquid at the upper layer into a new vial and
stored the vials at -80°C.

The second experiment would be isolating the DNA itself. For this
experiment we need ethanol absolute, SSC buffer, 70% ethanol, sodium acetate,
10% sodium dodecyl sulfate, tris EDTA buffer pH 8.0, phenol, and proteinase K.
First, we took the 0.5 ml of blood sample stored in EDTA vacutainer tubes which
is frozen later at -70°C. Then, we add 0.8 ml 1x SSC buffer and mix it. Later, we
centrifuged at 12000 rpm for one minute in the microcentrifuge. Took the vial and
then remove all the supernatant. After all the supernatant were drained, add 375
μl of 0.2M NaOAc to each pellet and vortex briefly then add 25 μl of 10% SDS
and 5 μl of 70% ethanol (10 mg/ml H2O). And then vortex briefly and incubate for
1 hours at 55°C. Later, add 120 μl pnehol and vortex briefly up to 30 seconds.
Then, centrifuge the sample for 2 minutes at 12000 rpm in a microcentrifuge.
After that, remove the aqueous layer to a new 1.5 ml microcentrifuge tube, add 1
ml of cold 100% ethanol, mix, and incubate for 15 minutes at -20°C. Collect the
vial and then centrifuged for 2 minutes at 12000 rpm in a microcentrifuge. Decant
the supernatant and drain. And then add 180 μl 1X TE buffer, vortex, and
incubate at 55°C for 10 minutes. And then, add 20 μl 2M sodium acetate and
mix. And then add 500 μl of cold 100% ethanol mix. Then, centrifuged for one
minute at 12000 rpm in the microcentrifuge. And then, decant and rinse the pellet
with 1 ml 70% ethanol. Last, resuspend with 200 μl of 1X TE buffer, incubate at
55°C for 30 minutes and store the sample at -20°C.

The third experiment would be measuring the quantitation of DNA

concentration. We need ultra pure water, DNA sample, Spectrophotometer
BIORAD, and a cuvette for this experiment. First, dilute the DNA sample in 1:10
ratio, 1 for DNA and 10 for water. Then, fill the cuvette with 50 μl !X TE buffer,
and place the cuvette into the cuvette holder. At the spectrophotometer, set the
type of nucleic acid we want to read. Then blank the spectrophotometer using the
cuvette filled with TE buffer. Then, put the diluted DNA into a new cuvette, then
read the cuvette usng the spectrophotometer. Record the concentration.

The fourth experiment would be detect the DNA using electrophoresis. For
this experiment, we need an electrophoresis chamber and power supply, gel and
castring trays, sample combs, electrophoresis buffer, loading buffer, ethidium
bromide, and DNA marker. First, prepare the gel tray. Then, we made the gel
using 0.5 gr agragose 10% gel in 50 ml TAE buffer. Boil the mixture until it reach
60°C, then add 1 μl ethidium bromide and pour the mixture into the casting trays.
Set the comb into the gel and let the gel become hard for about 30 minutes to
create wells. After the well is hard, fill the casting tray with TAE buffer. Then, we
prepare the DNA sample by mixing 0.5 μl of DNA and 1 μl of loading dye. Mix the
mixture with 6μl DNA marker and put the mixture into the wells which was
created by the comb and the gel. Close and turn on the electrophoresis chamber
for 30 minutes, at 100 V, 6 W, 0.6 A. Then, take out the gel and wash under tap
water. Using versa doc instrument, photograph the gel and save the picture and
the record of the data.
 The fifth experiment would be Polymerase Chain Reaction (PCR). For this
experiment we need DNA template, primers, 2.5 mM dNTP mix, taq DNA
polymerase, 5X taq buffer, 25 mM MgCl2, dH2O. First, prepare the PCR tube,
filled with 43000 μl dH2O, 8 μl of 2.5mM dNTP mix, 20 μl of 5X buffer PCR, 6 μl
of 2.5mM MgCl2, 6 μl of taq DNA polymerase, 3 μl of 10 μm forward primer, and
3 μl of 10 μm reverse primer, and 4 μl of DNA template. Then, put the PCR tube
into the PCR machine. Set the PCR machine, first step 95°C for 30 seconds,
second step 95°C for 30 seconds, third step 63°C for 30 minutes, and the fourth
step 72°C for 1 minute, and the fifth step 72°C for 10 minutes, and set the cycle
from second step to the fifth step for 40 cycles, and set the storage temperature
at 4°C, and run the machine.

The sixth and the last experiment would be DNA sequencing. For this
experiment, we need a computer, complete with the chomas lite software, a
bioinformatics software tools, and file of a DNA sequence and also fasta format.
First, turn on the laptop, install the chromas lite software, and open the software.
Then, select file, pick open and pick file DNA sequence fasta format. Then,
switch the DNA sequence to forward direction if it has reverse direction. Then,
analayzed the DNA sequence if there were some nitrogen bases then change it
accordingly to its fluorescence color. Save the file, and then choose file, click
copy sequence and choose TATA format. Open www.ncbi.nlm.nih.gov, click
BLAST menu, click Nucleotide blast, paste the data, and then click BLAST.
III. RESULTS

A. Blood Separation

Figure 3.1.1 image of blood in a vacuntainer without EDTA after centrifugation

Figure 3.1.2 image of blood in a vacuntainer with EDTA after centrifugation

This pictures was taken after the centrifugation. Figure 3.1.1 and figure
3.1.2 shows us that there were some difference on what kind of layers would
show up after centrifugation with or without the anticoagulant. On the figure 3.1.1,
there were 2 layers. The bottom layer named serum is a blood that is not mixed
with an anticoagulant resulting the blood clot, and the upper layer named plasma
is a blood without an anticoagulant contains many things inside. On the figure
3.1.2, there were 3 layers. The upper layer called plasma, is a transparent layer
usually yellowish which contain water, protein, hormones, nutrients and other
stuff. At the middle named buffy coat, is a white layer contain white blood cells
and platelets. And at the bottom layer, there were red blood cells that have duty
to transport CO2 and also O2. We use the plasma on figure 3.1.2 to find the DNA
because DNA only exist in the plasma and buffy coat, but not on the red blood
cells because red blood cells doesn’t have any nucleus.

B. DNA Isolation and Electrophoresis

Figure 3.2.1 vial after resuspended with 1X TE buffer an incubation

Sample number one Sample number two

More than 10000 bp

10000 bp
8000 bp
6000 bp
5000 bp 2500 bp
4000 bp 2000 bp
3500 bp 1500 bp
3000 bp 1000 bp
750 bp
500 bp
250 bp

Figure 3.2.2 electrophoresis imaging of DNA detection

 Fig 3.2.1 shows us that at the bottom of the vial there are some white
hank. It had a shape like a hank of string. Fig 3.2.2 shows us that our DNA
contains more than 10000 base pairs.

C. Quantitation of DNA Concentration

First DNA sample

First reading Second reading Average
A230 0.080 0.079 0.0795
A260 0.170 0.168 0.169
A280 0.110 0.121 0.1155

Second DNA sample

First reading Second reading Average
A230 0.134 0.126 0.1300
A260 0.359 0.354 0.3565
A280 0.191 0.188 0.1895

The purity of DNA was calculated by using formula of dividing the

Absorbance at 260 nm (A260) with Absorbance at 280 nm (A280) or in equation
A260
is . The purity index were 1.8 – 2.0 for a pure DNA, less than 1.8 means that
A280

the DNA sample was contaminated with protein and if the index are more than
2.0 it means that the DNA sample were contaminated with RNA.
0.169
The purity index of the first DNA sample = 0.1155 = 1.3884
0.3565
The purity index of the second DNA sample = 0.1895 = 1.8812

The Concentration of the DNA was calculated using formula of multiplying

Absorbance at 260 nm (A260) with the Dilution factor (Df) and divided with
A260
extinction coefficient (ɛ). The Concentration of the DNA = Df
ɛ

The dilution factor is 1 cm and ɛ for dsDNA was 20 L⁄g cm

 0.169 µg
Concentration of the first DNA sample = x 1= 84.5 ⁄mL
20

Pure Concentration of the first DNA sample = 8.45 x 5 x 10-6x 10-6

= 42.25 x 10-12
0.3565 µg
Concentration of the second DNA sample = x 1 = 17.825 ⁄mL
20

Pure concentration of the second DNA sample = 17.825 x 10-6 x 5 x 10-6

= 89.125 x 10-12

D. PCR

E. DNA Sequencing

Figure 3.5.1 the FASTA format of DNA sequence in chromas lite

Figure 3.5.2 Spesific DNA that was replicated the most by PCR
 Figure 3.5.3 Matching the code with the BLAST database

Figure 3.5.4 Matching the code with the gene of alpha-fetoprotein

Fig 3.5.1 shows us the sequence of the gene that was amplified by PCR
method. The bases showed up according to the color of the graph. Fig 3.5.2
shows us the specific sequence that was amplified before using PCR. Fig 3.5.3
shows us that the gene was correct and the gene expression was identified
which is alpha-fetoprotein in this case. There were no mutation identified which
could cause an error of gene expression.
IV. DISCUSSIONS

In the blood separation with the centrifuge, we found out the difference
between the blood that mixed with EDTA as an anticoagulant and the one that
hadn’t mix with EDTA. For the blood that wasn’t mixed with EDTA and then
centrifuged, there were a lot of clot at the bottom layer. This phenomenon shows
us that in the blood itself there were platelets coagulating the blood creating a
clot. In this case, not only the red blood cells are coagulated, but erythrocytes
including plasma, red blood cells, white blood cells and many others are
coagulated creating a clot at the bottom layer. At the upper layer there was blood
that was yet not have been coagulated. Later, all the blood will become a clot.
For the blood that mixed with EDTA and then centrifuged, we found out some
difference in layers. There were three clear layers, indicating platelets didn’t
coagulating the blood. There were three layers, the upper layer called plasma are
filled with water, amino acids, proteins, hormones, nutrition, platelets, and other
stuffs. At the second layer, the middle layer named buffy coat, there were white
blood cells in this layer and also platelets. We could use this layer to extract the
DNA but this layer was too thin and not that much so we use plasma for DNA
extracting. The bottom layer was red blood cells that have role on transporting
oxygen and carbon dioxide through the body. We didn’t use this layer to extract
the DNA because red blood cells doesn’t have nucleus.

At the DNA isolation, we were using 2 sample of DNA. First, we are using
the NaOAc which plays role as a salt that is responsible for clotting the DNA,
then we added SDS which is responsible to lysis the membrane cells, proteins,
and lipids. Then we added proteinase K which plays role as an enzyme to
destroy specific proteins like histone. Then, we added PCI which plays role in
separating the liquid into three parts, the polar layer, interphase layer and non
polar layer. Then we extracted the polar layer and move the layer into a new vial.
Then this layer were given with ethanol and then we centrifuged the mixture.
There were some clot in the bottom of the vial. We conclude that the clot on the
bottom of the layer is DNA materials. When we isolated the DNA itself, it turned
out to be a same structure from outside. We were sure that the structure we seen
only with eyes are both the same but we wasn’t sure that the genetic materials
were both the same. The result for quantitation of DNA concentration was not
good enough for us to use the sample. On the first DNA sample, the purity index
was very low. The number was 1.3 for the first sample. This number means that
our DNA sample were contaminated with proteins.