FUNDAMENTAL MEDICAL SCIENCE

FINAL REPORT (GENOMIC)

FRENDY

00000001642

Group A1-5

Universitas Pelita Harapan

Mochtar Riady Institute for Nanotechnology

Faculty of Medicine

2013
ABSTRACT

Genetics is one of the many important aspects of our life. By understanding it,
will help genetic disease treatment, specific antibiotic production, bioengineering, and
many more beneficial things. This experiment is done to obtain DNA sample from
human blood and determine the presence of the tumor protein gene p53 in the DNA by
comparing it to the database in the National Center for Biotechnology information
(NCBI). Blood sample is first obtained and then undergoes lysis of its components by
chemicals until what remains is only the DNA. The absorbance of the DNA is measured
next using spectrophotometer, followed by its purity and concentration. The presence of
the DNA is also confirmed using the Electrophoresis method. After that, tumor protein
gene p53 of the DNA is amplified during the PCR experiment. The DNA undergoes one
more electrophoresis. Finally, we have to access the database at the NCBI website. Our
DNA sequence will be submitted and the website will search for the DNA that are in
their database for a match. It will also show us how identical it is by percentage.

Although there are some errors during the process of the experiment, we still
managed to acquire the DNA sequence and compare it to the NCBI database. Also, the
result shows that there are DNA sequence in the database that are similar with the
samples we obtained from Frendy and Ivy.
I. INTRODUCTION

DNA or deoxyribonucleic acid, is the hereditary unit in humans and almost all
other organisms, it is what makes an organism so unique. In this experiment, we are
going to find out whether a person possess tumor protein p53 gene or not. (GHR, 2013)

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)

Plasma consist of water, sugar, fat, protein, and potassium and

calcium salts. It contains the chemicals necessary to for blood
clotting. More than 92% of plasma is water.

Red blood cells contain a special protein called hemoglobin, it

carries oxygen to all of the parts of our bodies. It then returns
carbon dioxide from our body to our lungs so we can exhale it.
Hemoglobin is responsible for the red color in blood.

White blood cells are clear round cells that are bigger than red
blood cells. White blood cells produce antibodies that help our
bodies fight infections.

Platelets are fragments of cells, platelets gather at the site of

the injury and stick to the edges of the wound. They are
responsible for blood clotting

Figure 1: Blood components (Utah, 2013)

 PCR (Polymerase Chain Reaction) is a method developed by Kary Mullis in the
1980s. PCR utilizes the ability of DNA polymerase to synthesize new strand of DNA.
DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, it needs
a primer to which it can add the first nucleotide. So, it is possible to amplify a specific
sequence of the DNA. At the end of the PCR reaction, the specific sequence will be
accumulated in billions of copies (NCBI, 2013)

There are several ways to quantitate nucleic acids. A spectrophotometer can be

used to measure the amount of ultraviolet radiation absorbed by the bases. DNA can
also be quantified by measuring the UV-induced emission of fluorescence from
intercalated ethidium bromide. (Carlos F. Barbas III, Dennis R. Burton, Jamie K. Scott
and Gregg J. Silverman, 2001)

Electrophoresis is a technique used to separate large molecules

like proteins and nucleic acids based on their size. DNA electrophoresis is done in
agarose gel, which is derived from seaweed. This is immersed in a solution of
a buffer which is responsible for conducting electricity and ensuring constant PH. (UQ,
2013)

The National Center for Biotechnology Information (NCBI) provides users with
DNA sequence database. BLAST stands for Basic Local Alignment Search Tool. We
can submit the algorithm of a sequence (DNA, RNA, Amino Acid Chain). It will then be
compared to the database. In this experiment, we use this to determine if a person have
the tumor protein p53 gene or not. (Davidson, 2008)
II. MATERIALS AND METHODS

All materials and methods that are going to be described in this lab report are
adapted from the laboratory protocol. Some modifications were made.

The experiment is started by drawing blood (6 mL each) from two appointed

students (Frendy and Ivy).Each obtained blood is transferred (3mL) into an empty
vacutainer and EDTA anticoagulant coated vacutainer, one mL of blood from each
vacutainer is transferred into labelled separate vials. The vacutainers will then undergo
centrifugation at 3300g (3700 rpm if using Alegra X15 centrifuge), 20°C, for 10 minutes.
After centrifugation, Serum will be separated into a new vial and all vials will be stored
at -80°C.

0.5 mL of the whole blood sample from the EDTA vacutainer is used for the next
step in the experiment, DNA isolation. 0.8 mL of 1X SCC buffer is added and mixed,
followed by centrifugation for 1 minute at 12,000 rpm (all centrifugation uses the same
rpm in DNA isolation step) in a microcentrifuge. This will result in the formation of pellets
on the bottom of the vial and supernatant (Clear Liquid) above it.1 mL of supernatant is
disposed. This step is repeated one more time with 1 ml of 1X SSC buffer. This time, all
of the supernatant is disposed. Then, add 375 µL of 0.2M NaOAc, vortex, then add 25
µL of SDS and 5 µL of Proteinase K, vortex and incubate for 1 hour at 55°C.After that,
120 µL of phenol/chloroform/isoamyl alcohol is added, vortexed for 30 seconds and
centrifuged at 2 minutes. The aqueous layer is removed and transferred into another
microcentrifuge tube, 1mL of cold 100% ethanol is added, mixed, and incubated for 15
minutes at -20°C and then centrifuged for 2 minutes. Supernatant is decanted and
drained.180 µL of 1X TE buffer is added, vortexed and incubated at 55°C for 10
minutes. After that, 20 µL 2 M Sodium acetate and 500 µL cold ethanol are added and
mixed, centrifuged for 1 minute. Decant supernatant, rinse pellet with 1 mL of 70%
ethanol. Centrifuge for 1 minute, decant supernatant, and air dry pellets. Pellets will
finally be re-suspended by addition of 200 µL 1X TE buffer, incubated at 55°C for 30
minutes, vortexing periodically to dissolve genomic DNA, and stored at -20°C.

Next, spectrophotometer BIORAD will be used to determine the DNA

concentration of the samples obtained. Samples are first diluted in 1:5 ratio. Cuvette is
filled with 50µL 1X TE buffer and is placed on cuvette holder of the spectrophotometer.
Type of nucleic acid and conversion factor is selected, and the spectrophotometer is
blanked beforehand using 1X TE buffer. After it’s done, DNA sample is inserted and the
absorbance (230, 260 and 280) and concentration are read and recorded. It is repeated
again for the other samples.

Electrophoresis is needed to actually detect the presence of DNA. Agarose gel

well was prepared beforehand (by MRIN lab assistant) by mixing 0.5 gr agarose in
50mL TAE buffer, boiling it, adding 1 µL ethidium bromide, pouring it into the tray,
setting the comb, hardening, and finally loading it.5 µL of DNA is mixed with 1 µL of
loading dye by pipetting. This process is done another time for the other sample. The
mixtures are then loaded into the agarose gel well, 6 µL DNA marker is also loaded
afterwards. The gel well is filled with TAE buffer until 1 mm above gel surface, lid is
closed, and power supply is connected. Electrophoresis is run at 100V, 6 Watts, 0.6 A
for approximately 30 minutes. After it’s completed, take out the gel, wash it, and save
the picture and record the data utilizing Versa Doc instrument.

The next thing to do is to set up the master mix consisting of 12.875 µL dH2O
which is then mixed with: 5 µL 5X buffer PCR, 2 µL 2.5 mM dNTP mix, 1.5 µL 25 mM
MgCl2, 0.125 µL Taq DNA polymerase, 0.75 µL 10 µM forward primer, 0.75 µL 10 µM
reverse primer and 2 µL DNA template, with the final concentration respectively: 1X,
200 µM, 1.5 mM, 0.625 units, 0.3 µM, 0.3 µM, and <250 ng. The volume per reaction
applied is multiplied by four because we need it for four PCR tubes. So, it will ultimately
yield a total of 100 µL. After the mix had been distributed evenly to 4 PCR tubes, the
tubes will put into PCR machine. The machine is set for 95°C (10 minutes) then,
denaturation at 94°C (30 seconds), annealing at 58.6°C (30 seconds), extension at
72°C (1 minutes), final extension at 72°C (10 minutes), and finally storage at 4°C
(indefinite time).

The last experiment is obtaining the DNA sequence of the samples that
have been processed carefully during the previous steps. In order to do that, first we
must turn on the computer and proceed by installing Chromas Lite software. Once
done, open the DNA file that will be used and switch it to forward instead of reverse.
Next, analyze the sequence, N bases should be changed according to the correct color
shown (C=Blue, Black=G, Green=A, Red=T), and save it afterwards. Then copy
sequence in fasta format, go to http://ncbi.nlm.nih.gov/, click “blast menu”, click
nucleotide blast menu, paste it into “enter query sequence area”, choose “human
genomic” database, and end the experiment by clicking “BLAST”.
III. RESULTS

A. Blood Separation

Figure 2: centrifuged blood inside a Figure 3: centrifuged blood inside a

vacutainervacutainer with anti-coagulant vacutainervacutainer without anti-
coagulant
B. DNA Isolation and DNA Electrophoresis

Genomic DNA of
DNA Marker Sample 1 (Ivy)

10000 bp

8000 bp

6000 bp

5000 bp

4000
4000bpbp

3500
3500bpbp

3000 bp

1500 bp

1000 bp

500 bp
Figure 4: Agarose gel electrophoresis of DNA isolated from blood sample
C. Quantitation of DNA Concentration

Frendy’s sample 1 Frendy’s sample 2 Ivy’s sample 1

A230 0.150 0.246 0.330
A260 1.829 1.941 1.982
A280 0.837 1.045 1.074
Table 1: Absorbance from DNA samples isolated from Frendy and Ivy

To calculate DNA concentration of a DNA sample, Optical density (A260) is

divided by extinction coefficient, the result obtained is then multiplied with the dilution
factor. Equation = [(OD : Ɛ) x Dilution factor]

DNA concentration from Frendy and Ivy’s samples with 1:5 dilution =

mg
Frendy’s sample 1 = 1.829 x 5⁄20 = 0.45725 ⁄mL

mg
Frendy’s sample 2 = 1.941 x 5⁄20 = 0.48525 ⁄mL

mg
Ivy’s sample 1 = 1.982 x 5⁄20 = 0.4955 ⁄mL

To calculate the purity index of a DNA sample, divide the absorption at 260 nm
with the absorption at 280 nm. Equation = [A260⁄A280]. DNAs are considered to be
pure when their purity indexes are in the range of 1.8 – 2.0.

DNA Purity Index from Frendy and Ivy’s samples with 1:5 dilution =

Frendy’s sample 1 = 1.829⁄0.887 = 2.062

Frendy’s sample 2 = 1.941⁄1.045 = 1.857

Ivy’s sample 1 = 1.932⁄1.074 = 1.845

 Another method can also be used to measure the purity of DNA, by dividing the
absorption at 260 nm with the absoption at 230 nm. Equation = [𝐴260⁄𝐴230]. DNAs are
considered to be pure when their purity indexes are in the range of 2.0 – 2.2.

Frendy’s sample 1 = 1.829⁄0.150 = 12.193

Frendy’s sample 2 = 1.941⁄0.246 = 7.890

Ivy’s sample 1 = 1.932⁄0.330 = 5.854

D. PCR

Ivy’s sample Frendy’s Sample

DNA 1000 bp
Marker
900 bp
Control
800 bp
Positive
700 bp
Sample 1
600 bp
Sample 2 500 bp

400 bp
Control
Negative 300 bp

200 bp
± 600 bp
100 bp

Figure 5: Agarose gel electrophoresis for PCR experiment

E. DNA Sequencing

Figure 6: Forward DNA sequence in Chromas Lite

Figure 7: reverse DNA sequence in Chromas Lite

Figure 8: Forward distribution of 16 BLAST hits on the query sequence

Figure 9: Reverse distribution of 16 BLAST hits on the query sequence

Figure 10: Forward sequences producing significant alignments

Figure 11: Reverse sequences producing significant alignments.

IV. DISCUSSIONS

A centrifuge is a machine that is utilized for separating heavy materials from light
materials. Centrifuge works by spinning the sample at high speed and uses the principle
of the “centrifugal force”. This force pushes against the bottom of the container causing
particles to clump at the bottom of the container. The solid clump is referred as a pellet,
and the solution above it is referred as the “supernatant”. (Tufts, 2011). The centrifuge
is used to separate blood in this experiment. After centrifugation, blood in a tube
containing anti-coagulant would be separated into 3 layers made up of upper plasma
layer, lower red blood cell layer, and a thin layer called the buffy coat which contains
white blood cells and platelets. But without the anti-coagulant, the result will differ. Blood
will be allowed to coagulate, therefore the final visible layers will be blood clot at the
bottom and clear serum above it. The results described in the reference are similar than
the results that we obtained from the experiment. We conclude that our experiment is
successful. (Rodney A. Rhodes PhD, David R. Bell PhD, 2011)

DNA needs to be isolated from the blood samples obtained from the previous
experiment in order for it to be viewed shortly after using the gel electrophoresis
method. Blood contains many components such as red blood cells, white blood cells,
etc.To only obtain DNA, these components must be removed completely. NaOAc is
involved in the lysis of red blood cells. SDS detergent works by lysing cell membrane
and forming complexes with lipid and proteins resulting them to be able to precipitate
out of the solution. Proteinase K will be used to degrade and denature the proteins
precipitated, followed by PCI alcohol solution to remove non-nucleic acid molecules.
Ethanol is used to precipitate DNA and once the pellet had dried, it will be re-suspended
using Tris EDTA buffer. DNA pellets will be visible. The final results described in the
reference is similar to what we obtained from the experiment. We conclude that our
experiment is successful. (Bruce A. Roe, 2001)

Unlike Ivy’s DNA, in the Electrophoresis procedure, specifically in the VersaDoc

imaging, Frendy’s DNA were not visible in the gel. This could be caused either by failure
of obtaining DNA during the DNA isolation experiment, or the DNA is not mixed well
enough with the agarose gel and ethidium bromide. However, the results of the
absorbance from the spectrophotometer indicate that Frendy’s DNA were present, it is
highly unlikely that we failed to isolate the DNA. The marker used in this experiment
range from 500 bp to 10000 bp. So, it can be interpreted that our DNA sample is larger
than 10000 bp. The thickness of the band depends on the DNA concentration in it
(Stefan Surzycki, 2003)

The DNA sample from Frendy 2 (1.857) and Ivy 1 (1.845) are both pure, since
their purity indexes are within the range of 1.8 – 2.0.The DNA sample from Frendy 1
(2.062) is however, not pure, because its purity index is larger than 2.0.This is most
likely cause by contamination. We conclude this experiment to be successful.
(NanoDrop, 2007)

AN electrophoresis is done one more time for the PCR experiment. The first lane
contains DNA marker. The second lane contains the control positive (DNA that is known
to work), which will be visible. The third and fourth lane contains the DNA samples
which shows the formation of bands at approximately 600 bp according to the DNA
marker. The fifth lane contains the negative control (DNA that is known not to work),
which would not be visible. Frendy’s DNA is again, not visible in this electrophoresis,
which could be a result from error when creating the master mix. (UNLV, 2007)

BLAST results confirms that our DNA samples were amplified correctly to the
gene homo sapiens tumor protein p53. The data that are used are obtained from the
database of http://blast.ncbi.nlm.nih.gov/Blast.cgi. Comparison shows that our amplified
DNA sample sequence match the sample in the website’s database. It was 99%
identical with the database. (NCBI, 2013)

From this series of experiments, we found out that there are some errors made
from the experiment, nevertheless we still managed to amplify the DNA and found a
match in the DNA sequence of the database.
V. REFERENCES

Books:

Anonymous. Laboratory Protocol for Fundamental Medical Science 1. Faculty of

Medicine Universitas Pelita Harapan. Tangerang; 2013.

Bruce A. Roe. Department of Chemistry and Biochemistry. The University of Oklahoma,

Norman, Oklahoma; 2001.

Rodney A. Rhodes PhD, David R. Bell PhD. Medical Physiology: Principles for Clinical
Medicine. 4th ed. North America: Lippincott Williams & Wilkins; 2012.

Stefan Surzycki. Human Molecular Biology Laboratory Manual. USA: Wiley-Blackwell; 1

edition; 2003.

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http://learn.genetics.utah.edu/content/begin/traits/blood/blood.html