FUNDAMENTAL MEDICAL SCIENCE 1

FINAL REPORT (GENOMIC)

JENNIFER ANGGAWAN

00000012984

Group C4-B

UNIVERSITAS PELITA HARAPAN

Mochtar Riady Institute Of Nanotechnology

Faculty Of Medicine

2015
 ABSTRACT

Human Genomic Project is one of the most popular trend in medical world
nowadays. It is a project that is based on human genomic, which is a set of code of
genetic material that determines the human itself, and the chances of predictable
characteristics of the next generation. It is already widely used to determine health
risks, helps in diagnosing, treating certain illness, and also predicting the prognosis
of a patient. That’s why this project is considered as useful and important in
medicine.

Our goal is to obtain gene p53 which is used as tumor supressor protein
transcript. In this experiment we used Jeremy and Jennifer’s blood as samples,
which was centrifugated to separate the content into layers. Then we isolated the
DNA, checked the DNA purity level and quantity of both samples with
spectrophotometer analysis, and also determined the size of the DNA by
electrophorensis. To make the sample sufficient, we multipled them using PCR
(Polymerase Chain Reaction). After that we did DNA sequencing by Sanger’s
method, and used Chromas Lite Software to edit the sequence. Finally, we
compared our results to the database in NCBI website using BLAST tool.

Our result was a bit of a blur because our subgroup made a mistake in
extracting the DNA sample, so that protein contaminant is present. But the other
subgroup did get the gene P53 which is 99% similar to the ones in NCBI’s database.
So overall, we could say that this experiment is quite sucessfull.
 I. INTRODUCTION

Blood is a vital component inside a human’s body, mainly produced by bone

marrow. There are four main components inside our blood, which is the red blood
cells, white blood cells, platelets, and blood plasma. The red blood cells is used to
carry oxyden and carbondioxide by binding it into haemoglobin and it’s critically
essential for our body’s respiration. They do not contain nucleus to broaden the area
of oxygen binding, and they made up 40%-50% of our blood. White blood cells
(leukocytes) made up only about 1% of our blood. They have nucleus, and could
also present in other organs. It’s essential for our defense mechanism against
pathogens. It is present in buffy coat layer when anticoagulated blood is centrifuged.
Platelets are small and irregular in shape, and used in blood clotting mechanism.
Lastly, plasma consists of 90% water, and 10% of metabolism products. If a blood
plasma doesn’t anticoagulant factor inside it, then it is called blood serum. To
prevent the blood from coagulating, usually anticoagulant factors are added inside
the tubes of which the blood will be stored.

DNA is a set of nucleic acid (Purines (Adenine and Guanine) and

Pyrimidines (Thymine and Cytosine)) joined together forming a double helix and
containing genetic informations that codes all of our body mechanisms and traits.
From the blood, DNA samples are present inside the nucleus or mitochondria, which
is contained inside the white blood cells. P53 gene is a gene that codes for tumor
suppresion. This is the specific region of genome that we want to obtain and
duplicate on PCR process. It is specifically regulated by Q17p13.1 in our body. DNA
is isolated by eliminating the nuclear membrane and non-DNA factors such as
proteins and RNAs, and it’s done by the addition of organic solvent.

PCR (polymerase chain reaction) is a process proposed and developed by

Kerry Miller in 1980. The main goal of this method is to produce more yet specific
DNA strand region by adding TAQ DNA polymerase (which is heat-stable) and a set
of primers. The strand produced is counted exponentially and could effectively be
repeated up to 40 cycles.

After duplication, we have to detect the presence of DNA by

electrophorensis. It is used to separate large strands to the small ones by using
agarose gel, and electricity which flows inside the electrophorensis chamber, which
was previously filled with buffer solution. DNA quantitation, on this experiment, is
done by spectrophotometry. It uses UV rays to record the absorbance of DNA, and
to count any contaminants (if they present).

The use of Chromas Lite and NCBI Database are used to compute the DNA
and check whether the gene has any anbormalities in its sequence. Beside that, we
could use the result to check the responses to a certain drug.
 II. MATERIALS AND METHODS

We do our experiment based on MRIN’s Laboratory Manual with some

adjustments to the procedures.

Blood samples (8 - 9 ml) were taken from both students and containes inside
two vacutainers, one of them has anticoagulants on its walls. Then the vacutainers
are centrifugated using Alegra x15 (330gr/3700rpm, 20⁰ Celcius) for 10 minutes. This
is called Blood Separation process. The blood plasma from the anticoagulated
vacutainer is then transferred into a new vial to be stored in -80⁰.

Next, we isolate the DNA contained inside the plasma by mixing each of it
with 0,8ml of 1x SCC buffer, then inserting it into micro centrifuge (1200rpm) for 1
minute, removing 1 ml of supernatant, then adding 1 ml of 1X SCC buffer, mixing it
inside the vortex for approximately 30 seconds, centrifuging the vial (1200rpm) for 1
minute, remove all of the supernatant. Add 375µl of 0,2MnaOAc then mix it briefly
with vortex. Add 25µl of 10% SDS and 5 µl proteinase K (10mg/ml H2O). Vortex the
vial briefly and incubate it for 30 minutes at 55⁰ Celcius. After that, add 120µl
phenol/chloroform/isoamyl alcohol, then microcentrifuge the vial (1200rpm) for 2
minutes. Carefully remove the aqueous layer to a new 1,5ml rube. Add 1ml of cold
100% ethanol, and mix it. Microcentrifuge (12000rpm) the vial for 2 minutes. Decant
the supernatant and drain the vial, then add 160 µl of 1X TE buffer, vortex it for 30
seconds. Microcentrifuge (1200rpm) the vial for 1 minute. Decant the supernatant
and dry the pellet for 10 minutes.

After that, we quantitize the DNA concetration to check its purity. First we
have to dilute the sample in ultra pure water for 1:5 (or 1:50 if the sample is not
diluted enough) then put the mixture inside a cuvette, add 50µl of 1X TE buffer, then
put it inside the spectrophotometer. Choose dsDNA type, and set the conversion
factor (no need to modify). Start the reading and record the result. Tabulate the data
and count the purity of DNA as well as the contaminant (protein and/or RNA)
quantities.

Next is electrophorensis. Prepare 1% agarose and fill the electrophorensis

chamber with TAE buffer, then mix 1µl of our sample with 1µl of loading dye on a
parafilm sheet, then put it inside the agarose’s 96 well plate. We also put 1 µl of DNA
marker as out control. The lid is then closed, and the electrophorensis ran at 100V, 6
Watts, 0,6A, for 25 minutes. Then the gel is washed, and put inside Versa Doc
Instrument where the result is read and recorded.

We continue with PCR, by mixing 12,875 µl dH2O, 5 µl 5X PCR buffer, 2 µl

mM dNTP mix, 25mM MgCl2, 0,125 µl TAQ DNA polymerase, forward and reverse
primer (10 µm each), and 2 µl DNA template from the samples. Then we put the tube
inside the PCR machine, with the programs set as such:

1. Step 1 : 95⁰ Celcius for 10 minutes

 2. A. Denaturation (94⁰ Celcius, 30 seconds)
B. Elongation (58,6⁰ Celcius, 30 seconds)
C. Annealing (72⁰ Celcius, 1 minute)
Do step 2’s A B C for 40 cycles in total
3. A. Final extension (72⁰ Celcius, 40 minutes)
B. Storage (4⁰ Celcius)

Then we keep the result in 4⁰ Celcius.

The last process is sequencing. Use Chromas Lite to read the DNA file in
FASTA format, make sure that the direction is forward, and analyse the sequence by
removing overlapping genes. Save and copy your work, then open
http://www.ncbi.nlm.gov/ and click the BLAST menu, click nucleotide blast menu and
paste your work into enter the query sequence area. Make sure that you chose
human genomic database to compare your data with, finally click BLAST and read
the result.
 III. RESULTS

A. Blood Separation
Figure 1. a) Vacutainer with Anticoagulant Figure 1. b) Vacutainer with no Anticoagulant

B. DNA Isolation and Quantitation Electrophoresis Result

A B C DE F GH I J

Figure 2.a) DNA Electrophorensis result (Agarose 1%) Figure 2. b) Details of 100-bp DNA Ladder

Lane Description:

A = DNA Marker F = DNA Marker

B = Jennifer’s DNA – C4B G - EMPTY

C = Jeremy’s DNA – C4B H - EMPTY

D = Jennifer’s DNA – C4A I - EMPTY

E = Jeremy’s DNA – C4A J - EMPTY

 C. DNA Purity Result

i. Jennifer

A 230 A 260 A 280

260 -0,198 -0,029 -0,033
280 -0,197 -0,033 -0,034
DNA purity -0,1975 -0,0031 -0,0335

A = - 0,031/20 x 5 = -0,00775

Contamination Index : A260/A230 = -0,031/-0,1975 = 0,1570

Purity Index : A260/A280 = -0,031/-0,0335 = 0,9254

i. Jeremy

A 230 A 260 A 280

260 - 0,208 -0,077 - 0,061
280 - 0,210 - 0,077 - 0,064
DNA purity - 0,209 - 0,077 - 0,0625

A = -0,077/20x50= -0,00385

Contamination Index : A260/A230 = -0,077/-0,209 = 0,3684

Purity Index : A260/A280 = -0,077/-0,0625 = 1,232

D. PCR Sequencing (Sanger’s Method) Electrophorensis Result

A BCDEFGH I J

Figure 3. a) DNA Electrophorensis Result (Agarose 1%) Figure 3. b) Details of 100-bp DNA Ladder
Lane Description:

A = Jeremy’s DNA – C4A F = Jeremy’s DNA – C4A

B = Jennifer’s DNA – C4A G = Jennifer’s DNA – C4A

C = Positive Control H = Positive Control

D = Negative Control I = Negative Control

E = DNA Marker J = DNA Marker

E. Chromas Lite Printscreen Result

Figure 5. a) BLAST Query Sequence – Color Key for Alignment Scores

Figure 5. b) Sample’s DNA sequence

F. NCBI Database Genome Similarity Printscreen - BLAST Result
 IV. DISCUSSION

The vacutainer with Anticoagulant shows three layers after centrifugated.

The first layer is plasma, and it’s yellowish yet translucent in color. The second and
thinnest layer is buffy coat, which is bright white. And finally at the bottom there’s red
blood cell layer, which is, of course, red in colour. The layers form because the blood
is not coagulating, unlike the ones inside the vacutainer without anticoagulant, where
the blood clot is visible (dark red) and irreversible. The colors described are the
same as the ones mentioned in the reference. (Blood Components, 2006)

DNA isolation is then done by taking the plasma samples, then the result is
supposed to be a clear liquid with some translucent strands visible inside it. But my
subgroup did something wrong on the sample-pipetting so that the buffy coat was
also taken accidentally. This causes our DNA samples to have too many
contaminants inside it, so the strands are not visible. The absence of our result is
then confirmed with the absence of bands in the agarose from electrophoresis
process. As expected, based on the previous results, our subgroup’s bands of DNA
electrophoresis result are invisible this may be caused due to the number of
contaminants which are too many, or worse, there may be no DNA at all in our
sample because when we calculated the purity, the result was all negative. Unlike
the other subgroup’s sample, the DNAs are clearly present, even though it’s not in a
bright gradient like the positive control. The total base pairs recorded for the visible
bands of the sample is higher than the first band present on the DNA Marker (which
indicates for 10 thousand base pairs), it means that we have got more than 10
thousand base pairs recorded. It’s a very good result though it’s a little bit more that
we expected. (Anonymous, 1988, Genes and Disease Inheritence)

The problem is, this result (DNA) is to be used for the whole experiment, so
if we fail this step, then it might lead to negative results on the other processes. And
that explains why we get an awful result in DNA Purity count, which was supposed to
range in about 1,8 to 2,0. More than 2,0 in A 260/230 count means there’s RNA or
other organic contaminant like RNA, which size is about 230nm, while less than 1,8
in A 260/280 means protein contaminant is present, which is around 280nm in size.
(Nanodrop, 1997)

Even after PCR is done, the electrophoresis still didn’t detect any DNA in our
subgroup’s sample – even the control is both absent. This worsening result may be
caused by the mistakes from the previous experiments, plus we might did something
wrong on the pipetting, which caused the agarose well to break and finally didn’t
show anything as the result. Other causes of this failure might be the lack of ethidium
bromide (which is essential to give colour to the sample), or inappropriate mixing.
Meanwhile, the other subgroup got around 600 to 700 basepairs on their sample.
That’s exactly what we want to obtain. Although, Jennifer’s band is bolder than
Jeremy’s, which is gradient-like, and that means Jennifer’s sample is more exact,
because only the DNA with 600-700 basepairs are present. (Nanodrop, 1997)
 Using the sequence of the other subgroup’s DNA, we record it using
Chromas Lite and the sequence was only present around 200 or 300 base pairs
(with a few SNPs), which is only a half from the target of 608 base pairs that we
wanted to obtain from PCR. Luckily, it’s enough to be detected by NCBI Program,
and it shows 99% similarity to the gene of p53 which is exactly the sequence that we
want to obtain. (NCBI, 2015)
 V. REFERENCES

Anonymous, FMS1 2006, Laboratory Manual and workbook. School of

Medical Science, University of Pelita Harapan, Tangerang

O’Neil, D.2006 Blood Components,

http;//anthro.palomar.edu/blood/blood_components.htm

Anonymous, 1988, Genes and Disease [Internet], National Center for

Biotechnology Information, United States of America
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Anonymous, 2015, An Introduction to Tumor Supressors, Winship Cancer

Institute, Emory University, Georgia, United States of America
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Biotechnology Information, United States of America
http://www.ncbi.nlm.nih.gov/probe/docs/techpcr/

Anonymous, 2015, Colorful Electrophoresis, University of Utah, United

States of America, http://learn.genetics.utah.edu/content/labs/gel/electrophoresis/

Narcissa, Vivien., 2014, What Is A Gene?, Nemours, Philadelphia, United

States of America, http://kidshealth.org/kid/talk/qa/what_is_gene.html

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Biotechnology Information, United States of America, http://www.genecards.org/cgi-
bin/carddisp.pl?gene=TP53

Nanodrop : William W. Wilfinger, Karol Mackey, and Piotr Chomczynski, 1997,

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http://www.nanodrop.com/Library/T042-NanoDrop-Spectrophotometers-Nucleic-
Acid-Purity-Ratios.pdf

National Center for Biotechnology Information. Basic Local Alignment Search Tool.
BLAST website 2009 [cited 2015 Nov 17], http://blast.ncbi.nlm.nih.gov/Blast.cgi