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FUNDAMENTAL MEDICAL SCIENCE 1

FINAL REPORT (GENOMIC)

FELICIA WIJAYA
00000001902
Group A2-4

Universitas Pelita Harapan


Mochtar Riady Institute for Nanotechnology
Faculty of Medicine
2013
ABSTRACT

In this era, genomics technologies are improving to establish knowledge in


human genome. Studying genomics can help us to know the roles from each gene in
human sequence and also to learn about how conditions of some disease caused by
the erorr or mutation in the celular level but also used to find the treatments of some
genetic condition. From all this experiments, the gene being studied is the p-53 gene.

This experiment was conducted to extract a blood sample and to find out if it
contains any mutation or errors in the sequence. We started from blood separation
experiments to know the component of the blood, isolation DNA from blood sample that
we took previously, determination of its concentration using spectrophotometer and the
size of the DNA using electrophoresis with agarose gel, the DNA was amplified using
PCR technique, DNA sequencing using Sanger Method, sequence DNA was edited
using Chromas Lite, compared to the database in NCBI using Basic Local Alignment
Search Tool (BLAST).

In the end of the experiment, we achieved the goal. Even though there are some
experiments that has some errors in the process, but it's still acceptable. There wasa
100% identities of p-53 gene compared to the database in NCBI site which located in
our sample's chromosome.
INTRODUCTION

Blood contains about 45% formed elements (blood cells) and 55% blood plasma
(liquid containing various dissolved subtances). Normally, more than 99% of the formed
elements are red blood cells and the remaining 1% occupied by pale, colorless white
blood cells and platelets. They form a very thin buffy coat layer between the packed
RBCs and plasma. If a sample of blood is centrifuged, the cells (which are more dense)
sink to the bottom of the tube while the plasma (which is less dense) forms a layer on
top (Tortora, 2011). The trombocytes along with coagulating factor in plasma will clot
the erythrocytes, results in only two layer formed inside the vial without anti-coagulant
factor. Clotted erythrocytes located at the bottom and serum (plasma without
coagulation factor) on the top (Schottenfeld & Fraumeni, 2006).

DNA forms the inherited genetic material inside each human cell which is located
inside a cell nucleus. In humans, each gene is a segment of a DNA molecule. When a
cell divides, its hereditary information passes on to the next generation of cells (Tortora,
2011). The nucleated blood component are leukocytes, so when the DNA was isolated
from blood, we actually isolating DNA from leukocytes. Cell lysing to release the
genomic DNA, removal of unrelated components, and recovery of purified DNA are the
steps that can complete using various enzymes and buffers (Zdanowicz, 2010).

Quantitation of DNA concentration was performed to measure the concentration


and the purity of the DNA, which can be done after the DNA isolation. Most optimal
result of DNA experiment obtained when DNA is pure and at a certain level. This
process need spectrophotometer to measure the absorption of the DNA at 260nm and
280nm (Fankhauser, 2007). Spectophotometric quantitation is the simplest method to
estimate DNA concentration. An absorbance ratio of 260nm and 280nm gives an
estimate for the DNA purity. Nucleic acids can absorb UV light maximally at 260nm. The
purity must be between 1.7 and 2.0. (Bazzi, 2009).

After isolated DNA, DNA have very long size and small concentration.
Polymerase Chain Reaction (PCR) is ), a technique to amplify specific nucleotide
sequence. The process requires a template DNA, oligonucleotide primers, and
thermostable DNA Polymerase. There are 3 important steps : template denaturation,
primer anneling, primer elongation by DNA polymerase (Rabinow, 1997).

Next are DNA sequencing, is the process of determining the nucleotide bases
(guanine, cytosine, adenine, and thymine) order. The patterns that make up genetic
traits can be determined by generating a DNA sequence for a particular organism (DNA
sequencing, 2011). The determined nucleotides sequence were saved in the database
called BLAST (Basic Local Alignment Sequence Tools ) in the bank genome on the
internet . BLAST save almost all the type of nucleotide and protein order, so we can
compare our nucleotide or protein order with the database in BLAST. The program
compares nucleotide or protein sequences to sequences database and calculates the
statistical significance of matches (http://blast.ncbi.nlm.nih.gov/).
MATERIALS AND METHODS

This steps are according to the Laboratory Protocol for Fundamental Medical
Science 1, Faculty of Medicine, Universitas Pelita Harapan, but with some modification
that being adjust while we're doing the experiments.

Blood samples were drawn from two appointed students (Vicko and Maranatha)
and transferred into separated vials. one vacutainer was empty and the other one
already had been added with EDTA (anti-cougulant) and labeled properly. The
vacutainers were centrifuged at 3000g using Alegra X15 centrifuge, 20°C, for 10
minutes. The upper transparent layer called serum was transferred into a new vial then
stored at -80°C.

0,5 ml of whole blood stored in EDTA was taken as a sample. 0,8 ml 1X SSC
buffer was added then mixed. Mixed sample was centrifuged for 1 minute at 12000 rpm
in a microcentrifuge. 1 ml of supernatant was removed and discarded into disinfectant. 1
ml of 1X SSC buffer was added and vortexed for a gentle mixing to vigorous
resuspension chemical pellets. The sample was centrifuged for 1 minute at 12000 rpm.
All of supernatant was removed. 375 ul of 0,2M NaOAc was added to each pellet and
vortexed briefly. 25 ul of 10% SDS and 5 ul of proteinase K were added. The sample
was vortexed and incubated for 1 hour at 55°C. 120 ul phenol / choloform / isoamyl
alcohol was added and vortexed for 30 second. The sample was centrifuged for 2
minutes at 12000 rpm in a microcentrifuge tube. 1 ml of cold 100% ethanol was added,
mixed, incubated for 15 minutes at -20°C. The sample was centrifuged for 2 minutes at
12000 rpm. The supernatant was decanted and drained. 180 ul 1X TE buffer was added,
vortexed, and incubated at 55°C for 10 minutes. 20 ul 2M sodium acetate was added
and mixed. 500 ul of cold 100% ethanol was added and mixed. The sample was
centrifuged for 1 minute at 12000 rpm. The supernatant was decanted and the pellet
was rinsed with 1 ml of 70% ethanol. The sample was centrifuged for 1 minute at 12000
rpm. The supernatant was decanted and the pellet was air dried for 10 minutes or until
dry. The pellet was resuspended by adding 200 ul of 1X TE buffer, incubated at 55°C
for 30 minutes, and vortexed periodically to dissolve the genomic DNA. The samples
were stored at -20°C.
The DNA sample was diluted (1:5). The cuvette was filled with 50 ul 1X TE buffer
and set on the holder of spectrophotometer. Nucleic acid type was selected (dsDNA).
The conversion was determined. The spectrophometer was blanked with 1X TE buffer.
DNA sample was inserted into cuvette to read the absorbance (50 ul final volume). The
concentration was recorded.

The agarose 1% gel was prepared in the gel tray which has been mixed with50
ml of TAE buffer in an erlenmeyer flask, boiled in the microwave, let the temperature
reached 60°C, and 1 ul ethidium bromide was added. The agarose was poured off into
the tray. The comb was set and let 30 minutes to allow gel become harden. The
electrophoresis chamber was prepared and the agarose gel was loaded. 5 ul of DNA
was taken and loaded into 0,5 ml tube. 1 ul of loading dye was added and mixed by
pipetting. The mixture sample was loaded into the agarose gel well. 6 ul DNA marker
was loaded. TAE buffer was filled until it reached about 1 mm above gel surface. The
electrode was connected to the power supply. The electrophoresis was ran at 100V, 6
Watts, 0,6 A for more or less 30 minutes. The gel was taken out and washed under tap
water. The picture was saved using Versa Doc instrument and recorded the data.

A reaction mixture was set up that contains : 43,5 ul dH2O, 20 ul 5X PCR buffer,
8 ul 2,5 mM dNTP mix, 6 ul 25 mM MgCl2, 0,5 ul Tag DNA polymerase, 3 ul 10 uM
forward primer, 3 ul 10 uM reverse primer. This mixture was divided into 4 separate
vials (21 ul for each tube) and 4 ul of DNA template was added so each tube has 25 ul.
The tubes were put in the PCR machine, the program was set. Step 1 was 95°C for 10
minutes. Step 2 was divided into 3 different processes : denaturation process took 30
second (94°C), annealing process took 30 second (58,6°C), and the extension process
took 1 minute (72°C). Step 2 was set for 40 cycles. Step 3 was divided into 2 step : final
extension took 10 minutes with temperature of 72°C, storage process at 4°C for
indefinite time. After the machine was ran, the PCR product was kept at 4°C until further
study.

Our computer was turned on and installed Bioinformatic Software tools (Chromas
Life), Chromas Lite was operated. The file DNA sequence FASTA format was opened. It
needed to be switch from reverse direction to forward direction. DNA sequence was
analyzed. The N base was edited with base according to appropriate color. Then the file
was saved. The sequence was copied in FASTA format. NCBI site
(http://www.ncbi.nlm.nih.gov/) was opened. Blast menu was clicked and Nucleotide
blast menu was clicked. The edited DNA sequence was pasted into Enter Query
Sequence Area. Search set was chose, Human genomic database was clicked, and
BLAST was clicked. The results were documented.
RESULT

A. Blood Seperation

Figure 1. Image of blood in vacutainer with anti-coagulant after centrifugation

Figure 2. Image of blood in normal vial (without anti-coagulant after centrifugation


B. DNA Isolation and DNA Electrophoresis

V VV V MM MM
10000 bp
9000 bp
8000 bp
7000 bp
6000 bp > 10000 bp
5000 bp
4000 bp
3500 bp
3000 bp
2500 bp
2000 bp
1000 bp
750 bp V = Vicko’s sample
500 bp
M = Maranatha’s
250 bp
sample

* = DNA Marker
Figure 3. Agarose gel electrophoresis of isolated DNA sample

C. Quantitation of DNA Concentration

Sample 1 I II Mean
A 230 0,142 0,144 0,143
A 260 0,304 0,305 0,3045
A 280 0,159 0,161 0,161

Sample 2 I II Mean
A 230 0,008 0,004 0,006
A 260 0,261 0,260 0,2605
A 280 0,154 0,156 0,155
The DNA concentration was calculated with the formula :
DNA concentration (µg/mL) = (OD : ) x dilution factor
DNA Concentration of Sample 1 = (0,3045 : 20) x 5 = 0,076125ng/mL = 76,125µg/mL
DNA Concentration of Sample 2 = (0,2605 : 20) x 5 = 0,065125ng/mL = 65,125 µg/mL

The purity index of the DNA sample was calculated with the formula :
Purity Index = A260/A280 (1.80 – 2.00)
Purity Index of Sample 1 = 0,3045 / 0,160 = 1,903125
Purity Index of Sample 2 = 0,2605 / 0,155 = 1,68064516
So the purity of sample 2 out of the range and be considered as there were protein
contamination in it and sample 1 is pure.

The purity index of DNA sample was calculated with the formula :
Purity Index = A260/A230 (2.00-2.20)
Purity Index of Sample 1 = 0,3045/0,143 = 2,12937063
Purity Index of Sample 2 = 0,2605/0,006 = 43,416667
So the purity of sample 2 is out of range as there was contamination in it. While the
sample 1 is pure.
D. PCR

- = Control Negative
* M M - + * V V + -
+ = Control Positive
V = Vicko’s sample
1517 bp M = Maranatha’s sample
1200 bp
1000 bp * = DNA Marker
900 bp
800 bp
700 bp
600 bp 600 bp
400 bp
200 bp
100 bp

Figure 4. Agarose gel electrophoresis after PCR experiment

E. DNA Sequencing
Figure 5. BLAST graphic summary and result of DNA sequencing experiment, FORWARD

Figure 6. Protein p53 sequence, FORWARD


Figure 7. BLAST graphic summary and result of DNA sequencing experiment, REVERSE
Figure 8. Protein p53 sequence, REVERSE
DISCUSSION

From the result of the blood separation, we have 2 condition one with anti-
coagulant and one without anti-coagulant. The result for the first, we got 3 layers :
plasma on the top, buffy coat (white blood cell and platelets, and red blood cell on the
bottom. The second, we got 2 layers that contain serum on the top and red blood cell on
the bottom. If we are compare with the references, our result was matched. And from
this result, we can concluce the experiment was success.

The sample was taken from the previous experiment. The result of DNA isolation
was white pellet at the bottom of the tube. There are some steps to isolate the DNA,
such as, separated red blood cell from blood cell, lysed the cell membrane by SDS
detergent, NaOAc to helped SDS detergent to lyse the white blood cells, proteinase K to
degrade the protein fraction, PCI to remove the non-nucleic acid molecules and Tris
EDTA buffer to resuspend the DNA.

The resulth of electrophresis experiment can show us how many base pair in our
isolated DNA samples. From our result of DNA electrophoresis, showed that the base
pairs are more than 10000 bp. In line number 2, there was no line because there was a
mistake when we used the pipet. In line number 1 and 3, that look very clear because
the concentration of that lines were high than the other. This experiment was succeed,
even though there was some errors.

From Quantitation of DNA Concentration, we are able to know the concentration


of the DNA and also the purity index of isolated DNA. The sample was measured at
A260 and A280 to know the purity index of DNA. The purity index is 1,80 – 2,00. If the
purity is < 1,80 it means there was protein or phenol contamination, and if the purity is >
2,00 it means there was RNA contamination. From the result we got, the purity of
sample 1 was 1,90, it means it was pure, the purity of sample 2 was 1,68 it means it
wasn’t pure maybe there was contaminated with protein or phenol during the DNA
isolation experiment. The result of the DNA concentration, sample 1 = 76,125 µg/mL
and sample 2 = 65,125 µg/mL.
After all PCR steps were done, the agarose DNA electrophoresis results were
600 base pairs for sample 1 and sample 2. If the PCR done correctly at the negative
control there will be no line in lane and in our result there was no line that means no
contamination in the sample. At the positive control lane we can see the line, it means
there was positive p-53 gene. Sample Vicko’s 1 band is ticker than sample Vicko’s 2
band, we indicates there were lack of DNA concentration when pipetting process. PCR
process was succeed.

The DNA sample was edited using Cromas Lite application that installed into our
computer. Firstly, we have to change the N sequence into DNA sequence according to
the graphic’s color (green for A (adenine), black for G (guanine), red for T (thymine),
blue for C (cytosine)). Copied the sequence in FASTA format, then DNA sequence was
BLAST-ed. The result showed that our DNA sequence matched 99% with Homo
sapiens tumor protein p53 (TP53), compared to the database in NCBI site. The
remaining 1% maybe occurred because of our mistake when we edited the N sequence.

Conclusion, all genomic experiments were done successfully. Even though there
were many errors during do the experiments, but our result still acceptable and the main
goal of this experiments were achieved.
REFERENCES

Bazzi. DNA Quantification; 2009; Available from: URL:

faculty.ksu.edu.sa/Bazzi-MD/LBCH435/12-DNA%20Quantitation.doc

DNA sequencing. United State; 2011; available from: URL: www.dnasequencing.org/

Medicine website [citied 2013 oct 29]; Available from: URL:


http://ghr.nlm.nih.gov/handbook/basics/dna

Rabinow P. Making PCR. A Story of Biotechnology. Chicago : University of Chicago


Press : 1997.

Schottenfeld D, Fraumeni JF. Cancer Epidemiology and Prevention. New York : Oxford
University Press; 2006.

Tortora GJ, Derrickson B. Principle of Anatomy and Physiology. 13th ed. San Francisco :
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Zdnowics MM. Concepts in Pharmacogenomics. Bethesda : America Society of Health-


System Pharmacists; 2010.