FUNDAMENTAL MEDICAL SCIENCE 1

FINAL REPORT (GENOMIC)

ELLA ANDREA WIDAGDO

00000004736
GROUP A1

UNIVERSITAS PELITA HARAPAN

MOCHTAR RIADY INSTITUTE OF NANOTECHNOLOGY
FACULTY OF MEDICINE
2014
ABSTRACT

Genomic science refers to the study of complete DNA sets of an organism.

The goal of these experiments was to extract and identified MDM2 gene from
human. The experiment chain was started with drawing blood from human for
samples, this blood was the main material of all of the genomics experiment. The
blood was separated by centrifuging. The blood with anticoagulan separated into
three layers; plasma, buffy coat, and whole blood. Then, pure forms of DNA were
obtained from DNA isolation with the help of certain enzymes, detergents and salts.
Concentration and purity of this DNA was calculated using spectrophotometer
analysis. Ellas DNA concentration was 74.4 g/ml and the purity was 1.77.
Esperanzas DNA concentration was 84.8 g/ml and the purity was 1.76. The size
was confirmed when DNA was detected using electrophoresis and Versa doc
machine showed that approximately more than 10.000 base pairs/10kb were present
in complete genome as compared to DNA ladder. After that, the gene in DNA sample
was amplified using polymerase chain reaction technique to multiply the DNA sample
until enough copies were made for further analysis. The approximate size of DNA
ladder was detected using gel electrophoresis and last experiment was done to
determine which gene it is by figuring out the nucleotides order with bioinformatics
software tool and bank genome NCBI database using Basic Local Alignment Tool on
the internet. The gene samples were indentical with MDM2 gene.
I. INTRODUCTION

Blood is the vehicle for long-distance, mass transport of materials between

the cell and external environments or between the cells themselves. Blood consist of
a complex liquid plasma in which the cellular elements erythrocytes, leukocytes,
and platelets are suspended. Erythrocytes are essentially plasma membrane
enclosed bags of hemoglobin that transport oxygen in the blood. Leukocytes, the
immune systems mobile defense units. Platelets are the stopping of bleeding from
an injured vessel. The constant movements of blood as it flows through the blood
vessels keeps the cellular elements rather evenly dispersed within the plasma.
However if it is put in a test tube and treat it to prevent clotting, there will be three
layers from the lightest in the top, plasma (55%), buffy coat that consist platelets and
leukocytes (<1%), to the heaviest in the bottom, erythrocytes (45%). (Sherwood,
2010)
DNA, which is present in the blood, is not produced by red blood cells
because RBCs do not have nuclei and lack of organelles. Plasma also doesnt
produce it as it only consists of water and water-soluble protein. Actually, white blood
cells produce our DNA (Sherwood, 2010). DNA is a long polymer of nucleotide,
normal DNA is approximately 3 million bps (Marks, 2008).
The polymerase chain reaction (PCR) is a scientific technique in a molecular
based on the ability of DNA pollymerase to amplify a single or few copies of a piece
of DNA across several order of magnitude, generating thousands to millions copies
of particular DNA sequences. There are three major steps involved in the PCR
technique: denaturation, annealing and extension. (Joshi, 2011)
DNA concentration is determined using Beers Law ; A = bc where A is
absorbance, is molar extinction coefficient, b is path length, and c is DNA
concentration. value for dsDNA is 20L/g cm (Farrell & Taylor, 2006).
Spectrophotometry is the principal method for evaluating quantity and quality
of nucleic acids. In aqueous solution, DNA has maximal absorbance near 260 nm
with an extinction coefficient of 50; protein absorbs light strongly near 280 nm. The
concentration of a sample can be read directly (in g/l) by diluting it 1:20 in water or
buffer; a practical lower limit of detection is 50100 ng DNA in a 50l00 l
microcuvette. The A260/A280 ratio provides an estimate of DNA purity; values of 1.7
2.0 predict clean DNA. (Susan J. Ahn et al, 1996)
The order of the nucleotide of a given DNA sequence is determined through
the process of DNA sequencing. The method that is often used is dideoxy method or
chain termination or Sanger method. The process is same like PCR, the only
difference is that there are dideoxyribonucleotide present in the reaction mixture that
halts the process. After incubation, the sequences are read from shortest strand to
longest strand. Then, our DNA sequence will be compared with the database by
using BLAST in the computer to identifies whether our gene is AFP or not.
(Cantor,1999)
II. MATERIALS AND METHODS

In blood separations, blood samples were drawn from the elected student
(Ella and Esperanza) at 8-9 mL by using a syringe. All samples were stored in the
vacutainer with anti-coagulant (K3-EDTA) and empty vacutainer. From each of the
sample, one mL of whole blood was transferred into a separated labeled vial. After
that, all of the vacutainer would be centrifuged at 3000g using Allegra x15 centrifuge
on 20C, for 10 minutes. Then, the plasma (transparent liquid which were present in
the upper layer of a vial coated with K3-EDTA) would be transferred into a new vial
and stored at -80C
SCC buffer 1X 0,8 ml was added to blood samples (prepared by assistant),
mixed and centrifuged (1 minute, 12000rpm in microcentrifuge). One ml of
supernatant was removed. One ml of 1X SCC Buffer was added, vortexed, and
centrifuged (1 minute, 12000rpm). Supernatant was removed. NaOAc 0,2 M 375 l
was added to each pellet, vortexed, then 10%SDS 25 l and Proteinase K (10mg/ml
H2O) 5 l were added, vortexed, and incubated (1 hour, 55C).
Phenol/choloroform/isoamyl alcohol 120 l was added, vortexed (30 seconds), and
centrifuged (2 minutes, 12000rpm). Aqueous layer was removed to 1,5 ml
microcentrifuge tube, cold 100% ethanol 1 ml was added, mixed, and centrifuged (2
minutes, 12000rpm). Supernatant was removed. TE Buffer 1X 180 l was added,
vortexed, NaCOOH 20 l 2M was added, mixed, cold 100% ethanol 500 l was
added, mixed and centrifuged (1 minute, 12000rpm). Supernatant was removed,
pellet was rinsed with 1 ml of ethanol 70% and centrifuged (1 minute, 12000rpm).
Supernatant was removed, pellet was air dried till dry and resuspended (1X TE
Buffer 200 l was added, incubated [55C, 30 minutes], vortexed), and then stored at
-20C.
Spectrophotometer BIORAD was used to determine the quantitation of DNA
concentration. Firstly, dilute the DNA sample (1:2) and filled the cuvette with 50 L of
1X TE buffer. Then, set the cuvette on the cuvette holder of spectrophotometer. In
this experiment, we would use double strand DNA as a type of nucleic acid. The
conversion factor will be determined to calculate the nucleic acid concentrations from
absorbance data and the spectrophotometer with 1X TE buffer was blanked. The
cuvette inserted along with DNA sample to read the absorbance (50 L final volume).
Then, the results of Absorbance 230,260, and 280 were recorded.
Each DNA sample 5l was loaded into agarose gel well (prepared by
assistant). DNA marker 6l was loaded as well. Lid was closed and electricity was
run from (-) end to (+) end (100V, 6W, 0.6A, 30minutes). Gel was taken out and
washed. Result was recorded using Versa Doc.
PCR reaction mixture of total 25 l containing 12.875 l dH2O, 5l 1X Buffer
PCR, 2l dNTP mix 200M, 1.5l MgCl2 1.5mM, 0.125l of Taq DNA Polymerase,
0.75l Forward Primer 0.3M, 0.75l Reverse Primer 0.3M, 2l DNA template for
each sample was set up in PCR tube. It was then put in PCR machine that has been
programmed (Predenaturation : 95C for 10 minutes, Denaturation : 94C for 30
seconds, Annealing : 62C for 30 seconds, Extension : 72C for 30 seconds, set for
35 cycles). Product was kept at 4C.
Cromas Lite was installed, DNA file was opened, edited and copied. BLAST
website was opened, then Blast Menu, Nucleotide Blast Menu were clicked
respectively. DNA Sequence was paste into Enter Query Sequence Area, Human
Genomic Database was chosen in search set. BLAST was run.
III. RESULTS

A. Blood seperation

Figure 1.1 vacutainer with anticoagulan

Without anticoagulan

Figure 1.2 vacutainer without anticoagulan

B. DNA Isolatian and DNA electrophoresis

A B C D E F G Lane A : DNA marker

H Lane B : Sample Esperanza
Lane C : Sample Ella
Lane D : Sample Esperanza
Lane E : Sample Ella
Lane F : Sample Esperanza
Lane G : Sample Ella
Lane H : Sample Esperanza

Figure 2. Agarose gel electrophoresis of DNA isolated from whole blood samples
C. Quantitation of DNA Concentration
DNA Concentration
1. Ellas Sample
A260 = (0.747 + 0.741) / 2 = 0.744
Concentration = (OD : ) x dilution factor
= (0.744 : 20) x 2
= 74.4 g/ml.

2. Esperanzas Sample
A260 = (0.858 + 0.838) / 2 = 0.848
Concentration = (OD : ) x dilution factor
= (0.848 : 20) x 2
= 84.8 g/ml.

DNA Purity
1. Ellas Sample
A230 = (0.292 + 0.291) / 2 = 0.2915
A260 = (0.747 + 0.741) / 2 = 0.744
A280 = (0.423 + 0.419) / 2 = 0.421
A260/A230 = 0.744 : 0.2915
= 2.55
A260/A280 = 0.744 : 0.421
= 1.77

2. Esperanzas Sample
A230 = (0.323 + 0.311) / 2 = 0.317
A260 = (0.858 + 0.838) / 2 = 0.848
A280 = (0.489 + 0.475) / 2 = 0.482
A260/A230 = 0.848 : 0.317
= 2.67
A260/A280 = 0.848 : 0.482
= 1.76

D. PCR
 Figure 3 The PCR product of agarose gel electrophresis

E. DNA Sequencing

Figure 4.1 Color key of alignment score

MDM2

Figure 4.2 Blast result

IV. DISCUSSION

From the blood seperation result, blood which placed in EDTA vials, an
anticoagulan factor, clearly visible seperates into three layers. The first yellowish
transparant liquid layers is plasma, followed by thin layers called buffy coat which
consisted of leukocytes and platelets. And the third layer is whole blood consisted of
erythrocytes. This result was corresponding with the theory of classification of blood.
Those 3 layers were visible because centrifugation separates mixture based on its
weight. The seperated blood in no EDTA vials shows only two layers. The upper
layers is serum because there was a part of plasma called fibrinogen, which
combined with platelets, part of buffy coat, caused the blood to be coagulated. And
bellow is whole blood consisted of erythrocytes, leukocytes, and platelets. So, this
experiment was successfully done and supported the stated theory in Sherwood
(2010).
DNA was isolated from leukocytes because leukocytes have nuclei where
DNA is located. DNA isolation procedures was using whole blood because even the
blood was already separated into layers, leukocytes in buffy coat was too little to be
extracted. To get DNA only, other components in the whole blood must be removed
first. Unwanted protein could be removed by using proteinase K by degrading and
denaturating the proteins. After that using SDS detergent, cell membrane was lysed
so DNA in the nuclei could be extracted. PCl was then added to remove most of non
nucleic acid molecules and unwanted components. DNA then have to be precipitated
using Ethanol and resuspended using TE buffer. The result in this experiment was
whitish color pellet DNA in vial beacause the sample was really clean.
Electrophoresis was done to know the length of the sequences. Human
genome have more than 10.000 base pairs. In figure 2, it shows that our DNA
samples are slightly above the band formed by the DNA ladder. The first band in the
DNA ladder indicates the size of 10.000 base pairs. So, our DNA samples had more
than 10.000 base pairs. The unclear thin band was also appeared which meant that
the concentration of DNA of the sample was low. Lane 6, which was filled with
Esperanzas DNA was thicker compared to lane filled with Ellas DNA. These results
proved that higher concentration of Esperanzas DNA caused her DNA band was
thicker when it was seen in electrophoresis.
Quantitation of DNA concentration showed significant calculation for DNA
purity. DNA is considered pure if the purity index is between 1.8 2. Result below
1.8 indicates that the DNA sample was contaminated with protein and result above 2
indicates that the DNA sample was contaminated with RNA. Our DNA sample had
purity value 1.77 for Ellas DNA and 1.76 for Esperanzas DNA. Both purity index of
DNA sample calculation results was below 1.8 which meant that both samples were
contaminated with protein. Consentration of Esperanzas DNA (84.8 g/ml ) was
higher than Ellas DNA (74.4 g/ml ). This could happen due to error during pipetting
of DNA.
After the process of PCR, electrophoresis was performed once again to
confirm that we had amplified the right target DNA. Then, the sample was compared
with the DNA ladder to measure the size using Versa Doc Machine. Our gene target
was MDM2 with the size of 622 base pairs. Unfortunatelly in our result, no samples
DNA were shown. This could be due to error in PCR process or making the mix of
PCR reaction. Some materials might be forgotten to be added or not perfectly mixed
so couldnt be read by electrophoresis.
Lastly, BLAST result was to show that the amplified DNA sequence was really
MDM2 gene that we wanted. BLAST is used to compare our DNA sample with the
database. Identification of gene by BLAST was based on the local similarity between
the sequences. By using these tools, the sample of gene was 84% identified as
MDM2 gene.
Conclusion from our experiments, although there was some error we were
succeed to reach the goal of all of these experiments.

V. REFERENCE

Lauralee S. Human Physiology: From Cells to Systems. 8th ed. Brooks/Cole:

Canada; 2010.

Marks DB, Marks AD, Smith CM. Basic Medical Biochemistry : A Clinical Approach.
 3rd ed. Lippincott Wiliams and Wilkins : Philadelphia; 2008

Farrell SO, Taylor LE. Experiments in Biochemistry: A Hands-On Approach. 2 nd ed.

Thomson Brooks/Cole: USA; 2006.

Cantor CR, Smith CL. Genomics: the science and technology behind the Human
Genome Project. John Wiley & Sons: Canada ; 1999.

Mohini Joshi, J. D. Deshpande. Polymerase Chain Reaction: Methods, Priciples and

Application. 2nd ed. International Journal of Biomedical Research: India;
2011.

Susan J. Ahn, Jose Costa. PicoGreen Quantitation of DNA: Effective Evaluation of

Samples Pre or Post PCR. 24th ed. Oxford Journal: England; 1996.