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FUNDAMENTAL MEDICAL SCIENCE 1

FINAL REPORT (GENOMIC)

JESSICA WIRYANTO
000 0000 5569
GROUP A2-B

UNIVERSITAS PELITA HARAPAN


MOCHTAR RIADY INSTITUTE OF NANOTECHNOLOGY
FACULTY OF MEDICINE
2014

ABSTRACT
The series of experiments performed are designed to help us understand more
about DNA and its structure. In these experiments we extracted and identified human
MDM2 gene. A whole blood sample was taken and separated through centrifugation.
The centrifugation showed clear differentiation of results.
The whole blood was then used for DNA isolation. The DNA was extracted with
the help of certain enzymes, detergents and salts. The result showed a white-tangledstrands structure of pure DNA. The obtained DNA was further used for quantitation of
DNA concentration and DNA electrophoresis.
DNA quantitation is a method used to determine DNA concentration using
spectrophotometer. DNA quantitation was performed to calculate the purity of DNA
obtained, while the DNA electrophoresis was performed to confirm the size of DNA by
comparing the result to DNA ladder. The results showed that approximately more than
10.000 base pairs were present in complete genome.
Once the concentration of DNA was calculated, we undergo DNA Polymerase
Chain Reaction in order to amplify the amount of MDM2 gene. Then we undergo
electrophoresis. The amplified gene sample will then be used for DNA sequencing.
DNA sequencing was done with Sanger method in order to determine the
nucleotides sequence of the DNA. DNA sequence was edited with the help of Chromas
Lite program, the result was then compared to NCBI database using Basic Local
Alignment Search Tool (BLAST). The nucleotide sequence was 96% matched and
identified as human MDM2 genes located on chromosome 12.

I. INTRODUCTION
Blood is a connective tissue that consists of blood plasma (liquid) and formed
elements (RBC, WBC, and platelets). The red blood cells have biconcave disk structure,
without nuclei. They transport most of gases. White blood cells provide protections
against disease, they have nucleus (contains DNA). Platelet is responsible for blood
clotting. As blood clots, serum (liquid part of blood without clotting proteins) is separated
out. Centrifugation in vacutainers without anti-coagulant will separate blood into 2 layers
of serum (straw-colored liquid) and coagulated blood (RBCs, WBCs and platelets).
K3EDTA is an anti-coagulant that will bind to the clotting factor. This anti-coagulant
prevents blood from clotting. In vacutainer with anti-coagulant, blood components are
clearly separated; blood plasma, buffy coat, and red blood cells. (Gerard J. Tortora,
2012)
Deoxyribonucleic acid is the genetic material of all cellular organisms which can
be found in the nucleus. Therefore, DNA cannot be obtained from red blood cells as it
doesnt have nucleus but it can be obtained from the white blood cells. Each nucleotide
of DNA consists of 3 parts, nitrogenous base, phosphate group, and pentose sugar.
DNA contains four different nitrogenous bases, which contain atoms of C, H, O, and N.
In DNA the four nitrogenous bases are adenine (A), thymine (T), cytosine (C), and
guanine (G). (G. J. Tortora, B. Derrickson, 2012)
DNA was isolated through 4 stages, disruption, lysis, removal of proteins and
contaminant, and DNA recovery. Salt was added and SDS detergent was used to lyse
cell membrane. Proteinase K and PCI alcohol help removes macromolecules such as
proteins and lipids. DNA was precipitated with organic solvent such as ethanol or
isopropanol. DNA is insoluble in ethanol, therefore, ethanol addition cause DNA to come
out of solution and stick together. At last DNA is eluted in a low salt buffer or elution
buffer. (MATER METHODS, 2013)
DNA concentration is determined by its absorbance which can be calculated
using the Lambert Beers law. Lambert Beers Law identifies the relation of sample

concentration to the intensity of transmitted light. The absorbance is the product of


concentration (c), extinction coefficient (), and optical length (l). value of dsDNA is
20L/g cm. DNA purity is determined by the ratio of A260/A280. DNA is considered pure
if the purity index is 1,8 - 2,0. When the purity index falls below 1,8 there might be
protein contamination, below 2,0 might be caused by phenol contamination, while above
2,0 shows RNA contamination. (Farrell &Taylor 2006)
DNA Electrophoresis separates DNA according to its size in a matrix using
electric current. Smaller fragments have more rapid rate of migration (Appleton &
Lange, 2004). The phosphate group carrying negatively-charged oxygen, gives DNA an
overall negative charge. In electric current, negatively charged DNA will move toward
the positive pole. (DNA Learning Center, 2014). The ethidium bromide will form a
brightly fluorescent adduct as it binds to DNA, enabling DNA fragments (in agarose gel)
to be photographed under UV lights (Appleton & Lange, 2004).
Polymerase Chain Reaction is used to amplify a specific gene from DNA
template. This process requires DNA Template, DNA polymerase, Primers, sNTPs, RTPCR, reverse transcriptase, buffer, and magnesium. PCR consist of 3 stages,
denaturation (separation of DNA double strand in high temperature), annealing (binding
of primers to DNA), and extension (Addition of complementary nitrogen bases by Taq
(Thermus aquaticus) polymerase thermophilic - heading toward 3 end). This cycle will
be repeated for several times until enough amounts of DNA copies have been
produced. (NCBI, 2014; Phillip Mc Clean, 1997)
DNA sequencing displays gene structure revealing regulatory region that control
gene expression. There are 2 methods developed, the Maxam Gilbert techniques relies
on the relative chemical liability of different nucleotide bonds, and Sanger method (the
use of dideoxitermination) which interrupts elongation of DNA sequences. The principle
is single-stranded DNA molecules that differ in length by just a single nucleotide can be
separated from one another using gel electrophoresis. The stages of reaction in DNA
sequencing are almost the same as those in PCR (denaturation, annealing, and
elongation). (Appleton & Lange, 2004).

By the help of Basic Local Alignment Search Tool we generate alignments


between a nucleotide or protein sequence (query) and the nucleotide or protein
sequences within a database (subject). (Humana Press; 2007)

II.

MATERIALS AND METHODS


In blood separation, 600 l of whole blood samples were collected from each

student (Felix and Gaby) and stored in 2 different vacutainers, vacutainer with and
without anti-coagulant. The samples were then centrifuged at 3700 rpm for 10 minutes.
Blood plasma was obtained from blood sample that had been centrifuged. Then it was
stored in the vials at -80C.
0,8 ml of 1X SSC Buffer was added. The sample was mixed and centrifuged at
12000 rpm for 1 minute. SSC buffer 1X 0,8 was added to the blood samples, mixed and
centrifuged at 12000 rpm for 1 minute using micro centrifuge. One ml of supernatant
was removed. One ml of 1X SSC Buffer was added, mixed, and centrifuged (12.000
rpm for 1 minute).Supernatant was removed. 375 l of 0,2M NaOAc was added to each
pellet and vortexed, then 25 l 10% SDS was added, followed by addition of 5 l of
proteinase K (10mg/ml H2O). The specimen was the vortexed and incubated for 30
minutes at 55C. 120 l of Phenol/chloroform/isoamyl alcohol was added, vortexed for
30 seconds, and centrifuged at 12000rpm for 2 minutes. Aqueous layer was removed to
1,5 ml micro centrifuge tube. One ml of cold 100% alcohol wad added, mixed, and
centrifuged at 12000 rpm for 2 minutes. Supernatant was drained. 180 l of warm 1X
TE buffer was added, and mixed by flicking. 20 l of NaCOOH 2M was added, mixed,
followed by addition of cold 100% ethanol 50 l, mixed and centrifuged at 12000rpm for
1 minute. Supernatant was removed. Pellet was rinsed with 1 ml of 70% ethanol and
centrifuged at 12000rpm for 1 minute. Supernatant was removed, pellet was air dried
and resuspended by adding 200 l 1X TE Buffer, incubated at 55C for 30 minutes,
dissolved (flicking), and then stored at -20C.
Spectrophotometer was blanked by running a 50 l of 1X TE Buffer. DNA sample
were then diluted in 1:2 and 1:4 ratio to distilled water. Samples were inserted into
cuvettes and set on the spectrophotometer. Absorbance at wavelength of 230nm,
260nm, and 280nm were read.
2 l of each DNA samples that had been mixed well with 2 l loading dye was
loaded into the agarose gel that had been well prepared previously by lab assistant.
Then 2 l of DNA marker was added. Electrophoresis chamber lid was closed and

electricity was run from (-) end to (+) ends (100V, 6W, 0.6A, 30minutes). Gel was taken
out and washed. Result was recorded using Versa Doc.
In PCR, a master mix was made of dH2O, 5X Buffer PCR, 2.5 mM dNTP mix, 25
Mm MgCl2, Taq Polymerase, and 10 M forward and reverse primer. The master mix
was divided into 4 separated labeled tubes. 2 tubes were added with DNA sample, while
the other 2 was used for positive and negative control. The samples were then put into
the PCR machine. The PCR cycle consist of initial denaturation step of 5 minutes at
95C, followed by 35 cycles of denaturation at 94C, annealing at 62C, and extension
at 72C, each for 30 seconds, followed by 5 minutes final extension at 72C. PCR
product was verified by electrophoresis.
Sample DNA was given by lab assistant. Bioinformatics Software tools (Chromas
Lite program) was installed, DNA file was opened, edited (reverse complement if
required and edit by color) and copied in FASTA format. NCBI website
(http://www.ncbi.nlm.nih.gov/) was opened, then Blast Menu, Nucleotide Blast Menu
were clicked respectively. The edited DNA sequence (query) was copied. BLAST was
run.

III.

RESULT
A. Blood Separation

Figure 1.1 Blood separation


vacutainer with anti-coagulant

Figure 1.2 Blood separation


vacutainer without anticoagulant

B. DNA isolation and DNA electrophoresis


>10 000
10000 bp
8000 bp

A B C D E F G
H I J

6000 bp
3500 bp

Figure 1.3 Agarose gel


electrophoresis of DNA
isolated from whole blood
samples

C. Quantitation of DNA concentration

Lane
Lane
Lane
Lane
Lane
Lane
Lane
Lane
Lane
Lane

A : DNA Marker
B : Sample Felix 1.1
C : Sample Gaby 1.1
D : Sample Felix 1.2
E : Sample Gaby 1.2
F : Blank
G : DNA Marker
H : Sample Felix 2.1
I : Sample Gaby 2.1
J : Sample Felix 2.2

DNA Concentration
OD
Concentration=
dilution factor

Felixs Sample:
A260 = (1,821 + 1,821) / 2 = 1,821
1,821
2=0,1821 mg/mL=182,1 g /ml
Concentration =
20
Gabys Sample:
A260: (0,826 + 0,947 + 0,939) / 3 = 0,904
0,904
4=0,1808 mg/ mL=180,8 g /ml
Concentration =
20

DNA Purity
Sample Felix: dilution 1:2
First Reading:

Second Reading:

Mean:

A230: 0,760

A230: 0,758

A230: 0,759

A260: 1,821

A260: 1,821

A260: 1,821

A280: 0,963

A280: 0,964

A280: 0,9635

Purity Index

Phenol (Solvent) Purity

A 260 1,821
=
=1,89
A 280 0,9635

Sample Gaby:

A 260 1,821
=
=2,339
A 230 0,759

dilution 1:4

First Reading:

Second Reading:

Third Reading:

A230:0,339

A230: 0.374

A230: 0,365

A260:0,826

A260: 0,947

A260: 0,939

A280:0,448

A280: 0,498

A280: 0,495

Mean:

Purity Index:

Phenol (Solvent) Purity:

A230: 0,359

A 260 0,939
=
=2,572
A 230 0,365

A 260 0,904
=
=1,88
A 280 0,480

A260: 0,904
A280: 0,480
D. PCR
100
0
900
800
700
600
500
400
300
200
100

DNA Purity Index = 1,8 -2,2 ; <1,8 contamination by protein ; >2,2


contamination by RNA
Phenol (Solvent Purity = 2,0 - 2,2 ; >2,2 contaminated by RNA

A B C D E F G H I
J

Figure 1.4 PCR


Electrophoresis

E. DNA Sequencing

622 bp

Lane A =
DNA
Marker
Lane B =
Sample
Felix
Lane C =
Sample
Gaby
Lane D =
Sample

Figure 1.5 Blast Result 1

Figure 1.6 Blast Result 2

IV.

DISCUSSION
In blood separation, centrifugation separates blood components. One of blood

component, plasma, contains albumin, globulin, and fibrinogen. In vacutainer with anticoagulant, blood was separated into 2 layers, serum and coagulated blood.
Centrifugation in vacutainer containing anti-coagulant separated blood into 3 layers,
plasma, buffy coat, and red blood cells. Buffy coat was formed in within the 2 layer
because they are less dense than red blood cells, however they are more dense that
plasma. This result supports the theory (introduction).
In DNA isolation, samples were added with SSC Detergent in order to remove
cell membrane. After centrifugation, blood samples were separated into 3 layers, a
layer of supernatant of transparent red color, a thin layer of white blood cells, and pellet.
After the addition of SDS detergent (lyse cell membrane), proteinase K (degrade
proteins), and salt (NaOAc to disturb pH and optimize the work of proteinase K and
SDS detergent) the sample turns into transparent brown colored liquid. The sample was
further added with phenol/chloroform/isoamyl alcohol, mixed and centrifuged resulting in
3 layers of aqueous phase which will be used for further experiment procedure (as it
contains DNA), interphase layer made of phospholipids, and a non-polar layer of phenol
(organic). Then the aqueous layer was obtained, tris EDTA (TE) buffer presented to
resuspend the DNA. Through this process, we could obtain pure white-like color pellet
DNA of thin tangled fiber like structure. (Surzycki, 2003) Referring to the result above
electrophoresis technique shown that according to DNA marker, both complete
genomes sizes were more than 10000 base pairs. This difference in bands thickness
appeared because the DNA sample could have different of concentrations of DNA
samples. (Rob Reed et al, 2007). Over all, the experiment was done accordingly and
the bands were successfully seen.
Using spectrophotometer we obtained data of DNA samples absorbance. These
data were used to calculate DNA concentration and purity. The isolated DNA was said
to be pure if the value of A260/A280 was between 1.8 2.0. Value of below 1,8 shows
protein contamination, while above 2,0 shows RNA contamination. The value of
A260/A230 was supposed to be between 2 - 2,2. Value below 2,0 might be caused by
phenol (solvent) contamination, above 2,2 shows RNA contamination. (Farrel &Taylor

2006). Both Felix and Gabys samples were similar in concentration (Felixs = 1,1821
g/ml , Gabys = 0,1808

g/ml ). Result shown RNA contamination (A260/A230

Felix= 2,428, A260/A230 Gaby=2,638). However, the results were pure from protein
contamination (A260/A280 Felixs = 1,89, A260/A280 Gabys = 1,88). DNA
concentration was well calculated, the calculation can be used to determine volume of
DNA sample that will be used for PCR. DNA quantitation was successfully done.
PCR results are different from the earlier electrophoresis as the PCR procedure
measures the size of a specific gene in our DNA sample, not the whole genome. PCR
was done on MDM2 gene using specific oligonucleotides (primers) for specific
amplification with the help of Taq DNA polymerase (thermostable enzymes). To reassure
our result there are 2 controls, positive sample and negative sample. Positive sample
will show a thick band with the same length as our DNA samples, while negative
sample will show no band at all in electrophoresis. Our electrophoresis results for
Felixs, Gabys, and positive samples (compared to DNA ladder) were about 622 bp
long. For negative sample, there was no band shown during electrophoresis. We can
conclude that our results were trust worthy (no fatal error) as the positive and negative
samples both shown the expected results. The difference in band thickness was due to
the difference in concentration. (Caldwell et al, 2006) Electrophoresis helped us verify
that the MDM2 gene has been successfully amplified.
The amplified samples were then compared to those in the data base with the
help of Bioinformatics Software tools (Chromas Lite). Based on BLAST, we discovered
that our DNA samples were valid and has match Homo sapiens MDM2 oncogene, E3
ubiquitin protein ligase (MDM2) RefSegGene on chromosome 12 data in the data base
(96% matched). Red color shows 200 alignment score, this indicates the similarity of
our gene sample to those in the database. (NCBI, 2014) To conclude, our experiment
was satisfactory. This can be seen when the result matched the gene-bank database.

V.

REFERENCES

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Ncbi.nlm.nih.gov. Polymerase Chain Reaction (PCR). 2014 [cited 4 November 2014].
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