FUNDAMENTAL MEDICAL SCIENCE 1FINAL REPORT (GENOMIC)

JESSICA WIRYANTO000 0000 5569GROUP A2-B

UNIVERSITAS PELITA HARAPANMOCHTAR RIADY INSTITUTE OF NANOTECHNOLOGY

FACULTY OF MEDICINE2014

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-tangled-

strands 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

doesn’t 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 Beer’s law. Lambert Beer’s Law identifies the relation of sample

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

concentration (c), extinction coefficient (ɛ), and optical length (l). Ɛ 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, RT-

PCR, 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 µl 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 -80°C.

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 µl of 0,2M NaOAc was added to each

pellet and vortexed, then 25 µl 10% SDS was added, followed by addition of 5 µl of

proteinase K (10mg/ml H2O). The specimen was the vortexed and incubated for 30

minutes at 55°C. 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 µl 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 µM 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

95°C, followed by 35 cycles of denaturation at 94°C, annealing at 62°C, and extension

at 72°C, each for 30 seconds, followed by 5 minutes final extension at 72°C. 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

B. DNA isolation and DNA electrophoresis

10000 bp

8000 bp

6000 bp

3500 bp

A B C D E F G H I J Lane A : DNA MarkerLane B : Sample Felix 1.1Lane C : Sample Gaby 1.1Lane D : Sample Felix 1.2Lane E : Sample Gaby 1.2Lane F : BlankLane G : DNA MarkerLane H : Sample Felix 2.1Lane I : Sample Gaby 2.1Lane J : Sample Felix 2.2

>10 000 bp

Figure 1.3 Agarose gel electrophoresis of DNA isolated from whole blood samples

Figure 1.1 Blood separation vacutainer with anti-coagulant

Figure 1.2 Blood separation vacutainer without anti-coagulant

C. Quantitation of DNA concentration

DNA Concentration

Concentration=ODε×dilution factor

Felix’s Sample:

A260 = (1,821 + 1,821) / 2 = 1,821

Concentration = 1,82120

×2=0,1821mg /mL=182,1g /ml

Gaby’s Sample:

A260: (0,826 + 0,947 + 0,939) / 3 = 0,904

Concentration = 0,90420

×4=0,1808mg /mL=180,8 g/ml

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 260A 280

= 1,8210,9635

=1,89 A 260A 230

=1,8210,759

=2,339

Sample Gaby: 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: 0,359A 260A 280

=0,9040,480

=1,88 A 260A 230

=0,9390,365

=2,572

A260: 0,904

A280: 0,480DNA 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

D. PCR

622 bp

E. DNA Sequencing

A B C D E F G H I J Lane A = DNA MarkerLane B = Sample FelixLane C = Sample GabyLane D = Sample

1000900800700600500400300200100

Figure 1.4 PCR Electrophoresis

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 anti-

coagulant, 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

genome’s 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 Gaby’s samples were similar in concentration (Felix’s = 1,1821

g/ml, Gaby’s = 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 Felix’s = 1,89, A260/A280 Gaby’s = 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 Felix’s, Gaby’s, 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.

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