FUNDAMENTAL MEDICAL SCIENCE I

FINAL REPORT (GENOMIC)

DEVINA
07120110064
GROUP A1-3

Universitas Pelita Harapan

Mochtar Riady Institute for Nanotechnology
Faculty of Medicine
2011

ABSTRACT

DNA, or deoxyribonucleic acid, is the hereditary material in humans and almost

all other organisms. An important property of DNA is that it can replicate, or make
copies of itself. Each strand of DNA in the double helix can serve as a pattern for
duplicating the sequence of bases. This is critical when cells divide because each new
cell needs to have an exact copy of the DNA present in the old cell. In this experiment
we want to replicating the DNA to knew it sequencing by taking white blood cells first
which contain DNA because it has nucleus, isolating DNA to not being contaminated by
other materials, counting the purity of the DNA which the index should be between 1.72.0, replicating DNA by using PCR and looking for DNA sequence using BLAST in NCBI
web to comparing with the sequences from NCBI database. The DNA extract which has
been taken should not be less or more. Because if it was less then it wont be enough
for this experiment. On the other hand, to much DNA extract will make it waste. So in
this experiment, accuracy is one of the most important things between the precision and
the thoroughness.

I. INTRODUCTION

Blood is a liquid tissue. Suspended in the watery plasma are seven types of cells
and cell fragments.
Red blood cells (RBCs) or erythrocytes, platelets or thrombocytes, five kinds of white
blood cells (WBCs) or leukocytes. Three kinds of granulocytes which are neutrophils,
eosinophils, and basophils. Two kinds of leukocytes without granules in their cytoplasm
are lymphocytes and monocytes (2011).
DNA is material that governs inheritance of eye color, hair color, stature, bone
density and many other human and animal traits. DNA is a long, but narrow string-like

object. A one foot long string or strand of DNA is normally packed into a space roughly
equal to a cube 1/millionth of an inch on a side. This is possible only because DNA is a
very thin string.
A strand of DNA is made up of tiny building-blocks. There are only four, different
basic building-blocks. Scientists usually refer to these using four letters, A, T, G, and
C. These four letters are short nicknames for more complicated building-block chemical
names, but actually the letters (A,T, G and C) are used much more commonly than the
chemical names so the latter will not be mentioned here. Another term for DNA's
building blocks is the term, "bases." A, T, G and C are bases (Donald E. Riley, Ph.D.,
2005).
PCR (short for Polymerase Chain Reaction) is a relatively simple and
inexpensive tool that you can use to focus in on a segment of DNA and copy it billions of
times over. PCR is used every day to diagnose diseases, identify bacteria and viruses,
match criminals to crime scenes, and in many other ways (Kevin Taylor, 2011). Kary
Mullis was the founder of this PCR technique. PCR less than 1 mm can made a
hundred billion identical copy within two hours and it was doing in in vitro.
One of the most common methods for nucleic acid detection is the measurement
of solution absorbance at 260 nm (A260) due to the fact that nucleic acids have an
absorption maximum at this UV wavelength. Although a relatively simple and timehonored method, A260 suffers from low sensitivity and interference from nucleotides
and single-stranded nucleic acids. Furthermore, compounds commonly used in the
preparation of nucleic acids absorb at 260 nm leading to abnormally high quantitation
levels(1). However, these interference and preparation compounds also absorb at 280
nm leading to the calculation of DNA purity by performing ratio absorbance
measurements at A260/ A280. A260/A280 = 1.7 to 2.0 for pure DNA (Dr. Thomas
Raebiger, 2011).
Gel electrophoresis can be used to separate DNA fragments. Electrophoresis
uses an electric current to separate different-sized molecules, in a porous, sponge-like
matrix. Smaller molecules move easily through the gel pores than large molecules.
While at Cold Spring Harbour Laboratory, Phil Sharp, Joe Sambrook, and Bill Sugden
develop the DNA electrophoresis technique using an agrose gel, made from highly

purified seaweed. This could be used to separate DNA molecules ranging from several
hundred nucleotides in length to over 10,000 nucleotides (CSH).
In the late 1970's, two DNA sequencing techniques for longer DNA molecules
were invented. These were the Sanger (or dideoxy) method and the Maxam-Gilbert
(chemical cleavage) method. The Maxam-Gilbert method is based on nucleotide-specfic
cleavage by chemicals and is best used to sequence oligonucleotides (short nucleotide
polymers, usually smaller than 50 base-pairs in length). The Sanger method is more
commonly used because it has been proven technically easier to apply, and, with the
advent of PCR and automation of the technique, is easily applied to long strands of DNA
including some entire genes. This technique is based on chain termination by dideoxy
nucleotides during PCR elongation reactions (Theresa Phillips, 2011).
The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity
between sequences. The program compares nucleotide or protein sequences to
sequence databases and calculates the statistical significance of matches. BLAST can
be used to infer functional and evolutionary relationships between sequences as well as
help identify members of gene families.

II. MATERIALS AND METHODS

Blood sample were drawn from the appointed student which is myself (Devina)
using vacutainer coated with K3EDTA and empty vacutainer. Each of the four vial
properly was labeled and then one ml of whole blood was transferred into separated
vial. The remaining whole bloods in each sample were centrifuged at 3700 rpm using
Alegra X15 centrifuge, 20o C, for 10 minutes. the serum was transferred into a new vial
and stored at -80o C.
10X TE buffer was made by adding 1.21 g/L Tris (100mM Tris), 0.292 g/L EDTA
(10mM EDTA), and dH2O up to 70ml. pH was adjusted to 8.0 and dH 2O against added
until 100ml.

Blood sample typically were obtained as 0.7 ml of whole blood stored in EDTA
vacutainer tubes, which was frozen later at -70 o C. 0.8 ml 1X SSC buffer was added and
mixed. Then, was centrifuged for 1 minute at 12,000 rpm in a microcentrifuge. 1 ml of
supernatant was removed and discarded into disinfectant. 1X SSC buffer was added,
vortexed and then centrifuged for 1 minute at 12,000 rpm in a microcentrifuge.
Supernatant need to be removed.
375 ul of 2.0 M NaOAc was added to each pellet and vortexed. Then 25 ul of
10% SDS and 5 ul of proteinase K (10mg/ml H2O) also added and need to vortex briefly
and incubate for 1 hour at 55o C. Next step is 120 ul phenol was added and vortexed
for 30 second, centrifuged the sample for 2 minutes at 12,000 rpm in microcentrifuge,
descanted the supernatant and drained.
180 ul 1X TE buffer was added, vortexed, and incubated at 55 oC . 20 ul 2M
sodium acetate was added and mixed. 500 ul of cold 100% ethanol was added to the
next step and mixed. All of the materials was centrifuged for 1 minute at 12,000 rpm in
a microcentrifuge and the supernatant need to be descanted, the pellet rinsed with 1 ml
of 70% ethanol.
The sample was centrifuged again for 1 minute at 12,000 rpm in a
microcentrifuge, supernatant descanted, and air dry the pellet 10 minutes. The pellet
was resuspend by adding 200 ul of 1X TE buffer, incubated at 55 oC for 30 minutes,
periodically vortexed to dissolve the genomic DNA and stored the samples at -20 oC.
The DNA sample was diluted and filled in thte cuvette with 50 ul of 1X TE buffer
which was setted on the cuvette holder of spectrophotometer.
We used dsDNA which represented the concentration of a solution with an A260 of
1.000. we blanked the spectrometer with 1x TE buffer in inserted the cuvette with DNA
sample to read the absorbance. And then we recorded to make sure our absorbance fall
between 0.1 to 1.0 for accurate concentration measurement.
NanoDrop Software were used to detect DNA and nucleic acid was selected.
1.5uL dH2O was placed on lower pedestal then the sampling arm closed. The system
was turn on by clicked OK button. After that, upper and lower pedestal were wiped with
soft lab wipes. On the lower pedestal, 1.5 uL 1X TE buffer was placed and sampling
arm closed. At those time, BLANK button which chosen to be clicked. And the upper

and lower pedestal were wiped again before 1.5 uL of sample were placed on lower
pedestal. Sampling arm wea closed, MEASURE button was clicked and the result was
ready to read and being recorded.
In the PCR tube, we setted up a reaction mixture including 10.875 dH 2O, 5 5X
buffer PCR, 2 2.5mM dNTP mix, 1.5 25mM MgCl2, 0.125 taq DNA polymerase, 0.75 10
miuM forward primer, 0.75 10miuM reverse primer, and 4 DNA template. The total
volume was 25 miuL. Then we putted the reaction mixture in the PCR machine, and set
the program 95oC, 10 minutes. Denaturation 94oC, 30 seconds, Annealing 58.6oC, 30
seconds, and extension 72oC, 1 minute for step 2. The cycled was setted for 40 times.
On step 3, 72oC, 10 minutes and storage the PCR product at 4 oC.
For DNA sequencing we used Chromas Lite program and file DNA sequence
fasta format. If DNA sequence had reverse direction, it switched to forward direction.
DNA sequence was analyzed to changed if there were found N base along DNA
sequence. After editing, we saved it and copied sequence in fasta format. In NCBI home
page, BLAST menu was clicked and after that Nucleotide blast menu was chosen as
well. Edited DMA was pasted into Enter Query Sequence Area. Our DNA was from
human genome so it must be Human genomic database which being chosen. For the
result, BLAST need to be clicked.

III. RESULT

A. Blood Separation
Plain Vial

EDTA Vial (with anticoagulant)

B. DNA Isolation and DNA Electrophoresis

From left to right : 1 and 15 = leader

2 to 14 = Devinas DNA sample

C. Quantitation of DNA Concentration

DNA concentration (mg/mL) = (OD :

) X dilution factor

Purity index = A260 / A280

Dna is considered pure if the purity is 1.80 2.00
A131

A131

Average A131

A230 0.039

A230 -0.004

A230 0.0175

A260 0.098

A260 0.084

A260 0.091

A280 0.063

A280 0.038

A280 0.0505

A132

A132

Average A132

A230 0.152

A230 0.092

A230 0.122

A260 0.105

A260 0.066

A260 0.0855

A280 0.078

A280 0.038

A280 0.058

A131
A260/A280 = 1.47
A260/A230 = 0.70

[DNA] =

0.0855
20

X 1000 = 4.275

A132
A260/A280 = 1.80
A260/A230 = 5.20

[DNA] =

0.091
20

X 1000 = 4.5

D. PCR

From left to right : 1 and 6 = marker

2 = negative control
3 = positive control
4 and 5 = Devinas DNA sample

E. DNA Sequencing

V. DISSCUSION

Blood separation experiment result was appropriate with the references. There
are plasma on the upper layer, which is contained 55% of whole blood, buffy coat which
contained white blood cells and platelets in the middle, which is used in the experiment
because it is the only part of blood which contained nucleus. DNA is the sample we
need to know it sequence, and it is in nucleus which is in the white blood cells, and
there are red blood cells on the bottom appeared in our experiment. In plain vial, there
are no anti-coagulant being added so after it was centrifuged, it was coagulated. But in
the vial whch has been added TRIS-EDTA buffer the anti-coagulant, the sample was not
coagulated. The plasma have been taken also to be used in proteotomic which
analyzed protein in blood. But in this experiment, we take buffy coat because we want
to know about quantitation of DNA concentration by isolating DNA first, DNA
electrophoresis, PCR, and sequence DNA.
On the DNA isolation and gel electrophoresis, it can be seen at the result that the
image of gel electrophoresis from DNA isolation experiment has moving. It was because
the phosphate groups in the DNA backbone, carry negatively-charged oxygens, giving a
DNA molecules an overall negative charge. In an electric current, the negativelycharged DNA moves towards the positive pole og the electrophoresis chamber. The
DNA molecules move through the gel by reptation, a reptile like snaking action through
the the pores of the agarose matrix. Smaller DNA fragments migrate faster and further
over a given period of time than do larger fragments. This is how DNA fragments can be
separated by size in agarose gel.
After doing the DNA isolation and tested DNA electrophoresis, we were checking
the quantitation of DNA concentration to know how pure our DNA which have been
isolated. Based on the references, DNA purity by performing ratio absorbance
measurements at A260/ A280 which the index 1.7 2.0 is counted as pure DNA. But in
our experiment, result showed the index of A131 sample was 4.275 and from sample
A132 was 4.55. In this case, we assume that our DNA has been contaminated with the
other ingredients.

In PCR experiment, there are three steps which are denaturation, hydrogen bond
broken and strand of DNA also separated. In step two which is annealing, allow the
primers to bind again, and on step three which is extension, replication was started.
Negative control was used in PCR experiment to checked wether something amplify
which mean there are contamination of reagents because it has no template DNA. It
was also to indicates that the bands in the other samples are likely contaminated as
well. Positive controls are needed for the verification of negative amplification results
and the positive control reaction should contain the same components as the sample
but include a template that is certain to amplify if the reaction goes as planned.
Our last experiment is to sequencing DNA. In our DNA sequences we had obtain
from BLAST result, the beginning part of sequence DNA was undetectable. Its only
appear N, which mean, the nitrogen base of the DNA cant be read because it was
contaminated or degradable. It was also appeared in the last scene of sequence DNA.
But, sequence DNA in the middle we took to be BLAST-ed, was good and only few of
N alphabet appeared but it soon repaired. As the result as we had input, the gene of
DNA has no mutation and a hundred percent same with the NCBI database of human
nucleotide.

V. REFERENCES

Book:
Sambrook, J., Fritsch, E.F. and Manniatis, T. Molecular Cloning. A Laboratory Manual.
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbour; 1989.
Steffan, R.J., Goksoyr, J., Bej, A.K. and Atlas, R. Recovery of DNA from Soils and
Sediments. Appl. Environ. Microbiol; 1988.

Websites:
Biology Pages. Blood. 2011 September 24; Available from: URL:
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html#neutrophils/08_No
vember_11.
Kevin Taylor. Virtual PCR. Learn Genetics. 2011; Available from : URL:
http://learn.genetics.utah.edu/content/labs/pcr//2011_November_09

Cold Spring Harbour Laboratory. Biology Animation Library. DNA Learning Center.
Available from: URL:
http://www.dnalc.org/resources/animations/gelelectrophoresis.html/2011_November_09.
Therese Phillips. DNA Sequncing Method. Biotech/ BioMed. 2011. Available from: URL:
http://biotech.about.com/od/pcr/a/sequencing.htm/_2011_November_09.
Nick. Tech Tips. BitesizeBio. 2010 June 01. Available from: URL:
http://bitesizebio.com/articles/the-pcr-controls-you-must-use//2011_November_10.
Journal:
Donald E. Riley, Ph.D. DNA Testing: An Introduction For Non-Scientists An Illustrated
Explanation. Scientific Testimony. 2005 April 06.