Beruflich Dokumente
Kultur Dokumente
FRENDY
00000001642
Group A1-5
Faculty of Medicine
2013
ABSTRACT
Genetics is one of the many important aspects of our life. By understanding it,
will help genetic disease treatment, specific antibiotic production, bioengineering, and
many more beneficial things. This experiment is done to obtain DNA sample from
human blood and determine the presence of the tumor protein gene p53 in the DNA by
comparing it to the database in the National Center for Biotechnology information
(NCBI). Blood sample is first obtained and then undergoes lysis of its components by
chemicals until what remains is only the DNA. The absorbance of the DNA is measured
next using spectrophotometer, followed by its purity and concentration. The presence of
the DNA is also confirmed using the Electrophoresis method. After that, tumor protein
gene p53 of the DNA is amplified during the PCR experiment. The DNA undergoes one
more electrophoresis. Finally, we have to access the database at the NCBI website. Our
DNA sequence will be submitted and the website will search for the DNA that are in
their database for a match. It will also show us how identical it is by percentage.
Although there are some errors during the process of the experiment, we still
managed to acquire the DNA sequence and compare it to the NCBI database. Also, the
result shows that there are DNA sequence in the database that are similar with the
samples we obtained from Frendy and Ivy.
I. INTRODUCTION
DNA or deoxyribonucleic acid, is the hereditary unit in humans and almost all
other organisms, it is what makes an organism so unique. In this experiment, we are
going to find out whether a person possess tumor protein p53 gene or not. (GHR, 2013)
Blood is the red fluid that circulates in our blood vessels to the most remote part
of the body. It mainly functions as the body’s transport system, but is also responsible
for immune responses. (America’s Blood Center, 2012)
White blood cells are clear round cells that are bigger than red
blood cells. White blood cells produce antibodies that help our
bodies fight infections.
The National Center for Biotechnology Information (NCBI) provides users with
DNA sequence database. BLAST stands for Basic Local Alignment Search Tool. We
can submit the algorithm of a sequence (DNA, RNA, Amino Acid Chain). It will then be
compared to the database. In this experiment, we use this to determine if a person have
the tumor protein p53 gene or not. (Davidson, 2008)
II. MATERIALS AND METHODS
All materials and methods that are going to be described in this lab report are
adapted from the laboratory protocol. Some modifications were made.
0.5 mL of the whole blood sample from the EDTA vacutainer is used for the next
step in the experiment, DNA isolation. 0.8 mL of 1X SCC buffer is added and mixed,
followed by centrifugation for 1 minute at 12,000 rpm (all centrifugation uses the same
rpm in DNA isolation step) in a microcentrifuge. This will result in the formation of pellets
on the bottom of the vial and supernatant (Clear Liquid) above it.1 mL of supernatant is
disposed. This step is repeated one more time with 1 ml of 1X SSC buffer. This time, all
of the supernatant is disposed. Then, add 375 µL of 0.2M NaOAc, vortex, then add 25
µL of SDS and 5 µL of Proteinase K, vortex and incubate for 1 hour at 55°C.After that,
120 µL of phenol/chloroform/isoamyl alcohol is added, vortexed for 30 seconds and
centrifuged at 2 minutes. The aqueous layer is removed and transferred into another
microcentrifuge tube, 1mL of cold 100% ethanol is added, mixed, and incubated for 15
minutes at -20°C and then centrifuged for 2 minutes. Supernatant is decanted and
drained.180 µL of 1X TE buffer is added, vortexed and incubated at 55°C for 10
minutes. After that, 20 µL 2 M Sodium acetate and 500 µL cold ethanol are added and
mixed, centrifuged for 1 minute. Decant supernatant, rinse pellet with 1 mL of 70%
ethanol. Centrifuge for 1 minute, decant supernatant, and air dry pellets. Pellets will
finally be re-suspended by addition of 200 µL 1X TE buffer, incubated at 55°C for 30
minutes, vortexing periodically to dissolve genomic DNA, and stored at -20°C.
The next thing to do is to set up the master mix consisting of 12.875 µL dH2O
which is then mixed with: 5 µL 5X buffer PCR, 2 µL 2.5 mM dNTP mix, 1.5 µL 25 mM
MgCl2, 0.125 µL Taq DNA polymerase, 0.75 µL 10 µM forward primer, 0.75 µL 10 µM
reverse primer and 2 µL DNA template, with the final concentration respectively: 1X,
200 µM, 1.5 mM, 0.625 units, 0.3 µM, 0.3 µM, and <250 ng. The volume per reaction
applied is multiplied by four because we need it for four PCR tubes. So, it will ultimately
yield a total of 100 µL. After the mix had been distributed evenly to 4 PCR tubes, the
tubes will put into PCR machine. The machine is set for 95°C (10 minutes) then,
denaturation at 94°C (30 seconds), annealing at 58.6°C (30 seconds), extension at
72°C (1 minutes), final extension at 72°C (10 minutes), and finally storage at 4°C
(indefinite time).
The last experiment is obtaining the DNA sequence of the samples that
have been processed carefully during the previous steps. In order to do that, first we
must turn on the computer and proceed by installing Chromas Lite software. Once
done, open the DNA file that will be used and switch it to forward instead of reverse.
Next, analyze the sequence, N bases should be changed according to the correct color
shown (C=Blue, Black=G, Green=A, Red=T), and save it afterwards. Then copy
sequence in fasta format, go to http://ncbi.nlm.nih.gov/, click “blast menu”, click
nucleotide blast menu, paste it into “enter query sequence area”, choose “human
genomic” database, and end the experiment by clicking “BLAST”.
III. RESULTS
A. Blood Separation
Genomic DNA of
DNA Marker Sample 1 (Ivy)
10000 bp
8000 bp
6000 bp
5000 bp
4000
4000bpbp
3500
3500bpbp
3000 bp
1500 bp
1000 bp
500 bp
Figure 4: Agarose gel electrophoresis of DNA isolated from blood sample
C. Quantitation of DNA Concentration
DNA concentration from Frendy and Ivy’s samples with 1:5 dilution =
mg
Frendy’s sample 1 = 1.829 x 5⁄20 = 0.45725 ⁄mL
mg
Frendy’s sample 2 = 1.941 x 5⁄20 = 0.48525 ⁄mL
mg
Ivy’s sample 1 = 1.982 x 5⁄20 = 0.4955 ⁄mL
To calculate the purity index of a DNA sample, divide the absorption at 260 nm
with the absorption at 280 nm. Equation = [A260⁄A280]. DNAs are considered to be
pure when their purity indexes are in the range of 1.8 – 2.0.
DNA Purity Index from Frendy and Ivy’s samples with 1:5 dilution =
DNA 1000 bp
Marker
900 bp
Control
800 bp
Positive
700 bp
Sample 1
600 bp
Sample 2 500 bp
400 bp
Control
Negative 300 bp
200 bp
± 600 bp
100 bp
A centrifuge is a machine that is utilized for separating heavy materials from light
materials. Centrifuge works by spinning the sample at high speed and uses the principle
of the “centrifugal force”. This force pushes against the bottom of the container causing
particles to clump at the bottom of the container. The solid clump is referred as a pellet,
and the solution above it is referred as the “supernatant”. (Tufts, 2011). The centrifuge
is used to separate blood in this experiment. After centrifugation, blood in a tube
containing anti-coagulant would be separated into 3 layers made up of upper plasma
layer, lower red blood cell layer, and a thin layer called the buffy coat which contains
white blood cells and platelets. But without the anti-coagulant, the result will differ. Blood
will be allowed to coagulate, therefore the final visible layers will be blood clot at the
bottom and clear serum above it. The results described in the reference are similar than
the results that we obtained from the experiment. We conclude that our experiment is
successful. (Rodney A. Rhodes PhD, David R. Bell PhD, 2011)
DNA needs to be isolated from the blood samples obtained from the previous
experiment in order for it to be viewed shortly after using the gel electrophoresis
method. Blood contains many components such as red blood cells, white blood cells,
etc.To only obtain DNA, these components must be removed completely. NaOAc is
involved in the lysis of red blood cells. SDS detergent works by lysing cell membrane
and forming complexes with lipid and proteins resulting them to be able to precipitate
out of the solution. Proteinase K will be used to degrade and denature the proteins
precipitated, followed by PCI alcohol solution to remove non-nucleic acid molecules.
Ethanol is used to precipitate DNA and once the pellet had dried, it will be re-suspended
using Tris EDTA buffer. DNA pellets will be visible. The final results described in the
reference is similar to what we obtained from the experiment. We conclude that our
experiment is successful. (Bruce A. Roe, 2001)
The DNA sample from Frendy 2 (1.857) and Ivy 1 (1.845) are both pure, since
their purity indexes are within the range of 1.8 – 2.0.The DNA sample from Frendy 1
(2.062) is however, not pure, because its purity index is larger than 2.0.This is most
likely cause by contamination. We conclude this experiment to be successful.
(NanoDrop, 2007)
AN electrophoresis is done one more time for the PCR experiment. The first lane
contains DNA marker. The second lane contains the control positive (DNA that is known
to work), which will be visible. The third and fourth lane contains the DNA samples
which shows the formation of bands at approximately 600 bp according to the DNA
marker. The fifth lane contains the negative control (DNA that is known not to work),
which would not be visible. Frendy’s DNA is again, not visible in this electrophoresis,
which could be a result from error when creating the master mix. (UNLV, 2007)
BLAST results confirms that our DNA samples were amplified correctly to the
gene homo sapiens tumor protein p53. The data that are used are obtained from the
database of http://blast.ncbi.nlm.nih.gov/Blast.cgi. Comparison shows that our amplified
DNA sample sequence match the sample in the website’s database. It was 99%
identical with the database. (NCBI, 2013)
From this series of experiments, we found out that there are some errors made
from the experiment, nevertheless we still managed to amplify the DNA and found a
match in the DNA sequence of the database.
V. REFERENCES
Books:
Rodney A. Rhodes PhD, David R. Bell PhD. Medical Physiology: Principles for Clinical
Medicine. 4th ed. North America: Lippincott Williams & Wilkins; 2012.
Websites:
America’s Blood Centers. What is blood. (Cited on 2013 Nov 9) Available from: URL:
http://learn.genetics.utah.edu/content/begin/traits/blood/blood.html
Cold Spring Harbor Protocols. Quantitation of DNA and RNA. (Cited on 2013 Nov 9)
Available from: URL: http://cshprotocols.cshlp.org/content/2007/11/pdb.ip47.full
Davidson. How to Use NCBI Blast. (Cited on 2013 Nov 9) Available from: URL:
http://www.bio.davidson.edu/courses/genomics/2008/simpson/tutorial.html
Genetics home reference. DNA. (Cited on 2013 Nov 9) Available from: URL:
http://ghr.nlm.nih.gov/handbook/basics/dna
NCBI. Reagents for functional genomics. (Cited on 2013 Nov 9) Available from: URL:
http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml
The University of Queensland. Agarose Gel Electrophoresis for DNA. (Cited on 2013
Nov 9) Available from: URL: http://www.di.uq.edu.au/sparqdnaelectrophoresis
University of Nevada, Las Vegas. PCR Lab. (Cited on 2013 Nov 9) Available from: URL:
http://faculty.unlv.edu/wmojica/PCR_LAB2.htm