ANGELINE TANCHERLA
01071170034
GROUP A2-2
Genes are found in living organisms and are passed on to their offspring.
They are in charge of producing proteins. Proteins are needed by the body in order
to work normally. Genes are also responsible for various traits in our body. Genes
have been studied by researchers to identify similarities or differences that may be
associated with diseases. Application of technology to analyze genes has been
beneficial for human health and medical knowledge.
I. INTRODUCTION
After DNA is isolated, it is important to find out the concentration and purity of
DNA, which are important factors for a good genetic analysis. DNA concentration
and purity can be quantified using their absorbance. The relationship between
absorbance and concentration of DNA is shown in Beer-Lambert Law [5]. It shows
that: Absorbance (A) = absorption coefficient (ε) x concentration (c) x length of light
path (l). Therefore, concentration can also be expressed as C = (A ÷ ε) x dilution
factor. The purity of DNA can obtained through A260 ÷ A280 or through A260 ÷ A230.
DNA can be considered pure if the purity ranges from 1.80-2.00 for A260/A280 or 2.00-
2.20 for A260/A230.
In order to gain enough desired DNA but also to minimize the wasting of
excess DNA samples, PCR is conducted. PCR (Polymerase Chain Reaction) is an
easy way to amplify a small amount of DNA. Firstly, the DNA sample is denaturated
to form single stranded DNA (template DNA). Next, the short and specific DNA
sequences (oligonucleotide primers) are annealed with DNA template. New
nucleotide bases are added to end of primer with the help of Taq DNA polymerase
enzyme (thermostable), which produces new double stranded DNA. This process is
repeated for numerous cycles to obtain desired amount of DNA strands copies [7].
To identify the type of gene, we need to know the DNA sequence. Sanger or
dideoxy sequencing is a sequencing method in which target DNA undergoes
denaturation, annealing to an oligonucleotide primer, and extension by DNA
polymerase [8]. The process will be stopped using dideoxynucleotide triphosphates
(ddNTPs), which does not have 3' OH group. Each of the four ddNTPs shows
different colors. ddATP is green, ddGTP is yellow, ddCTP is blue and ddTTP is red.
The computer will read the sequence (from smallest to largest fragment) and
interprets the colors. Cromas Lite software will be used to edit the sequence for
BLAST.
The stored blood samples were then used for DNA isolation. Firstly, 0.8 ml 1X
SSC buffer were mixed in the samples. The samples were centrifuged for 1 minute,
at 12,000 rpm in a microcentrifuge. One mL of supernatant was removed and 1 ml of
1X SSC buffer was added into samples. The samples were then vortexed and
centrifuged at 12,000 rpm in a microcentrifuge for 1 minute. All of the supernatant
were removed. Next, 375 μl of 0.2M NaOAc were added to each pellet, which is then
vortexed. Then, 25 μl of 10% SDS and 5 μl of proteinase K were added and the
samples were vortexed and incubated at 55oC for 30 minutes. Phenol/chloroform/
isoamyl alcohol (120 μl) were added and vortexed for 30 seconds. The samples
were centrifuged at 12,000 rpm in a microcentrifuge for 2 minutes. The aqueous
layer was removed carefully to a 1.5 mL microcentrifuge tube and 1 ml of cold 100%
ethanol were mixed. They were centrifuged at 12,000 rpm in a microcentrifuge for 2
minutes. The aqueous layer was moved to a new 1.5 ml microcentrifuge tube and 1
ml of 100% ethanol was mixed. The supernatant was decanted and 180 μl 1X TE
buffer was mixed. Then, 20 μl of 2 M sodium acetate and 500 μl of cold 100%
ethanol were mixed in the samples and they were centrifuged at 12,000 rpm in a
microcentrifuge for 1 minute. The supernatant was decanted and the pellet was
rinsed with 1 ml of 70% ethanol. The pellet was centrifuged at 12,000 rpm in a
microcentrifuge for 1 minute. The supernatant was decanted and the pellet was
dried. The pellet was resuspended by addition of 200 μl of 1X TE buffer. Lastly, the
samples were incubated at 55oC for 30 minutes and were stored at -20oC.
To calculate DNA concentration, DNA sample was diluted 1:5 (but C2 sample
was then changed to 1:2 as instructed). Next, 50 μl 1X TE buffer was added to the
cuvette, which was then set on the holder of spectrophotometer. The type of nucleic
acid was set as dsDNA and the conversion factor was modified. The spectrophoto-
meter was then blanked with 1x TE buffer. The cuvette was inserted to read the
absorbance (50 μl final volume). The absorbance at 230, 260 and 280nm were read
and recorded to calculate purity and concentration.
A. Blood Separation
DM A2 C2
>10000 bp
DNA Ladder
A2 Sample
First measurement Second Measurement Mean
(1:5)*
A230 0.032 0.03 0.031
A260 0.178 0.183 0.1805
A280 0.112 0.116 0.114
C2 Sample
First measurement Second Measurement Mean
(1:2)**
A230 -0.030 -0.032 0
A260 0.154 0.151 0.1525
A280 0.106 0.107 0.1065
* Dilution
** Dilution of C2 was changed from 1:5 to 1:2 as instructed
A2 Sample
Quantitation Result***
Purity Index
A260 0.1805
P1 = = 𝟓. 𝟖𝟐𝟑 > 2.2 RNA Contamination
A230 0.031
A260 0.1805
P2 = = 𝟏. 𝟓𝟖𝟑 < 1.8 Protein Contamination
A280 0.114
C2 Sample
Quantitation Result***
Purity Index
A260 0.1525
P1 = =∞ > 2.2 RNA Contamination
A230 0
A260 0.1525
P2 = = 𝟏. 𝟒𝟑𝟐 < 1.8 Protein Contamination
A280 0.1065
DM A2 C2 (+) (-)
DNA Ladder
Description
Lane A2 : A2 DNA Sample Lane (+) : Positive Control
Lane C2 : C2 DNA Sample Lane (- ) : Negative Control
Lane DM : DNA Marker
E. DNA Sequencing
IV. DISCUSSION
The result of the blood separation experiment shows that there are different
layers formed after centrifugation. The layers are developed due to the different
densities of blood contents. After centrifugation, heavier blood components will sink
to the bottom of the tube, while lighter components will appear on the upper layer.
For blood sample in EDTA vacutainer, three layers are formed. They are
plasma (yellowish and transparent), buffy coat (thin and white) and RBC layer. Buffy
coat is made up of WBCs and platelets. The appearance of buffy coat is due to the
anticoagulant in EDTA tube that prevents WBCs and RBCs from clotting together.
EDTA vacutainer contains anticoagulant that maintains blood in the fluid state. Since
the empty vacutainer has no anticoagulant, blood components (such as RBCs,
WBCs and platelets) will clot together and forms only two layers, which are serum
(yellowish transparent layer without coagulating factor) and blood clots. From the
results above, we can conclude that our experiment results match with the
references. Blood is composed of various components with different sizes and
functions.
Through DNA electrophoresis, we can analyze the size of our DNA samples.
By comparing the samples with the DNA marker (see Figure 3), we can conclude
that our DNA samples have a size of more than 10.000 bp. However, the band
shown in the picture is very thin and unclear. The thickness of the band is associated
with DNA concentration. Our DNA samples have low concentration, which results to
the appearance of thinner bands. In the previous experiment, we found out that A2
sample has greater concentration than C2 sample. In electrophoresis, this
relationship is shown, wherein A2 sample has a band, which is slightly more visible
than C2 sample.
In the last experiment, we blast our DNA sequence in the NCBI website. The
blast result shows that our sample is the most similar with the Homo sapiens
transforming growth factor beta receptor 2 (TGFBR2). The graph in the alignment
scores section (Figure 7) shows alignments of our sample (purple colored lines) that
matches to the database (red lines). From the graph, we can know that our sample is
an amplified DNA fragment, because the purple line (our sample) is quite short,
compared to the long red line (sequence in database). The alignment score is
approximately 80 and is labeled as purple color. The more the length of a sequence
matches the database, the greater the alignment score is. In Figure 9, we see that
our sequence has identities of 80/81 matches, which is 99% similar. This means that
our DNA sequence is very similar to TGFBR2 gene in the database. Meanwhile, the
single different identity may be due to error in editing the sequence using Cromas
Lite.
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