FUNDAMENTAL MEDICAL SCIENCE I

FINAL REPORT (GENOMIC)

CINDY CLARISSA THANDY

01071180028
GROUP A6-1

Universitas Pelita Harapan

Mochtar Riady Institute for Nanotechnology
Faculty of Medicine
2018
ABSTRACT

Genetic study has been one important enquiry in

our lives as understanding of genomics could take us to
its application in the medicine world. Over the years,
technologies genetic analyzation have been rapidly
developing. Genomes containing the entire hereditary
information that builds and maintains an organism can be
isolated using technology to be further studied of certain
possible disease developments. Possible mutations may
be examined then finding cures are the next steps.
The human genome project was started by blood
separation from whole blood followed by DNA isolation.
The isolated DNA was then determined its concentration,
purity from phenol, protein, and RNA contaminations by
measuring its absorbance with spectrophotometer
BIORAD, as well as its size through electrophoresis using
agarose gel read by Versa Doc Instrument. Amplification
of the desired gene was done through Polymerase Chain
Reaction (PCR) and sequencing by Sanger method.
Chromas Lite software was used to examine the order of
the nucleotide sequence and add some missing ones.
The resulted sequence was then compared to NCBI
database to find out the gene drawn from our sample.
The experiment was a success in detecting
ERCC2 gene from DNA. Electrophoresis bands were
clear showing appropriate concentration. Sizes of DNA
isolation showed more than 10000 base pairs. One of the
samples experience phenol and RNA contamination while
the other was only RNA contaminated. Sample DNA
showed 99% similarities of Homo sapien ERCC excision
repair 2, TFIIH core complex helicase subunit (ERCC2)
transcript variant 3, mRNA.
I. INTRODUCTION (350-450)

Development of genetic technology can be used to

identify specific genes like ERCC2 that codes a protein
called XPD, basic of gathering proteins, the general
interpretation factor IIH (TFIIH) complex which is
associated with translation and helps fix harmed DNA.
Blood is liquid existing as tissues as it vary with
functions. 55% composed of plasma, 45% erythrocytes
(RBC), less than 1% buffy coat containing leukocytes
(WBC), and rest are thrombocytes (platelets) (Rogers,
2011). The genetic material to be passed onto offsprings,
which is DNA (deoxyribonucleic acid) contained in
nucleus exists in the leukocytes.
To isolate the DNA, centrifugation is done to
separate blood based on their density. With
anticoagulant, non-coagulated blood separated as the
bottom contained the most dense (RBC) and top
contained the less dense (plasma), separated by buffy
coat. Without anticoagulant, the coagulated blood
separated to serum on the top, which has same
components as plasma except for coagulant factors and
clotted blood cells containing erythrocytes, leukocytes,
and thrombocytes at the bottom (Dean,2004). Whole
blood is used to obtain the DNA with several chemicals to
discard other components: proteinase K to degrade
proteins, ethanol to precipitate the DNA, SDS detergent
to precipitate lipid, proteins also to lyse the cell
membrane, PCI (phenol, chloroform, isoamyl alcohol)
solution to remove most non-nucleic acid organic
molecules, and Tris EDTA (TE) buffer to resuspend DNA.
(Schottenfeld & Fraumeni, 2006)
 Two ways to confirm DNA isolation success. First,
analyzing concentration and purity of DNA. Absorbance is
measured at 260 nm, 230 nm, and 280 nm using
spectrophotometer. DNA concentration was calculated
with formula according to Lambert-Beer’s law: light
absorbed in a sample is proportional to the concentration.
[DNA concentration (mg/mL) = (OD/ ε) x dilution factor.
The absorbance value at A260/A280 is to examine
protein contamination, the results must be between
1.8-2.0 and A260/A230 to examine phenol contamination,
the results between 2.0-2.2 to be categorized pure
NanoDrop, 2007). Second, applying DNA for
electrophoresis, to separate DNA fragments due to their
sizes using electric charge from negative to positive
electrodes. Negative charge of DNA from a detached
phosphate group. DNA sample mixed with loading dye
makes visual monitoring of migration in gel possible.
According to Surzycki, 2003, exposure to UV light emits
light showing bands in agarose gel.
DNA isolation results small amount. For further
analyzation, PCR is needed for amplification in
exponential manner. PCR has 4 steps: pre denaturation,
denaturation, primer annealing, and elongation. As PCR
mimics DNA replication, polymerase is essential, so Taq
DNA polymerase was used because it’s heat resistant,
unaffected to high temperature during denaturation.
DNA sequencing is to determine nucleotide order
of a given DNA fragment through Sanger method by
dideoxynucleotide to stop elongation. Chromas Lite
program used to edit DNA sequence to be compared to
gene NCBI database. ERCC2 gene resemblance was
expected.
II. MATERIALS AND METHODS (300-400)

During blood separation, samples were drawn

from two individuals (Edgard and Ririn), each were
placed in two labeled vacutainers, one K3EDTA-coated
(anticoagulant) and normal vacutainer. 800 µL of whole
blood samples were transferred into separated vials and
remaining whole blood was centrifuged at 3000rpm, 20 ̊ C,
for 10 minutes. The plasma (transparent liquid) was
moved to new vials, stored at -80 ̊ C.
0.5 µL of whole blood in EDTA vacutainers were
samples for DNA isolation and each was added by 0.8 µL
of 1xSSC buffer, mixed, and centrifuged for 1 minute at
12000rpm. 1mL supernatant were discarded, and another
1mL 1xSSC buffer were added, vortexed. Solution was
centrifuged at 12000rpm for 1minute and all supernatant
were removed. 375 µL of 0.2 M NaOAc were added to
each pellet and vortexed. 25µL of 10% SDS and 5µl of
proteinase K were added and briefly vortexed, then
incubated for 30 minutes at 55 ̊ C. 120µL PCI were added
and vortexed for 30 seconds. Samples were centrifuged
at 12000rpm for 2 minutes. Aqueous layers were
removed to new 1.5 mL micro-centrifuge tubes, 1mL of
cold 100% ethanol was added, mixed, and centrifuged at
12000rpm for 2 minutes. Supernatant were gradually
poured and drained.180 µL 1X TE buffer was added, and
vortexed. 20 µL 2M sodium acetate was added, mixed,
and 500 µL of cold 100% ethanol was added, mixed too,
then centrifuged at 12000rpm for1 minute. Supernatant
was decanted, pellet rinsed with 1mL of 70% ethanol, and
centrifuged for 1 minute at 12000rpm. Supernatant
decanted, and pellet was air-dried until dry. 200µL of
1xTE buffer was added for resuspension, incubated at 55
̊ C for 30 minutes with periodical vortexing to dissolve the
genomic DNA, then stored at -20 ̊ C.
For dilution, cuvettes were filled with 50µLof 1xTE
buffer, set on their holders in spectrophotometer. Nucleic
acid dsDNA type was selected and conversion factors
were determined. The spectrophotometer was blanked by
the 1x TE buffer. Then DNA samples were diluted 1:10
(5µL DNA 45µL water) and was inserted into the cuvettes
for absorbance reading. Concentrations and purities were
calculated using absorbances at 230nm, 260nm, and
280nm.
For DNA electrophoresis, agarose 1% gel was
prepared by mixing 0.5gr of agarose in 50mL TAE buffer
in an erlenmeyer flask and boiled in the microwave. 1µL
of ethidium bromide was added when the temperature
reached 60 ̊ C. The agarose were poured in gel tray, comb
were set, and was left 30 minutes in room-temperature to
let gel harden. Electrophoresis chamber were prepared
and agarose gel was loaded. 3µL of each DNA sample
were added 2µL loading dye and mixed by pipetting on
parafilm sheet. Wells loaded with mixtures with 2µl of
DNA marker and filled with TAE buffer until 1mm above
gel surface. Electrophoresis was run at 100V, 6W, 0.4A
for 30 minutes. Gel was taken out, washed under tap
water. Versa Doc instrument was used to save the
picture.
In PCR, reaction mixture was made containing
12.375µL dH2O, 5µL 1xBuffer PCR, 0.5µL 2.5mM dNTP
mix resulting 10µM concentration, 1.5µL 25mM MgCl2
resulting 1.5µM concentration, 0.125µL Taq DNA
polymerase resulting 0.652 concentration, 0.75µL each of
10µM forward and reverse primers resulting 0.3µM, and
equally divided to 4 vials. 4µL of DNA template for each
sample and controls were loaded into respective vials
resulting 25µL each tube and put to PCR machine set as
followed: Step 1 at 95 ̊ C, 10 minutes, step 2 of 40 cycles
with denaturation (95 ̊ C, 30 seconds), annealing (60 ̊ C,
30 seconds), and extension (72 ̊ C, 1 minute), step 3 final
extension (72 ̊ C, 7 minutes), and storage (4 ̊ C for
indefinite time) for keeping the PCR product until further
study.
In DNA sequencing, DNA sequence file was
opened using Chromes Lite and edited by changing N
bases to appropriate bases according to color, and
copied in FASTA format. Data was compared with NCBI
database using BLAST. BLAST was chosen then
nucleotide blast menu. Copied sequence was pasted into
the Enter Query Sequence area, search set was human
genomic database, then BLAST was clicked and results
were documented.
III. RESULTS
A. Blood Separation

Figure 1. Blood in vacutainer coated with anticoagulant (K3EDTA) after centrifugation

Figure 2. Blood in vacutainer not coated with anticoagulant factor after centrifugation

B. DNA Isolation and DNA Electrophoresis

Note
Above
10000bp 1 2 3 4 5 6 7 8 9 10 11 12
Lane 1
10000 bp DNA ladder
8000 bp Lane 2
6000 bp Sample EA61
5000 bp Lane 3
4000 bp Sample RA61
Lane 4
Sample EA62
Lane 5
Sample RA62
Lane 6
DNA ladder

Figure 3. 1%Agarose gel electrophoresis of DNA isolation from whole blood

C. Quantitation of DNA Concentration

Table 1. Absorbance and DNA purity calculation

DNA DNA Absorbance DNA Purity

Sample A230 A260 A280 A260/A230 A260/A280

R-A61 0 0.087 0.033 0.090

0.0365
0 0.093 0.040 0
average 0 0.090 0.0365 = 2.466

E-A61 0 0.087 0.033 0.432 0.432

0.035 0.2165
0 0.093 0.040
average 0.035 0.432 0.2165 = 12.343 = 1.995

Formula for DNA purity index to check phenol or RNA

contamination : A260/A230
Range : 2.0-2.2 (<2.0 is organic compounds
contamination, >2.2 is RNA contamination)

Formula for DNA purity index to check protein or RNA

contamination : A260/A280
Range :1.8-2.0 (<1.8 is protein
contamination, >2.0 is RNA contamination)

Formula for calculating DNA concentration:

DNA Concentration (mg/mL) = (Optical Density/ ε).dilution
factor
Note
optical density :absorbance when
spectrophotometer has path length of 1 cm
ε : extinction coefficient
(dsDNA = 20 g-1cm-1L for A260 = 1)


dilution factor :5

Sample R-A61
DNA Concentration (mg/mL) = (0.090 / 20) x 5 = 0.0225
mg/mL = 22.50µg/mL
Sample E-A61
DNA Concentration (mg/mL) = (0.432 / 20) x 5 = 0.108
mg/mL = 108µg/mL

D. PCR

Note
Lane 1
DNA ladder
Lane 2
1 2 3 4 5 6 7 8 9 10 Sample EA61
Lane 3
1500bp Sample RA61
1200bp Lane 4
1000bp Positive control
900bp Lane 5
800bp Negative control
700bp Lane 6
600bp DNA ladder
500bp Lane 7-10
350bp Other subgroup
approximately samples
300bp 330bp

Figure 4. 2%Agarose gel electrophoresis after PCR

E. DNA Sequencing

Figure 5. Color key alignment scores- Graphic summary

Figure 6. Result of DNA sequencing, showing match with

Homo sapien ERCC2 transcript variant 3, mRNA.

Figure 7. The nitrogen bases comparison of sample with

NCBI data base
IV. DISCUSSION (550-650)

In blood separation, the first tube coated with

anticoagulant factor resulted 3 layers: plasma at top with
highest quantity and least density containing hormones,
proteins, antibodies, vitamins, and minerals, RBC at
bottom with highest density, separated by thin layer of
buffy coat containing leukocytes. The thrombocyte
reacting with EDTA that binds with Ca results no blood
coagulant. Second tube without anticoagulant factor
resulted 2 layers: serum and blood clot. Serum does not
contain coagulant factor as plasma does. Results were
correct based on theories (Roger’s book).
DNA isolation was done from whole blood and
several chemicals to isolate the DNA. Sodium acetate is
added to instabilize environment for SSC buffer that
protects WBC, resulting quantity of salt higher in
environment than in the cell causing WBC to lysis. 10%
SDS is added as detergent that binds with lipid of WBC
cell membrane causing it to lysis and expose its content.
Proteinase K is used to degrade protein from WBC’s
content that is exposed before so pure DNA can be
gained. Phenol is a non polar that binds with lipid and
proteins so that the organic phase (phenol, chloroform,
and proteins) can be removed.
Electrophoresis results bands of sample compared
to DNA marker in 1% agarose gel. Figure 3 lane 2 and 3
showed result of subgroup 1, Edgard’s and Ririn’s DNA
respectively. Thicker banding showed more concentrated
DNA which Ririn’s was more thick than Edgard’s. This
difference may be caused by human error of micro-
pipetting DNA making one has fewer than the other or
difference in chemicals mentioned above affecting
amount of DNA isolated. However, both DNA sample
yielded decent amount proving minor errors. DNA marker
showed 10000 base pairs in highest band which the
samples positioned above it, concluding the size of
complete genome samples are above 10000 bp.
The normal range of A260/A280 for checkin protein
contamination must be between 1.8-2.0 and A260/A230
for checking phenol contamination must lie between
2.0-2.2 to be considered pure. Absorbance at A260/A230,
both Edgard’s (12.343) and Ririn’s (0) DNA sample was
not within the range, which showed RNA contamination
for Edgard’s and phenol contamination for Ririn’s (range
2.0-2.2). Phenol contamination may be from errors
addition of it during DNA isolation and imperfect
supernatant decanting. Absorbance at A260/A280, Ririn’s
(2.466) sample was more than range, which showed RNA
contamination, and Edgard’s (1.995) still within range
(1.8-2.0) showing purity from protein contamination.
Based on the concentration, Edgar’s is more
concentrated (0.108mg/mL) than Ririn’s (0.0225mg/mL)
which contradicts electrophoresis banding result. This
differences possibly caused by errors in decanting
supernatants by micro-pipetting that took more DNA
samples than intended or poor DNA-TEbuffer mixture
making some has fewer DNA.
PCR technique amplify specific segments of DNA
using forward and reverse primers. In figure 4, the DNA
markers and all samples showed clearly in desired
thickness and clearness. However, the positive control for
in lane 4 was too faint to be noticed due to human error in
micro-pipetting that results well destroying causing it to
not flow through the gel and show thick banding. The
negative control in lane 5 also showed faint banding
when it’s supposed to show none at all. Contamination
during work takes responsibility for this part. Overall, PCR
was a success which the size of DNA sample showed
approximately 330 bp.
DNA sequencing was to see the ERCC2 gene
fragment amplified to be compared the similarities with
database NCBI. The graphic summary overall shows high
similarities in gene 1-5. Gene 6 and so on were not 100%
similar to the database as several nitrogen bases went
slightly off from what they’re supposed to be (T when
supposed to be A,etc) that is why the ERCC2 gene query
cover was 92%. However, he process was a success
because gene showed 99% identity with human ERCC2
gene as shown in figure 6, Homo sapien ERCC excision
repair 2, TFIIH core complex helicase subunit (ERCC2)
transcript variant 3, mRNA.
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