REFERENCES of carcinoma cervix (CaCx). Journal of clinical

 

 
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Bridge, J. A., 2017. Reverse
transcription–polymerase chain reaction molecular testing of cytology
specimens: Pre?analytic and analytic factors. Cancer Cytopathology, 125(1),
pp. 11-19.
Erlich, H., 2015. PCR technology: principles
and applications for DNA amplification. New York: Stockton Press.
Farrar, J. S. & Wittwer, C. T., 2015.
Extreme PCR: efficient and specific DNA amplification in 15–60 seconds.
Clinical Chemistry, 45(1), pp. 145-153.
Prakash, P. et al., 2014. Nested multiplex
(NMPCR) detection of human papillomavirus (HPV) 16 and 18 in pre-invasive
lesions and its implication in screening of carcinoma cervix (CaCx). Journal
of clinical and diagnostic research: JCDR, 8(2), p. 110.
Saha, S. K. & Khuda-Bukhsh, A. R., 2014.
Berberine alters epigenetic modifications, disrupts microtubule network, and
modulates HPV-18 E6–E7 oncoproteins by targeting p53 in cervical cancer cell
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Westermeier, R., 2016. Electrophoresis in
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5 ed. Germany: John Wiley & Sons.
 

 The experiment
was to use Agarose gel electrophoresis and PCR to amplify the HPV E6 genes and
examine changes of HeLa DNA levels. The experiment was performed using all the
safety measures and results obtained were much in accordance with the
standards.

CONCLUSION

There was a variation in the original result and
result obtained via experiment. This could be due to various errors, such as  unavoidable human errors, measurement errors
and instrument related errors. Also, the lesser would be the concentration,
more would be the clarity of the band (Bridge, 2017). The primary reason
behind this is that a higher concentration would result in restriction in
mobility of DNA. This results in condensation of this product and thus the
aggregation. As a result, the transfer and movement of the molecule through the
gel lane is reduced considerably. This would result in an unclear or a
distorted band. To avoid this, we can dilute the concentration and compare with
the standard DNA ladder. Another possibility is smearing of the DNA sample.
Moreover, if the sample of DNA is not properly dissolved, it would also result
in smearing. The incubation rate in this case would be about 37 an hour. The
purity of sample would also affect the outcome of the experiment.

On the other hand lane and the
lane  3 (treated HeLa DNA(T-2) of the
original picture was absent, the second figure in this band was a visible with
poor loading, lane 4 shows a   control
GAPDH – HeLa Untreated),  and the
lane  5 GAPDH – HeLa ATO 2 µWhile  lane  6
(GAPDH – HeLa ATO 5 µM) were all  shows a
visible and shining on the original, but, in the  second figure the  lane 
5,6 and 7 are  all visible and
shining while line 4 shows a  absent,
that means the second experimentation was contamination ,because last  lane  7
which contains   a Non Template Control
and  not contain DNA it used as
control  so it should not have  the band and there was not a contamination.

Agarose gel electrophoresis is
a useful process of determining if restraint digest procedure has been
successful. The said DNA ladder is used to determine the size of fragment of
DNA in electrophoresis gel (Saha & Khuda-Bukhsh, 2014).The experiment showed
that DNA had not only been cut into two smaller DNA fragments; it could also
determine sizes of both the initial samples of DNA. The size of the product of
restriction digest could also be determined. Here, it used DNA ladder to
determine the size of DNA fragments. By comparing the band in lane two with the
existing DNA standard, it can conclude that the
samples (figure 1) has 11 bands of the ladder which shows a visible compared to
the slandered ladder while practical samples in (figure 2) the ladder bands are
not very clear this because a small variation, which could possibly due to
human errors, errors in measurement. Using the set ladder standards, it can
judge if the DNA was cut in correct location or not. The lane 1 shows that the (control
HeLa DNA HPV18/E6) of the original band and shows visible and shining, where in
the lane 2 shows a treated Hela DNA(T-1)) band was a visible but not shows any
shining and same applies to the group experiment picture.

DISCUSSION

 

Figure 2.  shows of DNA gel
electrophoresis to investigate effect of ATO on HPV of viral E6 gene. Ladder is
PCRBIO DNA ladder, lane 1 shows the control HeLa DNA HPV18/E6, lane 2 shows treated
Hela DNA(T-1), lane 3 treated HeLa DNA(T-2) HPV18/E6, lane 4 shows control GAPDH
– HeLa Untreated, lane 5 GAPDH – HeLa ATO 2 µM, lane 6 GAPDH – HeLa ATO 5 µM, and lane 7shows  Non-template control.

 

 

 

Figure 1  above are represents
a  module  samples consistent to:

HPV18 – HeLa Untreated (line 1, 500bp)

HPV18 – HeLa ATO 2 µM (line 2, 500bp)

HPV18 – HeLa ATO 5 µM (line 3)

GAPDH – HeLa Untreated (line 4, 500bp)

GAPDH – HeLa ATO 2 µM (line 5,500bp)

GAPDH – HeLa ATO 5 µM (line 6,500bp)

GAPDH – NTC (H2O) (line 7)

PCRBio Ladder IV (line 8, 1500bp)

 

Figure 1. the result obtained to preforming of
DNA gel electrophoresis to investigate effect of ATO on HPV of viral E6
gene. Ladder is PCRBIO DNA ladder, lane 1 is control HeLa DNA HPV18/E6 ,lane2
 shows treated Hela DNA(T-1),lane 3) is
treated HeLa DNA(T-2) HPV18/E6, lane 4) is control GAPDH – HeLa Untreated
lane 5) GAPDH – HeLa ATO 2 µM, also lane  6) GAPDH – HeLa ATO 5 µM,  finally lane 7) shows a Non-template control.

 

              

RESULTS

 

The practical was
started and prepared to run on1%   with
agarose gel electrophoresis, then the gel has changed into the running
position, then the gel electrophoresis buffer was transferred into tank and
fully covered the gel. Then was start with 
two HeLa cell lines one normal and one normal and the second treated
with  ATO for days and DNA samples
was extract  from samples , The samples
are loaded into the eight sample ,with and 8.5 µL Nuclease free water and 12.5
µL were allowed all the  tubes with the 
NTC ,while  2.0 µL of 10µM primer
F and  R mixed E6/HPV18 (H) were added
tubes 1,2, 3 and 7 which shows  Non
Template control, also  2.0 µL of 10µM
primer F and R mixed gene TAPDH was added at tubes 4,5 and 6 while the  2.0 µL of Nuclease free water were
putted  on tubes 5 and 7, also, 2.0 µL of
Control untreated DNA Hela (250ng) (H-C) were added to tubes 1 and 4.   While the 2 .0 µL of 2uM ATO treated DNA
(H-T1) (250 ng) were loaded on tubes 2 and 5.Lastly the 2.0 µL of 5uM ATO
treated DNA (H-T2) (250 ng) were added on tubes 3 and 5 for the total of 25 µL
of each reaction. the unit was set on 90 V and the power was switched on to run
the gel and wait for 30 minutes.

METHODOLOGY

 

The aim of this
practical was determine the present of HPV 18 E6 DNA and to inspect if there
are arsenic trioxide (ATO) that  affect
HPV 18 E6 DNA levels of treated Hela cells.

The
molecular modus operandi called PCR is in vitro augmentation of a specific
segment of DNA using a thermostable enzyme. Basically, applications of PCR are vast and used in many research
applications (Erlich, 2015). PCR id used for Direct Cloning of DNA
and without the use of bacteria. Using PCR, we can create DNA fingerprinting
for forensic uses. We can diagnose genetic diseases prenatally. Using PCR,
evolutionary analysis is possible. We can detect allelic sequence variations.
Detection of chromosomal rearrangements is possible using PCR. PCR is also used
for DNA sequencing processes. We can detect viral and/or bacterial infections
with the help of PCR. There are certain requirements or conditions that need to
be fulfilled for PCR. Following are needed for PCR (Erlich, 2015):dsDNA,dNTP nucleotides (dATP, dGTP,
dCTP, dTTP)Magnesium ion ,Forward Primer ,Reverse Primer,Tag polymerase
(extracted from Thermus aquaticus) and BufferUsing the above a cycle is created
and in each cycle the gene is doubled in number. Since, there is doubling of
number, it is observed that more than a million copies of genes are generated
from a single DNA molecule in just 20 cycles.   

Agarose gel electrophoresis is a
very elementary and indispensable technique in molecular biology. It is usually
used for scrutiny of PCR products, plasmid DNA, and products obtained via
restriction enzyme digestion. It is the initial step in analysing specific DNA
and RNA fragments. It used northern and southern blots (Westermeier, 2016).

INTRODUCTION

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