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A lab Report outlining the Rhesus Factor using polymerase chain reaction
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A lab Report outlining the Rhesus Factor using polymerase chain reaction
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Tanzeel Ali-33480309A lab report on examining the Rhesus factor using the polymerase chain reaction
Summary
The main aim of the experiment after conducting both laboratory sessions was to first
obtain isolated human DNA from cheek cells; via a swab test. PCR was then carried out by
adding PCR reagents to the DNA before using an incubator and vortex to ensure replication.
Gel electrophoresis was conducted to help determine the Rhesus factor from isolated
human DNA.
Introduction
The Rh factor is known for being an antigen that fights unknown invaders, by stimulating
the production of antibodies. These invaders include bacteria, viruses etc. [1] An individual
either contains the antigen in their blood or they don’t, therefore indicating that they may
be RH positive or negative. A person’s Rh factor can determine whether they are capable of
handling blood transfusions or organ transplants. [1]. Landsteiner discovered that human
blood serum could be categorised into four groups; depending on their ability to clot red
blood cells. These groups came to be known as A, B, AB and O. However, Landsteiner came
across another blood antigen by the name RH. This was due to the presence of another
[1]
antigen (Rh factor). .The Rhesus blood group is currently typed beginning with RHD and
RHCE genes. Genetic rearrangements that are various between them has produced RH
hybrid genes that encode countless RH antigens. [2] Genes that encode 2 district Rh proteins,
carrying C or c together with E or e antigens and the D antigens, have been cloned. [3]
PCR/polymerase chain reaction is a technique carried out by scientists to produce several
copies of a sequence of DNA. [6] This process is a common tool as it enables the detection of
the presence or absence of the gene to help look for pathogens to fight infections, or
creating forensic DNA profiles from small DNA samples. [6] Template DNA is required to copy
a complementary strand of DNA. [6] Primers are bound to the template DNA with the
sequence being identical to the 5’ 3’ template DNA. DNA nucleotide bases are required in
order to manufacture the new strand of DNA, along with Taq polymerase enzyme enabling
the bases to be added in. A buffer is then included ensuring that the conditions are suitable
for the reaction to take place.[6] Three main steps are required for the PCR reaction to occur.
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, Tanzeel Ali-33480309
[5]
The denaturing stage occurs when double stranded DNA is heated in order for separation
to occur into single strands. The annealing stage is where temperature is decreased to
ensure that DNA primers can attach to the template strand. The extending stage occurs
once the temperature is raised and the new strand of DNA is created through the use of Taq
polymerase which adds the bases to the newly formed strand. Around 20-30 copies of these
three stages are replicated, producing several amounts of DNA replicates [6] Overall, these
come into hand for my experiment as our aim is to isolate human DNA via the swab test,
carry out the PCR technique and use gel electrophoresis to determine our results from the
PCR experiment.
Methods
For the first lab session, the isolation of human DNA was the aim to be achieved. It was
done using the swab test whilst working alone. A numbered tube was taken that contained
DNA extraction solution. The tissue sample was obtained via the swab test, with the swab
firmly placed within the inside of the cheek and rolled around 15 times. Once done, the
swab end was placed into the numbered tube and swirled 5 times. The cap was tightly
screwed on as it ensured no liquid to spill out when it was on the vortex for 10 seconds. The
tube was incubated for 30 minutes at 65oC, before placed on the vortex for 15 seconds. This
was then repeated at 98oC for 8 minutes twice before it was placed on ice.
The next lab session was done in pairs. Firstly, the gel mould was removed from the
electrophoresis tank, with each rubber block fitted on either side. 40ml of molten 1.5%
agarose gel was added to the mould before it was left to set for 20 minutes. This was
noticed once the gel had reached a cloudy appearance. Once set the comb and rubber
blocks were carefully removed, enabling the gel to be added into the electrophoresis tank.
250ml of TB buffer was added to the tank, with the mini gel being added to ensure the wells
were visible. Two tubes were taken, and labelled as ‘C’ and ‘S’ with the number on each. 5ul
of bromophenol blue loading buffer were added to each of the tubes, with 15ul of the
controlled PCR sample added to the ‘C’ tube and 15ul of the sample PCR to the ‘S’ tube. 15ul
was then loaded into the first well of the tank, shortly followed by the sample in the next
well. Once done, the next well was loaded with 15ul of control DNA, before it was covered
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