Summary

The aim of
the experiment was to take a sample of cheek cells and isolate the DNA within
the cells and then to amplify the RHCE and RHD genes by the means of PCR. Then gel
electrophoresis is used to separate the nucleic acids by their size to produce
bands.

The main
purpose of Polymerase Chain Reaction (PCR) is to amplify specific fragments of
a DNA sequence. This is useful if you have a small sample of DNA as it can be
multiplied into the amount you require. Rhesus is an antigen that can appear in
the blood, identifying this gene is the second aim of this experiment.

 

 

Introduction

If someone
is Rh-negative it means that the Rh antigen is not present. If the antigen is
present, the person is Rh-positive. (Martini
et al Visual Anatomy and Physiology)

The ABO
blood typing system is the most important and commonly used blood typing system
worldwide. It is based on what, if any, antigens are present on the surface of
your red blood cells. This decides what blood group you belong to. The
importance of knowing your blood type is to prevent the risk of receiving an
incompatible blood type when/if needed. Receiving the wrong blood type can have
disastrous effects and can lead to clumping of blood cells which can in turn be
fatal. Knowing your Rh type is especially important for pregnant women. If a
mother and a fetus have different Rh statuses it can initiate the production of
antibodies against the baby’s blood. This is known as Rh-sensitisation. (Wijayasinghe, 2017)Gel electrophoresis is
a technique that separates nucleic acids. DNA moves towards the positive pole
when an electrical charge is initiated as the DNA backbone is negatively
charged.

 

 

Method

The
practical was divided into 4 sections.

The first
intent was to isolate the DNA fragments from the sample of cheek cells
obtained.

This was
done by taking a cotton swab and firmly rolling it on the inside of both cheeks
approximately 20 times. The swab was then placed into a tube of DNA extract
solution. Then the swab was rotated 5 or more times to ensure it was evenly
coated in the solution. To ensure all the excess solution was removed from the
swab, it was pressed firmly against the side of the tube. The tube, containing
the DNA and the DNA extracting solution, then went through a series of vortexes
and incubation periods in order to separate the DNA. It was first vortexed for
10 seconds, incubated at 65°C for half an hour, then vortexed
again for 15 seconds and incubated 98 DEGREES for a further 8 minutes. Once again,
the tube was placed in the vortex for a further 15 seconds. Lastly it was
incubated at 98 DEGREES for 8 minutes and vortexed for a further 15 seconds. After
this the tube was placed in ice to preserve it.

The next
thing to be carried out was PCR experiment. A tube labelled ‘R’ containing 0.5ml
of the reaction mix had added to it 90ml of megamix blue. This solution
contained Taq polymerase, nucleotide bases, buffer and 2.5ml of both primer A and B. A tube labelled ‘S’ containing
the sample solution was the negative control and contained no DNA molecules.

This solution was made up of 47ml of reaction mix and 3ml of sterile water. A third tube labelled ‘C’
contained 47ml of reaction mix and 3ml of sterile water – this is the DNA is later
amplified during PCR.

After this,
both the ‘S’ and ‘C’ tube were placed in a microfuge for approximately 3 seconds
and then transferred to the thermal cycler.

The tubes
were placed in a freezer until electrophoresis is to be carried out to preserve
the solutions.

The third
section of the practical was Gel Electrophoresis. 40ml of molten 1.5% agarose
gel was added to the tank and then was carefully put and put aside to set – gel
will be set when it is slightly cloudy. When set, remove the rubber blocks and
the comb carefully and place the gel in the electrophoresis tank ensuring the
wells are closest to the negative electrode. 250ml of TB buffer was added to
the tank and the tank was placed over a black background to allow the wells to
be seen clearly. Label 2 new test tubes with ‘C’ and ‘S’. To both of these add
5ml of bromophenol blue. Add 15ml of control PCR to ‘C’ and 15ml of sample PCR to ‘S’. Using a pipette mix
both solutions sufficiently, ensuring no bubbles were blown into the sample and
contamination didn’t occur. If needed centrifuge tubes for 5 seconds if there
are droplets of liquid on the side of the tubes. 15ml of ladder was loaded into the
first well. 15ml of solution from ‘C’ was loaded
into the second well and 15ml of solution from ‘S’ was loaded
into the third well. Replace the cover on the tank and connect it to the power
pack – now it is ready for electrophoresis. `

Run 150
volts through the gel for 45 minutes. The process can be observed by following
the movement of the bromophenol blue. Whilst electrophoresis is occurring
measure the concentration and purity of your DNA. To do this take the sample of
DNA to the nanodrop spectrophotometer and record the 260/280 and 260/230 ratios
and the DNA concentration in ng/ml.

After completion,
photograph the gel using the GelDoc system.