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Single nucleotide polymorphisms (SNPs) are a common type of genetic variation that represents a difference in a nucleotide and it occurs once in every 100 to 300 bases on average. Therefore, there are approximately 10,000,000 to 30,000,000 SNPs occurring in the human genome.
SNPs are commonly used to locate disease-causing genes as they often occur within the genes or a region near the genes. SNPs are also used in the identification of distinctive genetic differences between persons. Although 99.9& of one individual DNA sequences would be identical to that of another person's DNA sequence, of the 0.1% difference in the DNA sequences, over 80% will be SNPs of the human DNA. Thus, SNPs can be easily used to identify gene differences between different individuals.
Polymerase chain reaction (PCR) is a highly sensitive process that amplifies DNA sequences so that changes in specific DNA sequences can be analyzed easily. One example of the advantage of using PCR for DNA sequence analysis is the detection of the human immunodeficiency virus (HIV) that causes the acquired immunodeficiency syndrome (AIDS). As PCR is a highly sensitive procedure, it can detect HIV in its early stages when only a few thousand infected blood cells are present in a patient.
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In PCR, genomic DNA (gDNA) is broken down into large fragments using restrictive enzymes and then denatured into single strands using heat. Two synthetic deoxyribonucleotide triphosphates (dNTPs) that are complementary to the 3' ends of the targeted DNA sequence are added excessively to the denatured DNA and the temperature is lowered to 50 Í¦C or 60 Í¦C. While the gDNA remains denatured, specific dNTPs that were added binds with the complementary sequences of the denatured gDNA. The dNTPs then serve as primers for the DNA chain synthesis which starts once temperature-resistant DNA polymerase is added and it can extend primers at temperatures up to 72 Í¦C. Once the synthesis is complete, the mixture is then heated up to 95 Í¦C to melt the DNA duplexes that are newly formed. The cycles are then repeated to ensure that the excess primers were fully synthesised, thus, allowing amplification of the targeted DNA sequence to be quickened and the amount of the targeted DNA sequence to be exponentially increased.
POLYMORPHISMS IN HUMANS
Polymorphism is a term used to describe a difference in a specific DNA sequence or natural genetic variations that has no adverse effects on individuals. Only polymorphisms that occur in more than 1% of a population would be useful for genetic linkage analysis.
Some examples of human polymorphisms are human blood groups, sickle-cell anaemia, Duffy system, and human taste morphisms. In this report, only the polymorphisms of the human blood group would be tested on to understand the
ABO BLOOD GROUP
There are more than twenty-nine blood group systems used in the world but the blood group system that is the most commonly used is the ABO blood group system as the ABO blood group is the most immunogenic of all the blood group antigens. The blood group was first discovered by Karl Landsteiner in 1900 to 1901 in the University of Vienna when he was investigating blood transfusions and their effects. Karl Landsteiner then received a Nobel Prize in 1930 for his work on the discovery of the ABO blood group.
There are four primary blood groups: A, AB, B, and O. There are two antigens, antigen A and B, and two antibodies, anti-A and anti-B that are responsible for the ABO blood group. (http://anthro.palomar.edu/blood/ABO_system.htm)
MATERIALS AND METHODS
OVERVIEW OF EXPERIMENT
Figure 1: An overview of steps taken during this experiment
ISOLATION OF DNA FROM CHEEK CELLS
To extract DNA from cheek cells, some mouth rinse was obtained from a person and 2ml of spittle sample was pipette to a 2ml centrifuge tube. It was then centrifuged at 5000rpm for 5 minutes. The supernatant was discarded and topped up with another 2ml of spittle. It was then centrifuged again at 5000rpm for 5 minutes and the supernatant was discarded.
The cell pellet found at the base of the centrifuge tube was then carefully transferred to a microcentrifuge tube. 180Î¼l of Buffer ATL was added to the tube and the tissue was mashed with a blue pestle. 20Î¼l of Proteinase K was added and the tube was vortexed. The tube was placed in a water bath at 55 Í¦C until the tissue was completely lysed after one to three hours.
200Î¼l of Buffer AL was added into the tube, vortexed, and placed in a water bath at 70 Í¦C for 10 minutes before another 200Î¼l of ethanol was added into the tube as well. The tube was vortexed thoroughly again and the contents in the tube was pipetted into a DNeasy mini column placed in a 2ml collection tube. The mini column was closed firmly, labeled and centrifuged at 8000rpm for 1 minute.
The flow through and collection tube was discarded and the DNeasy mini column was placed in a new collection tube. 500Î¼l of Buffer AW1 was added into the mini column and centrifuged at 8000rpm for 1 minute. The flow through and collection tube was discarded once more and the DNeasy mini column was placed in a new collection tube.
500Î¼l of Buffer AW2 was added into the minicolumn and it was centrifuged at 12000rpm for 3 minutes. The flow through was discarded and the DNeasy mini column was placed into a labeled, clean 1.5ml microcentrifuge tube.
200Î¼l of Buffer AE was added directly onto the DNeasy membrane carefully without touching or breaking it and was left to stand at room temperature for one minute after the mini column was closed firmly. Lastly, it was centrifuged at 10000rpm for 1 minute and the DNeasy column was discarded. The microcentrifuge tube now contains the isolated DNA and was stored safely for agarose gel electrophoresis.
AMPLIFICATION OF DNA SAMPLE USING PCR
PCR was carried out for exons 6 and 7. The forward (F) and reverse (R) primers used in PCR are as follows:
Primers (5' ƒ 3')
% annealing temperature
17-mer; 5.2.9 Í¦C
20-mer; 65 Í¦C
21-mer; 76.2 Í¦C
21-mer; 57.1 Í¦C
Table 1: Forward, reverse primers and conditions needed to amplify DNA sample
PCR for exons 6 and 7 were carried out in 50Î¼l volume container tubes containing exon 6 and 7 separately. The following components listed below were then added into the volume chronologically.
Green GoTaqâ„¢ reaction buffer
Table 2: Components and amount of components needed to carry out PCR
For the tube containing exon 6, PCR was carried out in a DNA Thermal cycler. Initial denaturation of the exon was carried out at 94 Í¦C for 2 minutes. The cycler was then programmed at 94 Í¦C for 30 seconds for further denaturation, 53 Í¦C for 30 seconds for annealing of the exon, 72 Í¦C for 60 seconds for extension of the exon and 72 Í¦C for 5 minutes for the final extension of the exon. The reaction was then repeated for 35 cycles. Lastly, it was stored at 4 Í¦C for holding of the exon.
For the tube containing exon 7, PCR was carried out in the same way as exon 6, except that annealing of the exon was carried out at 59 Í¦C instead of 53 Í¦C for 30 seconds.
AGAROSE GEL ELECTROPHORESIS OF POLYMERASE CHAIN REACTION PROUCT
1.0% agarose gel was made by adding 1g of agarose powder to 100ml of 1X TAE buffer. It was then swirled and placed in the microwave oven for the agarose to be melted and mixed thoroughly in the buffer. The liquid was then poured into a gel casting tray attached to the horizontal electrophoresis chamber. A sample comb was placed at one end of the gel casting tray to form sample wells.
Once the gel had been set, the comb and tape was removed and 1X TAE buffer was poured over the agarose gel until it fully covered the surface of the gel. 1Î¼l of 6X loading dye, 2Î¼l of PCR product, and 7Î¼l of sterile water was then mixed and pipetted into one of the wells of the gel. 10Î¼l of markers 1kbp and 100bp were also pipetted into the wells of the gel.
Electrodes for the electrophoresis chamber were then attached to the sides of the chamber and power was turned on at 80V. Electrophoresis was run for an hour and DNA was observed using a UV Transilluminator. Gel photographs were taken using Gel Docâ„¢ System after electrophoresis was complete.
RESTRICTION DIGESTION OF AMPLIFIED PCR PRODUCT
With the remaining DNA sample, it was separated into three tubes; two meant for exon 6 and one for exon 7. Enzymes were used in the process of restriction digestion to digest the PCR product of the exons. Enzymes Kpn1 and BstEII were used to digest the PCR product of exon 6 while enzyme HpaII was used to digest the PCR product of exon 7. A premix was prepared using enzymes and certain components needed for the digestion process and they were added chronologically in order as shown in the table below.
Table 3: Components and volume of components needed for preparation of premix
After the premix was prepared, the tubes were vortexed and incubated at 37 Í¦C, 60 Í¦C and 37 Í¦C for the DNA samples treated with enzymes Kpn1, BstEII, HpaII respectively. After two hours, they were stored and placed in a -20 Í¦C freezer to stop all reactions.
AGAROSE GEL ELECTROPHORESIS OF DIGESTED PCR PRODUCT
Agarose gel electrophoresis was carried out for exons 6 and 7. 1% agarose gel electrophoresis was carried out for exon 6 while 4% agarose gel electrophoresis was carried out on exon 7. 2Î¼l of 6X loading dye was loaded together with 8Î¼l of the digested PCR product into the wells of the agarose gel. Markers of 1kbp and 100bp were used to determine the sizes of the digested products of exons 6 and marker 100bp was used for exon 7. Electrophoresis was then run at 80V for 1 hour and observed under a UV Transilluminator. After electrophoresis was complete, gel photographs were taken using Gel Docâ„¢ System for easy comparison of markers and exons.
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Plate 1: Electrophoresis scan of amplified PCR product, third lane from left.
(Legend: Extreme left lane, marker 1kbp. Extreme right lane, marker 100b
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Plate 2: Second electrophoresis scan of digested PCR product, second (exon 6) and third lane (exon 7) from right.
(Legend: Extreme left lane, marker 1kbp; Extreme right lane, marker 100bp)