Polymerase Chain Reaction is a lab technique used to amplify DNA sequences. It involves using short sequences of DNA and primers to select a certain chromosome on the DNA to be replicated. This is a relatively modern form of DNA production. It was discovered in 1993 by Kary Mullis (An Introduction to Genetic Engineering. 2nd Edition. D. S. T. Nicholl. Cambridge University Press 2002). The temperature is changed to help the enzymes DNA Helixase to unwind the DNA and DNA Primase to copy each of the DNA sequences. It is carried out in vitro so the amount of DNA produced can be controlled and produced in a short period of time. There are three main steps in PCR Denaturation happens first, then Primer Annealing and finally Extension. Denaturation takes place to denature and separate the Template DNA. This is done by heating the solution to 92°C to weaken the hydrogen bonds and separate the DNA into separate 3′ and 5′ strands. Annealing the primers by reducing the temperature to around 60°C allows the primers to bind to the correct parts of the template DNA. The final stage, Extension, allows the enzyme DNA Taq Polymerase to bind the correct nucleotides to the correct base pair on the DNA. PCR usually repeats this for 30 cycles.
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PCR is only successful when all the conditions are correct. Temperature is one of the main parameters which can affect the results of the PCR. Each of the stages of PCR must be at the correct temperatures as it is essential for the PCR to have accurate and successful results. If the temperature is below, the DNA Taq Polymerase will not work. Taq is from the Archaeon Thermus aquaticus which is found in thermal springs. The template DNA is synthesised by the enzyme DNA Taq Polymerase, which is resistant to high temperatures. The enzyme is found in geysers which are naturally at temperatures around 110°C (Joshi et al, 2010-2011). Although it is genetically engineered to be more tolerant to a wide array of temperatures below, stop Taq from working properly. High temperatures affect the way in which proteins unwind.
The reaction mix of template DNA and primers affects the concentration of enzymes, DNA, dNTP’s and primers. If the concentration is too high, the primers will not be able to anneal to the template DNA because they will inhibit each other. The concentration of DNA affects the denaturation because they will not be able to separate. At a high concentration the single strands of DNA will anneal to each other instead of the Primers, thus stopping the PCR from completing properly. The wrong concentration of dNTP’s affects the extension period because if the concentration is too low, the wrong nucleotides will attach to the wrong base pairs.
Primers are used to amplify certain genes on the Template DNA. For this lab the required gene is D1S80 which is found on Chromosome 1, the largest human chromosome. The repeating sequence of this chromosome is 16 base pairs long and has 29 different alleles.
This lab will be carried out over a period of three consecutive weeks. Four different concentrations of the Buffer will be used each week, 2.5x, 5x, 7.5x and 10x. Each week the Annealing Temperature will be changed. Week one will be 58°C, week two will be 65°C and week three will be 72°C. This will be done to see what the optimum conditions are for carrying out PCR and gaining the best results.
Extraction of Chelex – Week 1
10% Solution of Chelex. 5ml of Chelex solution is needed. This consists of 5ml of TE Buffer and 0.5g of Chelex.
Mix the Chelex solution together and pipette €³°°l into a tube.
Using aseptic techniques take a cheek swab and add it to the Chelex solution. Mix them together.
Put the tube into the Vortex, which is on the setting touch, to mix for 20 seconds.
Place the sample into the centrifuge for 2 minutes at 12000rpm.
Incubate in the heat box for 5 minutes at 95°C. (Instead of 20 minutes).
Vortex the tube again for 20 seconds.
Place the tube into the Centrifuge again for 2 minutes at 12000rpm.
dNTP is 10mM (millimolar). 200M (micromolar) is needed.
200M = 0.2mM this will be 20% of 1ml. (1000l is equal to 1ml).
20 samples are needed so 4l will be needed per sample.
80l is needed, so 100l will be made.
0.2mM in 100l (50x)
This equation will be needed to work out the final amount.
(C1) V1 = (C2) V2
10mM x V1 = 0.2mM x 100l
V1 = 0.2 x 100
V1 = 20
V1 = 2l
Add 2l of 10mM dNTB’s to 100l of TE Buffer to give 0.2mM or 200M.
Measure 98l of TE Buffer into a tube.
Add 2l of dNTB into a different tube. Note: keep everything separate so they don’t begin to react.
To an empty sterile tube label 2.5x. To this add 7.5l of buffer and 22.5l of water.
To three separate sterile tubes label 5x, 7.5x and 10x.
5x tube add 15l of buffer and 15l of water.
7.5x tube add 22.5l of buffer and 7.5l of water.
10x tube add 30l of buffer.
To each of the four tubes add 24l of dNTP’s.
Add 228l of water to each of the four tubes.
Add 6l of both the forward and reverse primers to each of the four tubes.
Add 6l of Taq to each of the four tubes.
Into 5 smaller, sterile tubes add 45l of the 2.5x sample.
Using 5 tubes for each concentration add 45l of 5x, 7.5x and 10x into the tubes.
Place all 20 small tubes into the thermal cycler and run it at:
10s at 95°C for Denaturation
10s at 58°Cfor Annealing
30s at 70°C for Extension.
This will run for 29 cycles.
PCR – Week 2
Defrost all DNA and samples.
120l of Master Mix is needed, so there will be 20l in each sample.
Label a sterile tube Master Mix and add 6l of each the forward and reverse primers to it.
Add 138l of sterile water to the master mix tube.
Add 45l of master mix to four separate sterile tubes.
Add 5l of each sample to each tube.
Add 5l to a separate tube.
Put the tubes into the thermal cycler and run at:
10s at 95°C for Denaturation
10s at 65°C for Annealing
30s at 70°C for Extension
This will run for 29 cycles.
The gel is already prepared.
Fill the tray with the AE Buffer.
Add 3l of dye to each sample.
Red samples first and then blue samples.
Pipette 15l of each sample to the gel electrophoresis tray.
Run the gel for 15 minutes.
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