Karyan Yuen Yuen 0707470 Module: Modul e: BB1204
PCR and agarose gel electrophoresis Introduction PCR (polymerase chain reaction) is an in vitro technique enabling researchers to produce millions of copies of a specific DNA sequence in approximately 2 hours inside a test tube. This automated process bypasses the need to use bacteria for amplifying DNA. To perform a PCR, we will need a reaction mixture containing the target DNA sequence to be amplified, 2 primers (forward and reverse), heat-stable Taq DNA polymerase and deoxynucleotide deoxynucleotide triphosphates. The first step of the cycle is denaturation, brought about by heating the target DNA to about 95 C. This process separates the double-stranded DNA into 2 single °
strands. After strand separation, cooling of the DNA in the presence of a large excess of forward and reverse primers allows these primers to anneal and hybridise to complementary sequences sequences in the 2 DNA strands. The optimal temperature for this to occur varies between 40 C to 60 C. °
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The mixture is then incubated with Taq DNA polymerase at 72 C which enables °
nucleotides to be added to the 3' end of the annealed primers and extend in the 5' to 3' direction. The temperature is raised to denature the DNA into 2 single strands again and then lowered sufficiently to allow more primers to anneal. Taq DNA polymerase now synthesises another set of new complementary strands. As the procedure is repeated over and over again, the newly synthesised fragments serve as templates for the next cycle and within a short period of time, many copies of the original DNA can be produced. This results in an exponential accumulation accumulation of the specific target fragment, approximately 2 n where n is the number of cycles of amplification performed. Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Agarose gel electrophoresis electrophoresis is routinely used used for the preparation and analysis of DNA. Gel electrophoresis electrophoresis is a procedure that separates molecules molecules on the basis of their rate of movement through a gel under the influence of an electrical field. DNA is negatively charged and when placed in an electrical field, DNA will migrate towards the positive pole (anode). An agarose gel is used to slow the movement of DNA and separate by size. size. Within an agarose gel, linear linear DNA migrates inversely inversely proportional to the log10 of their molecular weight.
Aim The aim of this experiment is to identify the absence or presence of the human “transgene” in 2 samples of mouse genomic DNA using the PCR method.
Karyan Yuen 0707470 Module: BB1204
Materials and Method Target DNA – I will be using 2 different samples of mouse genomic DNA alongside a dH 2O control sample which will be used as a negative control. Buffer solution – This is used to provide a suitable chemical environment for optimum activity and stability of the DNA polymerase. Deoxynucleotide triphosphates – These are used in excess for which the DNA polymerase uses to synthesise a new complementary DNA strand. MgCl2 - MgCl2 is a cofactor of Taq DNA polymerase, the concentration of MgCl2 influences the productivity and fidelity of polymerases. At optimal concentration, that must be determined for each primer/template condition. It is typically between 1.0 and 3.0 mM. Two oligodeoxynucleotide primers - The two primers, each typically about 15-30 nucleotides in length, are usually designed so they are 200-2000 bp apart, one hybridizing to one strand of dsDNA, the other hybridizing to the other strand such that both primers are oriented with their 3' ends pointing towards each other. Taq DNA polymerase – This thermophilic DNA polymerase is thermally stable and is able to work at 100 C at which DNA is denatured into linear strands. Taq °
DNA polymerase has an optimum temperature of 72 C. °
1. The reagents are mixed together and centrifuged - dH 20, 10x NH 4 PCR buffer, 50 mM MgCl 2, 50uM Forward primer, 50uM Reverse primer, 10mM dNTPs, Taq polymerase (5u/ul).
2. 24ul of the mixture is aliquoted into 3 eppendorf tubes. 3. In the first tube, 1ul of genomic DNA1 was added. In the second tube 1ul of genomic DNA2 was added and in the third tube, 1ul sterile water was added. 4. The tubes are then left in ice to be placed into an MJ Research PCR thermal cycler and stored at -20 oC for the next part of the experiment. 5. During the next part of the experiment – gel electrophoresis, 1% (w/v) agarose gel was prepared by mixing 1g of agarose with 100mls of 1X TBE buffer in a 250ml flask.
6. This mixture is then heated carefully in a microwave up to its boiling point with occasional gentle swirling.
Karyan Yuen 0707470 Module: BB1204
7. The agarose gel is left to cool before 2.5ul of 10mg/ml ethidium bromide solution per 100ml gel was added and mixed gently. This allows the DNA to be visualised under UV light. 8. The ends of the gel former were sealed with tape and the agarose gel is poured into it and left to set. 9. Once the gel has set, tape is removed and placed into an electrophoresis tank and 1X TBE buffer is poured into the tank until it is about 1m above the gel. 10. 5ul of the loading dye is added to each of the 3 eppendorf tubes. This aids the loading into the wells by increasing the sample density and allows us to see how far the samples migrate. 11. 5ul of Hyperladder I DNA size marker is loaded into a well and 20ul of each of the PCR samples are loaded into the wells beside it. 12. The voltage on the tank is then turned on so that electrophoresis can occur. The electrophoresis is stopped before the dye runs of the end of the gel. Gels are then viewed on a UV transilluminator and a photographic image is captured to be analysed.
Karyan Yuen 0707470 Module: BB1204
Results A photographic image obtained from our group (Group 19)
Distance migrated from 200bp
Hyperladder I DNA molecular weight marker
dH2O (Negative
Control)
Sample 2
Sample 1
(Figure 1)
(Figure 2)
Discussion
Distance migrated by Sample 2 – 6.1cm
Karyan Yuen 0707470 Module: BB1204
From looking at the photographic image (Figure 1), we can see clearly that Sample 2 of mouse genomic DNA has produced visible PCR products of the correct size which is just slightly above the DNA molecular weight marker of 200bp. In this experiment, we have used human-specific primers (forward and backward) which amplifies a segment of human DNA at 227bp. Therefore we have identified that only Sample 2 shows the presence of a human “transgene” sequence, whereas in DNA Sample 1 there are no distinct amplications which therefore shows us the absence of such “transgene”. The negative control shows no result as expected as there was no DNA sample present; this also shows that there was no potential contamination which could have affected the PCR results obtained. Looking at the graph (Figure 2), a relationship between the DNA molecular weight and the distance it travels can be clearly seen. We can also see a negative correlation as the distance migrated increases, the molecular weight of the DNA decreases. The graph almost shows a straight line which also assumes that the relationship is also inversely proportional. I have measured the distance in which the PCR products of Sample 2 have migrated which is 6.1cm. This is indicated on the graph and the approximate molecular weight of the DNA can be determined. From the graph, it shows that the molecular weight in log10 is 2.4bp. To convert this, we take the inverse log of 2.4 and we get 251bp for DNA Sample 2. This is a rough estimate which is in close proximity to our value 227bp that we are looking out for. Therefore this has proved we have shown the presence of the human “transgene” of the correct size. The distance DNA molecules can travel through agarose gel depends on the size of DNA molecules in electrophoresis. The agarose gel acts as a sieve for DNA molecules so that larger molecules have difficulty moving through the gel matrix compared to smaller molecules which can move more freely. Therefore, smaller fragments of the DNA are able to migrate further through the gel towards the positive end as DNA is negatively charged. This process allows the separation of large and small DNA fragments which can then be analysed and identified. We have used a molecular weight marker alongside our DNA samples which is a mixture of DNAs with known molecular weights; we can therefore use this marker to estimate the size of our DNA fragments. In conclusion, I have successfully shown that the PCR had worked in which the sample of mouse genomic DNA containing a human “transgene” had been identified and that the electrophoresis results have given me a set of good reliable results.
