Pcr Mandatory Lab Report On The Basis Of Genomics And Proteomics PDF

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Mandatory Lab Report on the basis of genomics and proteomics. ...

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Abstract This experiment aimed to amplify cDNA samples from purified plasmid DNA via polymerase chain reaction, PCR. By amplifying cDNA inserts and comparing molecular weight, with data from a similar electrophoresis, we could also distinguish which cDNA insert was present in samples of DNA. As the chosen method of amplification was PCR the need for primers was essential. For this purpose, we used the T7 forward primer and the SP6 reverse primer to instigate the development of the new DNA strands. The way in which PCR can increase the amount of a sample is quite straightforward and is completed in three steps; denaturation, annealing and synthesis. The results of the electrophoresis seemed to show the experiment worked well by showing three distinct bands at molecular weights of which was varied but expected. They were expected due to an earlier restriction enzyme electrophoresis which was carried out with the same DNA samples and gave molecular weight to match the ones in this experiment.

Introduction In any living organism, there are hundreds or even thousands of genes expressing and interacting at any given time. Traditionally techniques, such as RT-PCR or Northern Blot, used to measure gene expression are limited by the amount of data that they could obtain from one experiment (Burgess, 2004). These techniques have now evolved allowing molecular biologists to express many genes at a time, an example of this is PCR. Not only does it speed up expression but this rapid expansion of data allows the field of genomics to delve deeper to discover how these genes interact with one another, using additional techniques like a DNA microarray. Where principally the microarrays use unknown RNA or DNA to match up with known DNA by complementary base pairing or hybridization (Burgess, 2004). This experiment used PCR to amplify cDNA inserts that had been purified in a separate laboratory practical. The basis of this method involves four key ingredients; the DNA target sample that will be amplified (or in this case cDNA), primers, nucleotides and polymerase. Each ingredient plays an important role in each process of the PCR procedure which has around three steps and is repeated for multiple cycles until the amount of the sample desired is achieved. Each cycle starts with the separation of DNA strands, the denaturation, this occurs by an increase in temperature to around 95 degrees. By increasing the temperature, the strands are able to break the hydrogen bonds holding the two them together via the bases Adenine, Tyrosine, Cytosine and Guanine. This leaves two template strands; the process of annealing can begin. The primers are an essential step for initiating the formation of the new bands. To allows the primers to anneal to the flanking regions on the template strands the temperature is cooled to 54 degrees. Finally, the addition of free flowing nucleotides along the template strand can begin. By complementary base

pairing, the new strands are an identical copy of the original strands. The process is complete and has produced two new strands. After this, the procedure can be repeated to form even more copies of the original strands, starting with the denaturation of all the double stranded DNA. Primers are a significant part of a PCR because they initiate the transcription. T7 is one of the promoters used in this procedure and is an efficient promoter that is regulated by lacUV5, hence considered a lac derivative (Yali, 2006). The second promoter, SP6, is the sixth member of the SP family (Hertveldt, 2007).

Methods PCR reaction Mixture Three 0.2 mL microcentrifuge tubes were labelled 1 – 3 carefully to ensure recognition. Each tube was loaded with 5 µL of the appropriate DNA sample, A, B or C to each tube as outlined in figure 1. The rest of the reagents were added and thoroughly mixed with the ‘MyTaq Red Mix’.

0.2 ng/µl DNA 2 µM Primer Sp6 2 µM Primer T7 dH2O MyTaq Red Mix

Tube 1: DNA Sample A 5µl 5µl 5µl 10µl 25µl

Tube 2: DNA sample B 5µl 5µl 5µl 10µl 25µl

Tube 3: DNA Sample C 5µl 5µl 5µl 10µl 25µl

Figure 1. The guidelines for the reaction mixtures that were made up in each microcentrifuge tube.

Once the reaction mixes were complete, the three tubes were placed in the PCR machine. The PCR reaction took around 50 min and followed the steps; initial denaturation, denaturation, annealing, DNA synthesis and the final DNA synthesis. These steps varied in temperature moving between 95°C to 72°C. Once the reaction was complete, the three tubes were removed from the PCR machine. 5 µl of each reaction mix was moved to one of three labelled 0.5 ml tubes and 5 µl of loading dye and 10 µl of dH20 were added to give a final volume of 20 µl. Preparation of a 0.8% agarose gel To prepare the agarose gel we firstly diluted 10x stock solution of TBE (Trisborate-EDTA) to 0.5x. We accomplished this by adding 475 mL of deionized water into a 500 mL measuring cylinder and carefully poured 25 mL of the 10x TBE buffer solution up to the 500 mL mark on the cylinder. We could then mix the Page | 2

solutions together by inverting the tube, using paraffin over the top to prevent spillage. 0.24 g of agarose was added into a 100 - 150 mL conical flask, after which 30 mL of 0.5x TBE buffer was also added. A piece of cotton wool was fitted loosely into the top of the conical flask. The agarose was dissolved by heating in a microwave oven, we were careful not to exceed the “Medium” setting on the microwave. Once the agarose had dissolved and there were no solid particles left, the mixture was left to cool to around 55°C. 30 µl of GelRed (a 1: 1,000 dilution) was supplemented to the liquid agarose and swirled in the flask to ensure thorough mixing. The gel was poured into the gel tray and the comb was inserted to create the wells. This was left to set for about 30 minutes. Preparing samples and loading on the gel The comb was removed from the agarose gel and the gel placed into the tray in the electrophoresis chamber, making sure the wells were at the negative electrode (The DNA will migrate towards the positive electrode as they are negatively charged). In the first lane, 10 µl of the DNA size marker was added, Hyperladder I. For the PCR reactions, we mixed the 20 µl samples in loading buffer and added 10 µl to each well. Once the lid was in place, we switched the power on and set it to deliver 100V. To check that current was flowing through the gel we looked for bubbles at the negative electrode. The electrophoresis took around 40 minutes.16. Once the electrophoresis was complete, the gel was placed under a UV-trans illuminator to visualise the DNA bands (Gel Red is excited [fluoresces] at 250 – 300 nm when bound to DNA).

Results PCR Gel

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Figure 2. Electrophoresis of the PCR The electrophoresis ran well, with each band surfacing very distinctly. As expected the electrophoresis shows three bands of various sizes, as the cDNA's were different sizes. Each tube only varied in DNA sample with tube one containing DNA sample A, tube two containing DNA sample B and tube three containing DNA sample C. Each tube also contained five microliters of each of the forward and reverse primers, ten microliters of dH20 and twenty-five DNA Hypp erlad der

Tub e1

Tube 2

Tub e3

Size BP 10,000 3,000 2,000 1,500 1.000 800

000

600 400

200

microliters of ‘MyTaq Red Mix’. From figure 2 above tube one seems to be 980bp in molecular weight, tube two is around 900bp and tube three is 1,700bp. and tube two has the target piece of DNA as it is the lightest and therefore can travel the furthest as the sample in tube two did. Restriction Enzyme Gel

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DNA Hyp er ladd Size BP

Hindlll

Ndel

A

B

C

A C

Ndel + Hindlll

B A

B

10,000 3,000 2,000 1,500 1.000 800 60000 400 200

Figure 3. Electrophoresis from varying restriction enzymes. Figure 3 shows the gel image obtained by electrophoresis. The plasmids in the last three lanes had been cut by restriction enzymes NedI and HindIII hence two bands were formed, they cut the plasmid in two places producing two strands of DNA. The top band indicates the plasmid and the bottom band indicates the cDNA insert cut from the plasmid. My approximations, using figure 3 and the hyperladder as a standard, of the size of each band are; sample A is 990bp, sample B is 910bp and sample C is 1800bp.

Discussion

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We can use these experiments as comparisons primarily because they have the same scale of measurement but also because the same DNA samples were used in both practical’s. By looking at the molecular weight that resulted from the restriction enzyme electrophoresis we can determine which samples contained which cDNA insert in the PCR. The basis of this comes from the knowledge that lighter segmentations of a sample travel further than heavier samples. The T7 forward promoter has the sequence, 5′ TAA TAC GAC TCA CTA TAG GG 3′ and the SP6 promoter has the sequence, 5′ ATT TAG GTG ACA CTA TAG 3′ (Yoon, 2016). The enzyme ‘Taq DNA polymerase’ was used to help the synthesis. As aforementioned, to decipher which cDNA insert was present in each DNA sample, in the polymerase chain reaction, we have to compare molecular weights. In the restriction enzyme experiment, the data used for comparison is the lanes that contain both the enzymes NedI and HindIII. This is because they are both required to cut the plasmid to attain the cDNA insert that we amplified for PCR. Hence the results in those lanes should be used as an accurate match for the results from PCR. Furthermore, it is important to state that we are looking at the second bands formed in the three lanes as these are the bands that contain the cDNA insert. The restriction enzyme gel gave the following results; sample A was measured at 990bp, sample B was 910bp and sample C was 1800bp. The PCR gel gave these results; tube one was at the 980bp weight, tube two was around 900bp and tube three was 1,700bp. In this case sample, A corresponds to tube one, sample B is the same with tube two and sample C with tube three. The information above corresponds with the expectations. Sample B has the cut segment of cDNA insert in it and travelled the furthest to around 910bp, the same as tube two, in the restriction enzyme electrophoresis, which travelled to 900bp. In both experiments, sample C had the heaviest sample and so travelled the least distance. Sample A is in between both but slightly closer to sample B. Showing that these bands are comparable has consolidated that the correct cDNA insert was isolated and therefore amplified. Thus the aims of the experiment were achieved.

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References 1. Burgess, J, Rapley, R, John Wiley &, S, & Harbron, 2004, Molecular Analysis and Genome Discovery, Chichester, West Sussex, England: Wiley, eBook Collection (EBSCOhost), EBSCOhost. 2. Hertveldt, V, C, De Mees, S, Scohy, P, Van Vooren, J, Spizer, C, Spizer, 2007, The Sp6 locus uses several promoters and generates sense and antisense transcripts, Biochimie, 89(11), 1381-1387. http://dx.doi.org/10.1016/j.biochi.2007.05.011 3. Yali, X, Rosenkranz, S, Chiao-Ling, W, Scharer, J, Moo-Young, M, & Chou, C 2006, 'Characterization of the T7 promoter system for expressing penicillin acylase in Escherichia coli', Applied Microbiology & Biotechnology, 72, 3, pp. 529-536, Business Source Corporate, EBSCOhost 4. Yoon, J.M, 2016, Genetic distances in Three Ascidian Species determined by PCR Technique, Development and Reproduction, 20(4), 379-385. http://doi.org/10.12717/RD.2016.20.4.379

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