Multiple results were obtained from taking part in the PCRexperiment in identifying from a number of samples which caused food poisoning.The migration for all bands were measured using a ruler and a picture of thegel electrophoresis (figure 1) this was measured consistently so the resultscould be precise and accurate. The band sizes were converted to log as thisreduces the amount of errors (Schaffer andSederoff, 1981). Overall the results were fairly reliable and nooutliers were seen this is backed up by the calibration curve that was plotted(figure 2) as this produced a R2 value of 0.9459 which is fairlyclose to 1 which would show total accuracy.
The calibration curve was used toidentify the cause of poisoning for the victim which showed a migration of 30mm(400kb) which matched sample Y this was confirmed by rearranging theequation which was (2.536-4.5359)/-0.0645=31mm.
The log band size received for X, Y,Z and the victim using thecalibration curve (figure 2) showed that X had a log of 1.891 (78.5 kb), Y hada log of 2.
600 (398 kb), Z had a log of 2.794 (622 kb) and the victims samplehad a log of 2.536 (343.5 kb).This confirms sample Y and the victims sample were theclosest in terms of visually on figure 1 and through calculations the victimsband spread to 343.5 kb and sample Y spread 398 kb which is a difference of54.5 kb which is far closer than any of the other samples (Devonshire, et al2014). This shows that the source of poisoning was caused by a drink given tothe victim Willy ‘the whale’ Thomlins at a bar by blonde bombshell Iva ‘notrust’ Inhera which was also seen celebrating with a player Vincent ‘hacker’Jones from the opposing team Grimthorpe’s after their victory.
This proves thefoul play that was reported. The results were compared to previously publishedliterature so that a better understanding of the results could be seen in whichthe previously published literature uses different blood splatter to match tothe positive control. When compared to the results on figure 1 the literatureshowed a distinct band that matched that of the positive control which was inline with the results received in figure 1 as sample Y matched the victims DNA.(Power, et al. 2010). Figure 3 shows how the PCRreaction would produce DNA fragments of different sizes using the same set ofprimers in each case.
The purity and yield of the products being formed may beaffected by many factors including the annealing temperature which may producenon specific products, primers that are too short may anneal at multiplelocations on a DNA template strand that is long. The melting temperature of aprimer will also need to be taken into consideration as this may producenon-specific products whether too high or too low in which the annealingtemperature is usually 5oC below melting temperature. If theannealing temperature is incorrect then this would mean primers may bindnon-specifically or may not bind at all (Rychlik, et al. 1990).
Denaturation, the step beforethermal cycling separates the two DNA strands so that the primers can anneal,and elongation is sufficient enough to occur. The reason as to why denaturationoccurs for only 1 minute is, if the DNA is denatured for too long then theprocess may inactivate the DNA polymerase (Taq) (Lorenz, 2012). The temperature at the denaturation will be95oC as this is the most optimum for both the DNA polymerase and theguanine cytosine content of the template strand of DNA. Thermal cycling willuse 3 temperatures which will be 94oC, 50oC and 72oCthis will be done for 15 seconds for the first two and 10 seconds for the lasttemperature a total of 30 cycles (Liew, et al. 2015).
Increasing the number ofcycles above 35 will mean that the a large amount of PCR products will beproduced alongside the production of unwanted secondary products. The reason asto why there are three different temperatures in thermal cycling is to denaturethe template strand, optimal annealing of primer and lastly the synthesisationof the PCR product by DNA polymerase binding to the template strand. Thetemperature and the time may be altered to produce certain amplicons. The firstdenaturation temperature and time is kept as this is optimal for the DNAtemplate. The next part includes a final extension which allows for thesynthesis of uncompleted amplicons and since Taq polymerase will be usedadenine residue will be added to the 3′ end of the PCR products. Lastly a holdis achieved by cooling to 4oC (Lorenz, 2012).