IntroductionPolymerase chain reaction (PCR)is technique in biochemistry to amplify a single or few copies of DNA. The invention of PCR was discovered in 1993by Kart Mullis due to it he won a Nobel prize in chemistry. Plasmid is a small circular DNAof a bacterium which is independent form chromosomal DNA and it can replicateitself. Bacterium can be manipulated by inserting a foreign DNA in to thesection of the plasmid. The three plasmids used for this PCR reaction arecSox1, cWnt1 and cMyod. It’s possible to amplify a certain of a plasmid sincethe primer binds to the vector they can used regardless of the insert sequencetherefore they are called “universal primers”.
Performing PCR requires 3 steps.The first one is denaturation which is specifically for breaking the hydrogenbonds between the double stranded DNA molecules, this is by applying some heatwith a temperature of 95°C for about 15 seconds. After separation of the doublestrand, the second process of PCR is annealing which binds the primers ontostrands of the DNA by cooling the heated solution to a temperature around 57°Cfor about 15 seconds.
The primers used in this experiment was M13Foward (5′-CCAGGGTTTTCCCAGTCACG-3′) and M13Reverse (5′-TCACACAGGAAACAGCTATG-3′). Primerattaches itself into the opposite strand of the vectors DNA by doing itspossible to amplify the selected area of the DNA. The step that ends theprocess is called extension, Taq polymerase can withstand high temperatures thatit is ideal for PCR. used nucleotides to the correct base pair on the DNA. NewDouble stranded DNA segment is created by Taq polymerase by building eachsingle strand of DNA marked by the primer. The polymerase begins DNA synthesison the primers and elongate in the 5′ to 3′ direction on both ends so thecooled solution is heated for 1min to a temperature of 72°C. The result of thePCR produces exponential amplification so at 36 cycle there is 68 billioncopies of the selected DNA.
Gel electrophoresis is used toseparate the DNA sample as it is negatively charged thus applying an electricfield will allow DNA to move through the gel, consequently smaller DNA fragmentmoves further away whereas larger DNA fragment travels less. MethodIn this experiment contaminationneeds to be avoided since PCR is a highly sensitive method so wearing glove isnecessity for this experiment.Firstly, PCR tube was labelled as1, 2 and 3.
In each tube 9.5µl of water, 12.5µl 2X Biomix red, 1 of M13F and M13Rprimers was added.
1µl of the appropriate plasmid DNA (1,2,3) where each PCRtube was containing only one type of plasmid)Secondly, A fresh tube waslabelled as N, the 1 µl plasmid DNA was replaced by 1µl of water. Thus thenegative control contained 10.5µl of water,12.5 µl 2X BioMix Red (Taqpolymerase, dNTPs, buffer),1 µM13R primer (50 µM), 1 µl M13F primer (50 µM) The tubes were then placed in thePCR machine and the following programme was running.
There was initialdenaturation for 1 minute at 95°C then 25 cycles of denaturation taking place15 seconds at 95°C, primer annealing of 15 seconds also at 57°C and extensionof 1 minute at 720C. Finally, the final extension of primer took 5 minutes at72°C The gel solution is poured ontothe electrophoresis tray and enough buffer was added to cover the gel. 12 ?l of DNA molecular weight marker of 1kb ladderis loaded onto the first well, pipette 8 ?lof each PCR samples onto gel in the following order of N,1,2 and 3. Electrodesare placed on each end, cathode being near the wells and anode being furtheraway from the sample, the Gel was run at 120V until the red dye was about 2cmfrom the bottom. Ethidium bromide is added to the solution so when the UV hitsthe sample the DNA fragments becomes fluorescent. Resultsand Discussion I decided to use linear regression to estimate thekilobase as some of the fragments are larger than the ladder due to thismigration from well to fragments midpoint was used to calculate kb.
Above is the graph between log10(kb) againstmigration although the R2 of the linear regression (R2=0.9886)is lower compared to 5-degree polynomial regression (R2=1), due tothe error caused by the fragment size height compared to the ladder, the linearregression model yielded a better result. Migration 1(mm) Log10(kb) Kb 27 0.347 2.22331 48.5 -0.
36465 0.431867 Migration 2(mm) 24 0.4463 2.794474 55 -0.5798 0.263148 Migration 3(mm) 24 0.4463 2.794474 38 -0.
0171 0.961391 47 -0.315 0.484172 55 -0.5798 0.263148 The result of the gel electrophoresis shows differentsize fragment due to the three plasmids used as template, cSox1, cWnt1 andcMyoD. The negative control is used to identify contamination in the resultingexperiment, since there is no fragment in the coulomb his is to be expected.The intensity of the band is due the abundance of the DNA fragments, this isthe result due the PCR amplifying the insert.
The amplified DNA fragment was much thicker which may cause it tooverlap with some fragment that are bit bigger or smaller. On the PCRproduct 1, band on 0.431867 kb is brighter than the band on 2.22331 kb, this shows that the insert isamplified by a lager factor and since the insert is ?0.6kb (600 base pairs) so it is cWnt1 plasmid DNA. Whereas PCR product 2, band on 0.
263148 kb is more fluorescent than the bandon 2.794474 kb, sothe insert at ?0.3kb (300 base pairs) has been amplified by PCR whichshows that this is cSox1 plasmid DNA.
On PCR product 3, there is an unexpected result as it wasexpected to be two bands but the PCR process amplified the correct insert 0.961391kb which is roughly 1kb(1000 base pairs) this show that this is plasmid DNA cMyoD. But pBluescript is expected to be ?3 kb long but there are few fragments in 0.3 kb and 6 kb,this could be due to various such as primer binding to other site where the itonly few cycle of amplification occurred, or nucleotide interaction in thesolution. ConclusionPCR can be used to amplify a wanted DNA segment, for thecase of this experiment we can amplify a certain wanted DNA fragment as aninsert which can be used to modify the plasmid of bacteria. This process isused to create insulin, which helps regulate blood glucose level, by adding acertain segment of human DNA which contributes to the production of insulin tothe bacterium’s plasmid and let bacteria grow into colonies to produce insulin.By using linear regression, the kb ladder was modelledwith respect to the migration of fragment from well, this was because of thecompressed spacing and faded band causing the band’s position relative to thekb ladder to be in accurate.
Using the modelled linear regression line the PCRproduct 1,2 and 3 was predicted which gave an fairly accurate result and helpidentify the size of the figments as well as the different plasmid DNAs usingthe size of the insert.The result consist of various error as some of the bandwas very faded and the space between were very compressed resulting in beinghard to identify the correct base pairs. The spacing between the bands can beincreased by increasing the electric field between the cathode and anode, byrising the voltage between them.