IntroductionFor this experiment, we will be doing crime scene DNA profiling to determine the suspect who did the crime.
We will have the DNA samples from the crime scene and the four suspects DNA. DNA profiling will be carried out by using Polymerase Chain Reaction (PCR). Like DNA replication process, PCR allows small DNA samples to multiply into vast amounts by making copies of a specific DNA region. PCR requires DNA primers followed by DNA polymerase known as Taq polymerase to make new copies of DNA. Hence, this will allow investigators to compare and study the crime scene DNA to the suspects DNA easily.
The DNA samples will be further analyzed by using Gel Electrophoresis. Gel electrophoresis will cause DNA fragments to be separated based on their size. It will allow us to quickly compare the suspect DNA to the crime scene DNA by finding a match.
ProceduresPCR ReactionCollect all equipments required for the experiment (CS Tube, A Tube, B Tube, C Tube, D Tube and MMP Tube) and label them accordingly with our group name (RJ). Using a pipette, pipette 20 microlitre of MMP and add it into each tube (CS, A, B, C, D) respectively. Mix the contents well. Use a new pipette tip after each tube has had MMP added and mixed in well. During this procedure, the other remaining 4 tubes have to be placed onto a rack to prevent any mix up.
After which, the tubes have to be placed into a thermal cycler for 35 cycles that would take about 3 hours. Note the position at which your tubes have been placed. Gel ElectrophoresisTo prepare the agar, add in 3 litres of TAE electrophoresis buffer with 60ml of concentrate 50x TAE buffer and 2940ml of distilled water. Through heating in the microwave, dissolve 1x TAE electrophoresis buffer with 10.5g of agarose powder.
The microwave heating should be set at 60°C In the meanwhile, set the casting tray with the comb located at 1cm for easy reading. Once the gel is ready, pour 30ml of it into the casting tray and leave it to solidify for 20 minutes. After which, remove the comb from the casting tray that has the gel and subsequently placing the tray into the DNA electrophoresis chamber. Ensure that the chamber is levelled with the table and the wells are facing the cathode (black) end.
Fill the chamber with 1 x TAE running buffer till it touches the maximum line.In order to have a balanced microcentrifuge, place your PCR tubes collected from the thermal cycler into the pulse-spin. Ensure that the liquid is collected at the bottom of the tube at the end of the procedure. Next, pipette 10 microlitre of the loading dye into every PCR tube. Ensure that the liquid is thoroughly mixed by the pipette tip by the action of up and down mix.
Followed by placing the tubes in pulse-spin for a quick mix. The crucial part of this experiment is loading the samples into the wells that would determine your final result. The samples have to be added into the wells in a systematic order as follows:Allele Ladder with orange loading dyeCS DNA with orange loading dyeSuspect A DNA with orange loading dyeSuspect B DNA with orange loading dyeSuspect C DNA with orange loading dyeSuspect D DNA with orange loading dyeOnce the samples have been added into the wells without any spillage, run the gels for 100 volts for 30 minutes or till it reaches 7.
5cm (whichever gives a better result)After 30 minutes, remove the gel and place it into the tupperware with 1x Fast Blast Staining Solution for Overnight Staining Protocol. Cover the gel totally with the staining solution. Let it rest for 5 minutes before de-staining it using distilled water to check for the stained gel.Lastly, when you are satisfied with your destaining result, take a photo of it and explain it in your report. Results (Picture of the destained gel + Picture of labelled gel)Figure 1 : Destained Gel (With labelling) Lane 1: Allele Ladder with orange loading dye (from left)Lane 2: Crime Scene DNA with orange loading dyeLane 3: Suspect A DNALane 4: Suspect B DNALane 5: Suspect C DNALane 6: Suspect D DNADiscussion (Referring to Figure 1)The DNA samples from the crime scene have two bands.
Suspect A, B, C, and D also have two bands. Suspect C have the two bands which are similar to the ones at the crime scene. (same sized bands) Hence, we can conclude that the suspect who committed the crime is suspect C. It is a perfect match to the DNA samples from the crime scene. Suspect A have only one of the bands which are similar to the crime scene DNA. Hence, he or she is not proven as the suspect. There are common pitfalls that can be avoided or take note in the experiment.
We need to ensure there is proper loading of samples into the gel during electrophoresis. This is to allow proper separation of DNA fragments and to allow us to see the DNA fragments clearly. We need to avoid the DNA bands from being digested for a long period and an incorrect voltage applied during electrophoresis. We need to ensure that we are using a new pipette tip for different samples to prevent contamination of DNA samples by using the same tip.
QuestionsWhat is the genotype of each of the DNA samples? Whose DNA is left at the crime scene? Lane of gelDNA sampleGenotype1Allele LadderN/A2Crime Scene DNA7-3 (allele 7 + allele 3)3Suspect A10-3 (allele 10 + allele 3)4Suspect B5-2 (allele 5 + allele 2)5Suspect C7-3 (allele 7 + allele 3)6Suspect D10-2 (allele 10 + allele 2) Suspect C DNA is left at the crime scene. Suspect C DNA is the same as the Crime scene DNA by matching them. Each of their DNA had two fragments that each migrated displaying that they were the same size.What components do you need to perform a PCR and what is the function of each component? What is contained in MMP?We need DNA template, DNA polymerase, primers, Nucleotides (dNTPs or deoxynucleotide triphosphates) and reaction buffer. DNA template: sample DNA which contains target sequence.
DNA polymerase: An enzyme which new strands of DNA complementary to the target sequence such as Taq polymerase. Taq polymerase is a thermostable enzyme that will not denature at high temperature.Primers: short oligonucleotides of DNA, vary from 8 to 60 base pairs in length. Sequences can be randomized if the goal is for general genomic studies. For amplifying individual sections of the DNA in the genome, primers of specific sequences will be used instead. Nucleotides: building blocks for DNA replication Reaction buffer: Provides a stable pH.
Master mix primer(MMP) contains Taq DNA polymerase( dATP, dGTP, dCTP, dTTP), dNTPs, salt buffer, MgCl2 and additives in a buffer at a favourable concentration which is optimized for PCR. A salt buffer will maintain a neutral pH for the PCR reaction and magnesium chloride is a cofactor needed by DNA polymerase. Why do you need to run a PCR before gel electrophoresis?DNA is amplified as there is little amount of the sample available, hence, PCR needs to be run through. This procedure can be applied for anything from the hair follicle, fingernail, teeth, saliva, skin cells and other biological materials. PCR works by replicating the DNA to more similar copies to perform a forensics test. Explain in your own words what is the difference between allele, locus and loci? The difference in an allele, locus and loci is that an allele is a variation of a gene found in the chromosome whereas a locus is the location of the particular gene found in the chromosome and a loci is basically a plural form of locus. Explain why you should load the DNA samples at the cathode end of the electrophoresis chamber.For the separation of the DNA fragments, gel electrophoresis is used according to their charge and size.
DNA samples are negatively charged. It will be placed at the cathode end of the chamber. By placing at the cathode end, the DNA will move towards the anode end which is also the positive electrode of the electrophoresis chamber.
There is the same amount of charge per mass for all DNA samples. This will allow small DNA samples to move at a faster speed through the gel compared to the large ones. Explain the principle of gel electrophoresis in separating DNA fragments.In order to separate the DNA fragments, gel electrophoresis is used. After creating wells by using a comb and by solidifying the gel, the DNA samples are added into the wells via a pipette. They are seen as stains on the gel. The electric current is then run through at 100 volts from the negatively charged DNA to the positively charged DNA.
Once the gel has been run through thoroughly, it can be viewed under ultraviolet light after staining and destaining. The smaller fragments include less mass of DNA, as a result they would absorb less of the dye and hence, when viewed under ultraviolet light, there would be less fluoresce. Furthermore, the DNA ‘ladder’ also known as the DNA fragments are located at the extreme right and are used to identify the unknown fragments and their sizes.
ConclusionOverall, it was a rather successful and enriching experiment for us. We were able to get our desired destaining results in a rather short period. We were able to accurately deduce the correct suspect which is suspect C through the destained gel. Through this experiment, we were able to learn a real world application of PCR and gel electrophoresis. Referenceshttp://passel.unl.edu/pages/informationmodule.php?idinformationmodule=968252315&topicorder=3&maxto=11