MD105 - Cellular Biology Laboratory Forensic DNA Fingerprinting (Fall 2024) PDF

MD105 - Cellular Biology Laboratory Lab Exercise 3: Forensic DNA Fingerprinting Fall Semester 2024  Forensic DNA Fingerprinting Theoretical background: a) Introduction to DNA Fingerprinting b) Restriction Digest of DNA Samples c) Agarose gel electrophoresis pr...

MD105 - Cellular Biology Laboratory Lab Exercise 3: Forensic DNA Fingerprinting Fall Semester 2024  Forensic DNA Fingerprinting Theoretical background: a) Introduction to DNA Fingerprinting b) Restriction Digest of DNA Samples c) Agarose gel electrophoresis principle Methodology and protocol: a) Sample and agarose gel preparation b) Electrophoresis of DNA Samples c) Analysis and interpretation of results  The Crime ▪ An EUC Medical student left for a lab session never to be seen alive again. ▪ He was found brutally stabbed at the parking lot of the university. ▪ The CSI Nicosia Department has requested from his fellow Medical students to support their efforts in finding the murderer. ▪ The students gladly agreed to aid.  The Clues ▪ Evidence indicate an intense fight. ▪ DNA has been collected from skin cells under the victims' fingernails.  The suspects There are five suspects as they knew the schedule of the victim: S1:The heartbroken ex-girlfriend S2:The jealous brother S3:The girlfriend S4:EUC campus security S5:The father of heartbroken ex-girlfriend  The plan ▪ The students discussed together as a team a way to solve the mystery. ▪ They recall all the experimental procedures they had learned and finally they found the way. ▪ DNA from suspects has been isolated and will be compared to the DNA found at the crime scene with RFLP technique. ▪ DNA fingerprinting is a laboratory technique used to establish a link between biological evidence and a suspect in a criminal investigation.  Restriction Fragment Length Polymorphism (RFLP) ▪ A type of polymorphism that results from variation in the DNA sequence recognized by restriction enzymes. ▪ A short genomic site of four to eight nucleotides long is polymorphic when it differs among individuals. ▪ The DNA is then treated with restriction enzymes that cut the DNA into fragments of various length. ▪ The resulting fragments are separated according to their lengths, and the pattern of fragment sizes will differ for each individual tested.  Experimental procedure CSI Nicosia has supplied us with 6 PCR products (containing the RFLP site of interest) from DNA that belongs to the suspects and the DNA found on the victim. Aim: To perform restriction digest with a set of enzymes and compare the pattern of the bands to reveal the murderer. RFLP markers inheritance & test validity  Agarose Gel Electrophoresis Principle ▪ Agarose gel electrophoresis is a technique to separate nucleic acids based on their size. ▪ The separation occurs when nucleic acid molecules migrate through an agarose gel under the influence of an electric field (cathode (-) → anode (+)). ▪ Agarose is a polysaccharide derived from seaweed. ▪ To visualize DNA, it needs to be stained with dyes such as RedGel, which binds between DNA bases and fluoresces under UV light. ▪ A tracking dye (e.g., loading dye) is mixed with the DNA sample, to monitor the progress of electrophoresis since DNA is colorless.  Experimental procedure PART A: Restriction Digest Using a fresh tip for each sample, pipet 10 µl of each DNA sample from the stock tubes and transfer to the corresponding-colored microcentrifuge tubes. Make sure the sample is transferred to the bottom of the tubes. Pipet 10 µl of enzyme mix (ENZ) into the very bottom of each tube. Use a fresh tip to transfer the ENZ sample to each tube. Pipet up and down carefully to mix well. Tightly cap the tubes and mix the components by gently flicking the tubes with your finger. If a microcentrifuge is available, pulse spin in the centrifuge to collect all the liquid in the bottom of the tube. Otherwise, gently tap the tube on the tabletop.  Now your DNA samples should contain: Incubate the tubes for 45 min at 37°C waterbath. Using a separate tip for each sample, add 5 µl of loading dye "LD" into each tube. Cap the tubes and mix by gently flicking the tube with your finger. Collect the sample at the bottom of the tube by tapping it gently on the table or by pulse-spinning in a centrifuge.  Experimental procedure PART B: Agarose Gel Electrophoresis and Visualization of DNA Fragments Prepare a 1% agarose gel by adding 100ml Tris-Acetic acid-EDTA buffer (TAE) to 1g agarose in an Erlenmeyer flask. Place the flask in a microwave or on the heat until agarose is melted. Stop periodically and swirl the solution. Do not permit to boil over. Assemble the casting tray by blocking the ends with tape or plastic baskets. Place the comb into the casting tray at the upper side. Add 10μl of GelRed. Pour the solution into the tray and wait until the gel is solidified. Remove the comb and place the casting tray into an electrophoresis chamber.  Cover the gel with TAE buffer. Using a micropipette, load 20μl dye samples sequentially into the wells. Cover the electrophoresis chamber with the lid and ensure good contact between electrodes. Set the power supply to 100-120V, press the Run button (you should see bubbles at each electrode), and allow to run for at least 40 minutes. After 40 minutes, stop the current and remove the gel in the casting tray. Slide the gels into the staining solution for visualization. The instructor will slide the gel onto a UV transilluminator behind a shield and show the results to the class. Document the findings of the gel by photographing with your phone. The instructor will discuss the results and ask for you to interpret the findings.  RESULTS Ladder CS S1 S2 S3 S4 S5  QUESTIONS Which of the following is FALSE? A. DNA is cut into fragments using restriction enzymes. B. Each restriction enzyme cuts DNA at a specific base sequence C. Fragments are separated based on charge using a process called agarose gel electrophoresis. D. DNA fragments are injected into the wells and an electric current is applied along the gel causing the negatively charged fragments to move.  QUESTIONS What is the purpose of Agarose Gel Electrophoresis? A. It helps cut the DNA B. It counts the genes in DNA C. It separates DNA based on the size D. All the above  QUESTIONS Which of the following is FALSE? A. Hydrogen bonds hold the DNA strands together. B. During gel electrophoresis, DNA moves from the negative end to the positive end. C. Gel electrophoresis enables scientists to combine DNA fragments. D. Restriction enzymes allow the strands of DNA to be cut into various lengths for testing.