Lab 5 – DNA Fingerprinting

Lab 5 – DNA Fingerprinting

Introduction to DNA Fingerprinting

  • Living organisms exhibit remarkable adaptations to survive and reproduce in their environments.

  • The relationship between organisms and their environments leads to evolutionary changes.

  • Evolution is evidenced by studies in taxonomy, comparative anatomy, and the fossil record, showing that species are not constant but evolve over time.

  • Environmental changes can lead to extinction, with 99% of species that have lived being extinct.

  • Charles Darwin proposed in 1859 that evolution occurs via natural selection, where environmental factors influence which traits are preserved.

  • This concept unifies many biological principles and offers explanations for various biological patterns.

Lab Learning Objectives

  1. Explain the concept of RFLPs (Restriction Fragment Length Polymorphisms) and how they are used to create DNA fingerprints.

  2. Extract DNA from onion cells.

  3. Use gel electrophoresis to create and separate DNA samples into RFLP bands.

  4. Interpret and analyze DNA fingerprint patterns from the experiments conducted.

Part I: DNA Fingerprinting

  • DNA fingerprinting involves identifying individuals based on their unique RFLPs.

  • Applications of DNA fingerprinting:

    • Forensic analysis of blood, hair, saliva, and semen at crime scenes.

    • Determining familial relationships in paternity disputes.

    • Tracking hereditary diseases.

    • Identifying organ transplant compatibility.

    • Assessing inbreeding levels in endangered species.

  • The technique of DNA fingerprinting relies on the different lengths of DNA fragments created by restriction enzymes.

    • Definition: RFLPs are variations in DNA sequences that result in fragments of different lengths after the DNA is cut with restriction enzymes.

Mechanism of DNA Extraction and RFLP Analysis

  1. Extraction of DNA

    • DNA is typically extracted from white blood cells or other tissues.

    • A restriction enzyme cuts DNA at specific sequences of 4-8 bases, producing fragments of varying lengths.

    • The polymorphism refers to the variations in length due to the different spacing of cut sites in various individuals.

  2. Research Background of RFLPs

    • Alec Jeffreys discovered that introns (non-coding regions of DNA) contain repeating base pair sequences that vary among individuals.

    • The number of these repeats can differ significantly, leading to unique DNA fragment lengths post-restriction enzyme treatment.

    • The technique of gel electrophoresis is utilized to separate these DNA fragments based on their lengths:

      • An electric field moves negatively charged fragments toward the positive end of a gel, allowing shorter fragments to travel further.

      • Resulting bands indicate fragments of similar length and can be visualized using ultraviolet light.

    • Jeffreys termed this process "DNA fingerprinting" due to the distinct banding patterns seen in individuals, except for identical twins who share the same patterns.

    • Important Note: Restriction enzymes are found in bacteria, evolved as a defense against viruses, with over 600 types commercially available.

Exercise 1 – Extraction of DNA from Cells
Summary of Extraction Method

  • Onion cells present challenges due to their cell walls:

    1. Blender Usage: Breaking the onion cells releases cellular contents.

    2. Preparation of Lysing Solution: Contains:

    • A detergent to disrupt nuclear membranes.

    • Sodium chloride to clump DNA for precipitation.

    • Citrate ions to inhibit DNAase activity on DNA.

    1. Heat Treatment at 65°C: This denatures DNAase (the protein) without harming the DNA.

    2. Filtration Process: Plant debris is filtered out, and chilled alcohol is added to precipitate DNA since DNA is soluble in water but not in alcohol.

    • Upon contact with alcohol, DNA precipitates, allowing for extraction using a spooling rod.

Detailed Extraction Procedure

  • Work in pairs to perform the following:

    1. Transfer 7 ml of liquefied onion to a 20-ml test tube.

    2. Add 7 ml of cell lysis solution, resulting in a two-thirds full tube.

    3. Invert the tube gently to mix.

    4. Heat in a water bath at 65°C for 15 minutes.

    5. Filter mixture through a cheesecloth into a larger test tube.

    6. Carefully layer 10 ml of chilled DNA precipitation solution on top of the lysate at a 45° angle.

    7. Gently turn a glass rod in the interface to extract DNA.

    8. Describe the extracted DNA for evaluation, ensuring it is not used in further exercises (specifically Exercise 3).

Exercise 2 – Practice Using Pipettes

  • Loading DNA into wells of a gel requires precision.

  • Practice using micropipettes on a petri dish containing agar wells, filled with loading dye, is necessary.

  • Proper technique involves:

    1. Using a fresh pipette tip for each dye sample.

    2. Carefully introducing the tip without puncturing the well.

    3. Slowly expelling the dye to ensure it sinks correctly.

Exercise 3 – DNA Fingerprints of Unknown Samples

Steps for Conducting DNA Analysis

  1. Preparing the gel for electrophoresis:

    • Acquire a sealed E-Gel.

    • Attach it to a power base for operation.

    • Ensure proper insertion indicated by a steady red light.

  2. Uncovering Wells:

    • Remove the plastic comb carefully to open the wells.

    • Dispose of the comb properly.

  3. Sample Loading:

    • Obtain and load:

      • 20 µl of DNA from Suspect #1 into well #2, and continue for Suspects #2, #3, and #4 in specified wells (4, 6, and 8).

      • Record the color of the crime scene sample.

  4. Running the Gel:

    • Initiate the electrophoresis run for 35 minutes.

  5. Stopping the Current:

    • The power base signals the end of the run; turn it off.

  6. Viewing Results:

    • Remove the E-Gel and observe under ultraviolet light for the banding patterns.

Questions for Analysis

  1. Which samples indicate that they are from the same individual based on RFLP banding patterns?

  2. Describe the implications of forgetting to add the restriction enzyme before loading into the gel on the banding patterns observed.
    Show the test tube containing the extracted DNA and the finished electrophoresis results to the teaching assistant (TA) for evaluation and feedback.