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DNA Fingerprinting

A modern tool for genetics testing, forensic science, and research. DNA Fingerprinting. Overview. DNA Fingerprinting is an applied genetic testing utility for issues in: Criminilistics (Forensic Science) Clinical Testing (Genetic disorders, cancer markers, and paternity)

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DNA Fingerprinting

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  1. A modern tool for genetics testing, forensic science, and research. DNA Fingerprinting

  2. Overview • DNA Fingerprinting is an applied genetic testing utility for issues in: • Criminilistics (Forensic Science) • Clinical Testing (Genetic disorders, cancer markers, and paternity) • Research (DNA purification and comparative analysis of DNA)

  3. Process • DNA Extraction and Purification • DNA is retrieved for analysis. • Ensures that only DNA is being analyzed. • DNA Fingerprinting (Electrophoresis) • DNA is fragmented. • DNA fragments are separated by mass. • Samples of the same DNA have the same pattern on the agarose gel.

  4. Extraction • Several steps are taken to retrieve the DNA and purify it. • The cell wall is destroyed and the plasma membrane is disrupted. • The nuclear envelope is disrupted. • Histones are broken to free the DNA. • Proteinaceous materials are salted out. • DNA is extracted and thus purified.

  5. Step I – Bust up the Cell Walls • Place the DNA source (green peas) in the blender and add some water. • Liquefying will break the cell walls and disrupt plasma membranes.

  6. Step II – Membrane Deterioration • Detergent will be added to disable the lipid interactions that create the plasma membrane and nuclear envelope. • The effect is much greater than the breaking of the cell wall for disruption. http://www.youtube.com/watch?v=Rl5EmUQdkuI&feature=related

  7. Step III – Denaturing of Histones • DNA is wrapped into a histone/DNA complex that forms chromosomes. • Protease (protein hydrolyzing) enzymes are added to destroy the histones and free the DNA molecules. • The proteinaceous debris is then salted out of solution (normally). http://www.youtube.com/watch?v=RMZis3OlWFk

  8. Step IV – DNA Retrieval • DNA is forced out of solution by adding isopropyl alcohol. • The white precipitate formed is the DNA from the cells. • It can now be used for analysis.

  9. DNA Fingerprinting • DNA Fingerprinting is a genetics utility used to compare the variable number tandem repeats (VNTR) in the genetic code of an individual to a sample of another individuals genetic code. • Two methods: • PCR (Polymerase Chain Reaction) • Electrophoresis with Restriction Enzyme Treatment

  10. Electrophoresis Method • We will use the electrophoresis method that uses a restriction enzyme on the VNTR genes. • There are five samples to compare with the sample collected at the crime scene.

  11. How it Works • We lyophilate (re-hydrate) the lyophilized (freeze-dried) DNA samples. • We treat them with a restriction enzyme that chops up the VNTRs into different sized chunks depending on the genetic code of the individual. • This is done in an incubation at 37°C.

  12. How it Works • Samples are then transferred to an agarose gel after being mixed with a dye. • The gel is subjected to electrophoresis which causes the different sized chunks to move opposite the electric field generated by the potential difference. • The gel is then stained and the bands are compared. The bands which line up the most are the ones which are the same DNA.

  13. Example Gel

  14. Note on the Gels • Not all gels have the same order of the bands. Therefore, you must compare the bands you see and note the ladder.

  15. Acknowledgements • American Society for Biochemistry and Molecular Biology – UAN Outreach Award • James Ventrice – Genetics Lab Coordinator, Department of Biology • Dr. Dale Ensor and Dr. David Crouse – QUANT and Organic Lab Coordinators

  16. Safety Orientation • Wear goggles at all times during the pea experiment. These will be provided. • Only touch those things that we tell you to for your own safety!

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