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

2.  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.  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.  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.  Place the DNA source (green peas) in the blender and add some water.  Liquefying will break the cell walls and disrupt plasma membranes.

6.  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=Rl5EmUQd kuI&feature=related

7.  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.  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 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.  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.  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.  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.

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

15.  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.  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!