DNA Structure and Analysis Activity 4.5: Forensic DNA Fingerprinting

Activity 4.5: Forensic DNA Fingerprinting Research Question: Which suspects can be eliminated from the investigation? Objectives: Digest DNA samples from the crime scene and the five suspects with EcoRI and PstI restriction enzymes Load and run samples on a 1% agarose TAE gel Generate a standard curve based on lambda HindIII DNA standard Determine the length of all DNA fragments by comparison to the standard curve Determine if the DNA from the crime scene matches the DNA from any of the suspects

Safety Reminders Follow all laboratory safety procedures Appropriate PPE should be worn at all times Attention should be given when using electricity in the presence of liquids. If there are leaks in equipment, notify teacher immediately Turn off power before removing lid and ensure that lid is in place before turning power on If ethidium bromide or SYBR® Safe stain is used, follow appropriate safety precautions

Skills to Master Perform a restriction digest (Activity 4.4) Perform agarose gel electrophoresis (Activity 4.3) Analyze an agarose gel (Activity 4.4) Generate standard curve using a DNA standard (Activity 4.4) Determine DNA fragment sizes using standard curve (Activity 4.4)

Student Workstation Materials

Setting Up a Restriction Digest Play video: Restriction Digestion of DNA

Running an Agarose Gel Play video: Agarose Gel Electrophoresis

Protocol Highlights/Tips When setting up restriction digests use fresh tips each time to prevent contamination Tubes can be incubated in a water bath, dry bath, or at room temperature overnight If incubating overnight, it is helpful to incubate for a short while at 37ºC first, then let come to room temperature overnight If left too long, some enzymes exhibit STAR activity, which means they will cut in places that are not a complete match. So digesting longer than recommended times could give suboptimal results

Protocol Highlights/Tips Once the gel is stained and imaged, measure the distance that the lambda HindIII standard bands moved from the well Record all information into the notebook using a table similar to Activity 4.4 Measure the bands in the other lanes and record in the table

Protocol Highlights/Tips Graph the lambda HindIII control, using distance traveled as the X axis and DNA base pairs as the Y axis This can be done on semilog graph paper or with Microsoft Excel If Microsoft Excel is used, adding a trend line to the linear part of the curve and displaying the equation allows one to mathematically solve for y given x Convert all of the distance measurements into base pairs using the graph Lambda HindII Standard

Summary Make sure to: Record all steps in your notebook Ensure all lane contents are recorded in an organized manner in your notebook Measure bands using a ruler that measures using millimeters Measure to the leading edge of the band as it travels away from the well Compare crime scene bands to all other suspects Who can be eliminated?