DNA Structure and Analysis

1. DNA Structureand Analysis Activity 4.5: Forensic DNA Fingerprinting

2. 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

3. 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

4. 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)

5. Student Workstation Materials

6. Setting Up a Restriction Digest • Play video: Restriction Digestion of DNA

7. Running an Agarose Gel • Play video: Agarose Gel Electrophoresis

8. 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

9. 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

10. 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

11. 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?