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 2 Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 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 3 Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 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) 4 Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com Student Workstation Materials 5 Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com Setting Up a Restriction Digest Play video: Restriction Digestion of DNA 6 Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com Running an Agarose Gel Play video: Agarose Gel Electrophoresis 7 Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 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 8 Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 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 9 Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 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 10 Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 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? 11 Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com