Biology High School students from Delaware Valley Friends School (DVFS) visited Bryn Mawr College on November 4 and 18, 2005 to take part in Molecular Biology/DNA Fingerprinting and Forensic Anthropology/Human Evolution labs. The Forensic Case introduced below tied the two labs together by giving the students an investigative missing persons/possible homicide case to try and solve using scientific methodologies. Photo's of the students in the Molecular Biology/DNA Fingerprinting and Forensic Anthropology labs.

The Forensic Case the students studied:

As members of a team of the forensic scientists working for Montgomery County, you and your colleagues have been contacted by local law enforcement agents to examine and identify human remains recovered at a potential crime scene. Two days after she was last seen leaving work in her 1994 Ford Escort, a woman was reported missing by her mother. Local and national searches failed to locate the woman or her car and the woman's disappearance remained a mystery. Two years later, the missing woman's car was discovered at a storage facility thirty miles from where she was last seen. When the rental payments for the storage facility became delinquent, an attendant entered the storage unit where he discovered a red 1994 Ford Escort. The attendant became suspicious due to the condition of the car- the windshield was cracked, the driver's window was missing and a large amount of glass and debris was uncovered inside the car. The serial number and license number revealed that the car belonged to the missing woman and her husband. Further records showed that the husband rented the storage space the same day his wife was last seen. In addition to the glass and debris on the floor, human remains were recovered in the car. The local law enforcement agents have turned over the human remains and debris to you in several small containers. The remains included thirty-four fragments of what appear to be human bone. Several of the fragments exhibit what appear to be dark areas on their surfaces that appear to be lead stains. Samples of dried bloodwere collected from the car.

Results:

Forensic anthropologist finds that the fragments are human and were most likely from the skull. The dark staining was in fact lead, probably resulting from impact of lead pellets from a shot gun. There were two bloodstains found and the DNA was extracted. One of the stains was found to match some DNA extracted from a hair follicle from the wife’s hairbrush, linking her to the car. The other bloodstain was able to exclude the father, but unable to exclude the husband.

Questions to be answered:

• Are the bones human? (Nov.18th)
• Biological profile (age, sex)
• Can DNA be recovered from blood?
• Comparative DNA sample (ie. no known sample of deceased's blood; deceased was adopted, so cannot use parents' DNA, but could get samples from defendant and deceased's two children).

("Small bones of contention" In Hard Evidence: Case Studies in Forensic Anthropology, D.W. Steadman, editor, pp. 234-243. Upper Saddle River, NJ: Prentice-Hall, adapted from Stout, S. 2003)deceased's two children).

Outline of the days events:

A. Laboratory Session One, 9:30-11:00 A.M.

1) Introduce the Case

2) Talk about different strategies for solving the case.

• molecular Biology, Nov 4
• forensic, Nov 18

3) Talk about the starting materials

• DNAextraction from Strawberries
• Highlight the case of DNA extraction using these "household" ingredients
• Can practice using a pipette (if time)

B) Lecture Session, Gene Regulation in Eukaryotes, 11.10 am-12:00 pm

C) Laboratory Session 2, 1 pm – 2:15 pm

• Continue with case
• See if the students can tell one strawberry’s DNA from another by looking at it in their respective tubes, lead discussion to discuss the concept that different people’s genomes are different sequence-wise
• Move onto discussion about restriction enzymes and how we can exploit them to show these differences in DNA sequence
• electrophoresis
• Talk about how it works
• Students load a sample (of BPB dye) onto a pre-made gels (one per group, max of four groups)
• Show students a completed run under UV light, discuss how we visualize the ETBr rather than the DNA
• Discuss Sizing of DNA using a Molecular weight standard (use wife’s hair identity to bone for example)
• Tie into case, as to who can be implicated. (Have students work on a virtual gel on board, give students list of band sizes (from various “individuals” (i.e. husband, wife, father etc… they put them up on board, then instructor put up blood stain profiles (for two stains). Discuss.

Laboratory Protocols, DNA Isolation using Strawberries:

1. Place 1 strawberry in the plastic bag. Remove air and seal.
2. Mash strawberry for 2 minutes.
3. Add ~10 ml DNA Extraction buffer (Pre-measured). Remove air and seal.
4. Mash for 1 minute.
5. Pour solution through coffee filter into paper cup
6. Pour ~2 ml of this solution back into the 15 ml tube. It will be very red.
7. Pour ~ 4 ml (2X the volume of strawberry solution) of cold ethanol (found on freeze door in 50 ml tubes) so that it lays on top of the strawberry solution. (NOTE: can use 1X volume of room temperature isopropanol, rubbing alcohol, as a substitute)
8. If the tube just sits, there will be DNA at the interface with bubbles in it. If you gently rock the tube, more will come out of solution and eventually float to the top. (NOTE: if the DNA doesn’t come out of solution at home, add a little more salt)

Extraction buffer:

100 ml shampoo without conditioner or dishwashing liquid

900 ml distilled water

15 grams NaCl

Supplies:

Frozen Strawberries: allow to come to room temperature, or at least un-freeze.

Sandwich bags (good ones with good zip-locks)

Cheese cloth or coffee filter

Funnel or aluminum foil (to make a funnel)

Cups, or flask, 15ml centrifuge tubesQuestions and Answers :

Strawberries? 8 times the amount of DNA of normal cells

Why frozen? Ice disrupts cells. Can use fresh strawberries, too. Cheap!

Detergent? To disrupt cell membranes to liberate the DNA

Salt? To shield the negative charges on DNA to allow precipitation

Alcohol? To cause DNA to precipitate

Friday November 18, 2005 Forensic Anthropology/Human Evolution taught by Professor Melissa Murphy, Department of Anthropology.