DNA evidence has been submitted to your lab for analysis. There is one sample of the victim's DNA, one sample collected from underneath the fingernail of the victim, one sample of DNA from suspect #1, and one sample of DNA from suspect #2. The samples have all been prepared (solid matter was pre-digested ) by  a DNA technician. Standard DNA analysis techniques will be used to determine whether the material found underneath the victim's fingernail belongs to the victim, one of the suspects, or can not be identified, yet.

Humans have approximately 3,000,000,000 (3 billion) base pairs of DNA, but only 2-3% of this DNA actually codes for genes.  The function of the other 97% (or 2.9 billion base pairs) is unknown.  Some of the non-coding DNA contains sequences that tend to repeat many times.  The repetitive DNA can mutate readily with no apparent ill effects to the organism.  The sequence, length, and number of repetitions varies greatly in the population (approx. 6 billion).  Restriction enzyme digestion produces a collection of fragment lengths unique to each individual ("polymorphism").  Often referred to as Restriction Fragment Length Polymorphism (RFLP), it utilizes the fact that a difference in only one base pair alters the restriction site location, and thus the size of the fragment produced.  DNA identification is, therefore, as unique as fingerprinting.  The likelihood of two persons other than identical twins having identical RFLP patterns is so low as to be beyond a reasonable possibility (1 in 30 billion).

Analysis of RFLP patterns in a criminal case is a comparative technique.  The DNA patterns generated from crime scene specimens are compared with DNA from the suspects.  This process begins with isolating the typical small amounts of DNA in biological evidence (tissue, hair root, blood, semen, etc.), duplicating or amplifying it millions of times, and cutting it with specific restriction enzymes.  The DNA fragments are then separated by size using gel electrophoresis.  This produces band patterns that are unique to each individual.  These band patterns are like human "bar codes" like those found in the grocery store. This separation is only the first step in the DNA profiling process.  Further analysis of exact fragment length and nucleotide sequence is necessary in order to be admissible as evidence in court. Visualization will be performed with a simple dye.

Procedure:

You will work in pairs.  Each pair will receive 4 samples of DNA:
Sample 1: Suspect 1
Sample 2: Suspect 2
Sample 3: Victim
Sample 4: Evidence

How to prepare Agarose Gel:   A group made up of two pairs will prepare one gel. Each gel holds 8 samples.

1. Using a 25 mL graduated cylinder obtain approximately 30 mL of agarose solution (from the water bath).

2. Insert the comb across the plate (this will create the wells in which the DNA samples will be placed, later). Pour agarose solution in a smooth motion, quickly.  Allow solution to fully cover the glass plate in the apparatus.  Insert the comb to make sample wells.

3. Allow the gel to set for at least 20 minutes and carefully remove the comb (lift from one side then the other to prevent lifting the gel out of the plate) rinsing with distilled water if necessary.
 

How to use Electrophoresis:

1. Using a micro-pipette set to 10 µL, load the contents of each vial into its own well in the agarose gel, NOTING WHERE YOU LOADED EACH SAMPLE.  Use a fresh pipette tip for each sample.  Slowly expel each sample into the well.  The dye placed in the DNA (blue color) serves two purposes.  First, it allows us to visualize the DNA which would normally be transparent.  Second, its high density helps the solution to sink and remain in the well.

2. Put the cover tightly on the chamber.  MAKE SURE THE POWER SUPPLY IS TURNED OFF.  Connect the electrical leads to the power supply.  Make certain the leads are matched to the appropriate color (red to red [positive] black to black [negative]).  The samples should start at the black cathode.  Turn on the power supply and set the voltage to 150 Volts.

3. You should see the loading dye flowing away from the origin soon after turning on the power supply.  If not, check with instructor.  Stop electrophoresis after the dye has moved 6-8 centimeters from the wells (40-50 minutes).  The dye travels faster than the DNA, so it is no longer mixed with the DNA.  Turn off the power supply.  Disconnect the leads.

How to Stain and Destain the Gel:

1. Remove the gel from the chamber and pinch off one corner opposite the wells. This will mark the orientation of the gel for identification.  Slide the gel into a staining tray in the fume hood.  PUT ON GLOVES!  Slowly add methylene blue to the tray until the gel is covered.  Stain for 20 minutes with occasional rocking of the tray.

2. When  gel is stained, drain the stain (using a funnel) into labeled storage bottle.

3. Rinse the gel with distilled water several times (still in the tray).  Then fill the tray with distilled water and allow the gel to "destain" for 20 minutes, again with occasional rocking.

4. Dry the bottom of the gel with a paper towel.  Place the gel on the plastic wrap on top of the transilluminator.  With the light on, you should see dark blue bands in the gel.

For more information on what  DNA is and it's uses in criminology see your textbook.

Fingerprint Analysis:  Just for fun, while waiting for DNA to process. No written objective, procedure, discussion.

There are three basic types of fingerprints that occur at crime scenes; visible, plastic, and latent.

Visible: visible prints made by fingers stained with colored material such as blood, ink, paint, grease, or dirt.

Plastic:  a visible indentation pattern formed by pressing onto a soft surface such as wax, putty, tar, soap, butter, clay.

Latent:  normally not visible prints that require some means of development. These are chemically  enhanced or lifted from various surfaces at the crime scene or from collected evidence.

You will find the resources to collect one of each of the above types of fingerprints.  Collect one visible, one plastic, and one latent fingerprint from yourself. Record observations from each type. Record your fingerprint (when possible) in your notebook, when not possible record key observations that make your fingerprint identifiable and unique.
 

Conclusion and Discussion:
Report all the findings of the DNA analysis. For clarity draw the results for your four samples tested. Clearly identify each sample in your illustration. How confident are you in your finding? Explain sources of error involved in DNA analysis.

Questions:

1.Why do we need to test the victim's DNA ?

2. A burglar working in a hurry cuts herself breaking into a safe. Police find no fingerprints at the crime scene. However a very astute officer collects a few scrapings of dried blood from the edge of the safe. What other DNA samples will the officer need to submit for DNA analysis in order to confirm who the burglar was?
 
 
 
 

Fingerprinting information from Forensic Science, De Forest,  P.R., Gaensslen,R.E., Lee.H.C., 1983
DNA general information from CH118 lab write-up, Jan 2000.
DNA processing information taken from: Fotodyne Safekit 106,  instruction manual, 1994

LMM, JAN2001