JS 113 Forensic DNA Laboratory103107

Forensic DNA Laboratory CaseA case was submitted to your laboratory to determine whether or not the blood stain left at the crime scene may have been left by a suspect. Reference samples were collected from the victim and the suspect and you also included a positive (known DNA sample) and negative reagent control.

SamplesSample # Description

1 Victim reference sample2 Suspect reference sample3 Evidence sample (blood stain left at the crime scene)4 Positive control5 Negative control

DNA extraction and QuantificationFollowing the screening of the biological evidence using presumptive tests the next step was to extract the DNA. A commonly utilized method of extraction is the Organic Extraction method. This method utilized chemicals and reagents to open (lyse) the cells, purify the DNA by removing proteins, other cellular material and other impurities that may be present in crime scene samples and concentrates the DNA. These 5 samples above were extracted before the laboratory.

Agarose Gel Electrophoresis (aka Yield gel electrophoresis)A yield gel may be used by itself or in concert with other methods in order to assess the quality and quantity of DNA. Note that this gel can also be used to evaluate the PCR products following amplification.

Preparation of the gel:1) All gels use 1% agarose in 1X TBE (Tris Borate EDTA) buffer supplemented with 0.5 mg/mL ethidium bromide (5 mL of 10 mg/mL EtBr per 100 mL of buffer). Alternatively, EtBr may be omitted and the gel stained after the run in a staining tray in the same concentration of EtBr.

Prepare the appropriate volume of 1X TBE buffer. Weigh out the appropriate amount of agarose into a flask or bottle.1 g Agarose in 100ml of 1X TBE= 1% gellAdd the 1X TBE.Bring to a boil in a microwave to dissolve agarose.Add EtBr (if using).Place an appropriate size comb into the gel tray.When the temperature is ~60-C, pour agarose into gel tray.Let stand until solid.

2) Pour 1X TBE buffer into electrophoresis tank. 3) Place the gel into the tank with the well comb at the cathodic end. Enough buffer should be present to cover the gel to a depth of ~3 mm (refer to appendix B). Remove the comb.

All of this has been done already before the laboratory. You will start here.

4) The DNA sample mixed with loading solution will be pipetted into the well with the gel submerged.

You will be loading gels. Each person in your team should pipette one of the DNA samples into the gel. For this laboratory we will add 3 ul of DNA with dye already added.

Load your gel as follows

Lane Sample 1 Ladder2 Quantification standard3 Victim4 Suspect5 Positive6 Negative control7 Ladder

5) Run at constant voltage of 100 volts. When the bromophenol blue tracking dye has moved ~2 cm from the origin, the run can be stopped. For 12 X 14 gels, this should be about 45 minutes.

6) Take a photograph of the gel on a UV transilluminator with a camera.

7) From the photograph, visually assess the quality and quantity of DNA in test specimens by comparison with the DNA standards.

Here is an example of a result from samples ran on an agarose gel. Note that the lanes and samples are different than the gel you loaded. You can see the sample 11 and 16 have some minor degradation as there is a smear of DNA that represents smaller sizes below the main band

Polymerase Chain Reaction (PCR)Polymerase Chain Reaction of PCR is an enzymatic process whereby a targeted DNA sequence is subjected to repeated rounds of DNA synthesis. Forensic DNA laboratories are now using multiplex PCR in which as many as 15 STRs are simultaneously amplified in a single tube with different fluorescent dyes. These multiplex kits contain everything except the DNA template (which is the DNA extracted from references, standards and crime scene evidence). The resulting STR PCR products may be separated and typed using capillary electrophoresis. The DNA from the samples were amplified using the Profiler Plus STR kit.

DNA samples from your case were amplified and then the STRs were separated and detected by capillary electrophoresis. The resulting electropherograms were produced for the 5 samples below.

Only 3 STR results are shown.

For 3 extra credit points answer the following questions.1. What is the genotype of the victim? The suspect? The evidence?2. Can you include the suspect as a contributor of the stain? Why or why not?3. Did your controls work? 4. What other controls might you include in your analysis? 5. After this test is there anything else you might do to help solve this crime?

For the laboratory tomorrow please prepare the following

1) 6 agarose gels with at least 8 wells per gel (normal 1%Agarose 1X TBE1 g agarose in 100ml TBE).

2) 6 sets of 1.5ml eppendorf tubes containing 30ul of bromophenol blue loading dye

Label tubes 1,2,3,4,5, and L for ladderThere will be a total of 36 tubesSet these up in separate boxes or racks each labeled with Team 1, Team 2…Team 6

3) Set up 4 of the gels into the buffer in the gel apparatus on the cart (the other two can be in some Tupperware containers with buffer (1XTBE).

4) Students will be loading dye into the gels5) The pipettes P10s and P20s with appropriate tips for them to load 3 ul each. We will

need at least 6 pipettes6) Gloves S,M and L 7) Lab coats8) Pre cut butcher paper for 6 stations9) Beakers for waste tips

Give me a call if you have any questions.Thanks for your help again.

Steve