CSI : AHS

Introduction

          DNA is essentially the blueprint to create a person, and as we are all unique, so our DNA is unique as well. This has a great number of uses in proving relatedness of individual organisms, identifying species, and studying ancient peoples. DNA is also particularly important in the field of forensic science. Although human beings are 99.9% the same, that 0.1% can be crucial in telling one individual from another. DNA evidence can be used either to incarcerate a man, or to set him free.

 Restriction Enzymes and DNA Analysis

          DNA analysis is carried out through Restriction Fragment Length Polymorphism, or RFLP. Restriction enzymes are normally used within bacteria to protect them from invading viruses, cutting a virus's DNA at the recognition sequence, or one specific point in DNA that the restriction enzyme is made to cut. If there are multiple recognition sequences, the restriction enzyme will cut at multiple points. However, restriction enzymes are not limited to the bacteria in which they originate. In the RFLP process, these "scissors" snip DNA into fragments, the resulting sequence unique to each individual because of his or her specific DNA.When compared to DNA evidence obtained from a victim or crime scene, the cut DNA patterns can be compared to determine from which suspect the DNA originated.

Agarose Gel Electrophoresis

          However, if the restriction enzyme is not aided in any way, the cut DNA will sit in an invisible tangle, undecipherable by forensic scientists. To give the DNA color and place it in the aforementioned patterns, the process of agarose gel electrophoresis is necessary. In electrophoresis, cut DNA fragments are loaded into an agarose gel slab, which is placed in a chamber containing a conductive buffer solution and two dyes, bromophenol blue and xylene cyanol. Electrodes are attached to the chamber, and direct current is passed through these electrodes, polarizing the it. Because DNA is negative, it will be drawn towards the positive pole in the field.The gel is filled with holes, which filter the DNA fragments according to the size of their base pairs. Smaller pairs will slip easily down through the gel, while larger ones will become stuck and take more time. Fragments of the same size ultimately stay together, forming the bands seen on a completed DNA test. While the DNA is filtering down through the agarose gel maze, so are the bromophenol blue and xylene cyanol dyes. These group around the bands of DNA, making them visible to the naked eye.

Procedure

Restriction Enzyme

1. Pipet 10 µl of restriction enzyme into each of the 6 micro test tubes containing 10 µl DNA from a suspect. Flick/tap the tubes to mix the contents.
2. Incubate for 45 minutes at 37°C. This is the temperature of the human body, and will help the restriction enzyme thrive.

Agarose Gel Electrophoresis

(Note: I was absent during this part of the lab, so I am paraphrasing the instructions in the lab packet.)

1. Place tubes in a centrifuge to bring all the liquid to the bottom
2. Add 5 µl of "loading dye" into each tube. The dye helps to make the samples denser, so they sink into the wells in the gel.
3. Place the agarose gel in the in the electrophoresis apratus, and cover it with 1x TAE buffer.
4. Load 10 µl DNA size marker and 20 µl of each of the DNA samples into separate wells, using a separate tip each time to prevent contamination.
5. Turn on the electrophoresis apparatus and let it run for 30 minutes.

Visualization of DNA Fragments

1. Add 120 ml of 100x Fast Blast DNA stain into a staining tray
2. Stain the gels for 2 minutes
3. Rinse with warm water

Results

         Because the restriction enzymes are made to cut at one specific sequence in DNA, the patterns formed by the DNA of different individuals are all unique. It is fairly simple to tell which one of the six samples is identical to the sample taken from the crime scene.