DNA fingerprinting can be used to identify individuals

As preparation for a lab where students will use DNA fingerprinting to demonstrate the concept that DNA fingerprinting can be used to identify individuals and solves problems, the following material is printed out: DNA fingerprinting is a technique that can be used to identify an individual from the DNA contained in a sample of the individual's cells. DNA fingerprinting relies on the ability of specific enzymes to cut the DNA into small pieces and the ability to analyze those pieces and compare them between samples. The DNA is cut by a type of enzyme called a restriction enzyme. Restriction enzymes occur naturally in bacterial cells as a line of defense, similar to our immune systems. The enzymes are very specific: each will recognize only a certain site on a DNA molecule and cut only at that site. These sites occur randomly anywhere on the DNA molecule. Therefore, every different DNA molecule (for example, DNA from two different people) will be cut into a different number of pieces and pieces of different sizes when incubated with a restriction enzyme. In addition, no two restriction enzymes will cut the same DNA molecule in the same way. Therefore, no two enzymes used to cut the same DNA molecule will give you the same number of pieces nor the same sized pieces of DNA. After the DNA is cut, it is necessary to determine exactly how the enzymes cut the DNA and to measure the size of the pieces of DNA that have been cut. This analysis is done by a process called gel electrophoresis. Electrophoresis is possible because DNA has a negative charge. Therefore, if an electric current is applied to the DNA, the DNA will move towards the positive pole. However, moving the DNA in this way will not separate the DNA pieces based on their sizes. That's where the "gel" in "gel electrophoresis" comes in. The gel is made of agarose, a substance that forms pores that the DNA has to move through. The larger the size piece of DNA, the more difficulty it will have moving through the pores in the gel and the slower it will move. In this way different sized pieces of DNA can be separated, and pieces that are the same size will move to the same position in the gel. After electrophoresis, the pieces of DNA will appear as bands in the gel. Each band contains thousands of DNA molecules, which are all the same size. This array of bands forms the pattern that is called a "fingerprint." It is also necessary to run a marker of known size on the gel. The size of each piece of DNA can be determined based on how it moved in relation to the marker.

Human DNA fingerprinting requires one further step: in humans, the amount of DNA is very large, and cutting DNA with restriction enzymes would result in indistinguishable bands on a gel. To obtain a clear fingerprint, the DNA is transferred from the gel to a special piece of paper, called nitrocellulose that still retains the fingerprint pattern from the gel. After this transfer, short, radioactive pieces of DNA, called probes, are incubated with the nitrocellulose. These probes will bind with specific regions of the DNA. The sizes of these regions vary greatly among the population. When the nitrocellulose is exposed to X-ray film, the regions of DNA that bound to the probe will appear. This X-ray film is the fingerprint of an individual. Another fast emerging technology in forensics is a technique called the polymerase chain reaction (PCR). This procedure also gives a fingerprint from the DNA but does not rely on using restriction enzymes. PCR involves the use of probes to target a region of DNA, but, unlike traditional fingerprinting, these specific regions of the DNA are copied millions of times to allow for analysis of very small quantities of DNA, for example from a single cell. Using either procedure, gel patterns are examined and the suspect's DNA is compared with the DNA sample found at the crime scene. Many patterns, resulting from different probes that can detect different areas of DNA, are examined. There is, of course, a random probability that anyone's DNA could match someone else's at one point (called a locus). But, the chance that two people's DNA will match exactly at four or five or six different points (loci) is highly improbable. You may have heard of statistics in the range of one in a billion as the chance two random samples could match at all loci. If the patterns do not match exactly, then the suspect could not have contributed the DNA. In the past few years, many individuals have been released from prison based on these new techniques. If the patterns do match, the suspect is said to still be included in the sample as the probable contributor of the DNA. However, statistically, it is very likely the sample did come from the suspect. Taken together with other evidence, such as motive and opportunity, DNA evidence has helped in convicting countless suspects. Today you will cut DNA with restriction enzymes and analyze these reactions through gel electrophoresis. You will be asked to consider a particular murder case and use DNA analysis to investigate the crime. You will ultimately determine if one or both of the suspects are exonerated based on no matching DNA patterns, or if the suspects are still "included" based on matching patterns. INSTRUCTOR SET UP

The procedures used here are similar to numerous other basic restriction enzyme analysis procedures. Any of these basic procedures can be adopted to fit this exercise. Equipment needed is as follows:

Micropipetes, capable of measuring between 1-10 uL. Waterbath set to 370 degrees Celsius. Electrophoresis gel chambers and power supplies. Microcentrifuge. One or more restriction enzymes and their respective buffers, available from various biotechnology supply companies, such as New England Biolab. Concentrations should be in the range of 1-2 U/uL. At least four types of purified DNA, also available from biotechnolgy supply companies. (It is recommended that you use small genomes, such as lambda DNA and plasmids, as these are much easier to analyze, as opposed to mammalian DNA. These DNA samples should give different restriction patterns when cut with the same enzyme.) Concentrations should be in the range of 100-200 ng/uL. Agarose gels (50 ml per group, 1.0% concentration, made up in TBE buffer, boiled in a microwave to dissolve agarose). Ethidium bromide, 10 mg/ml. Sample loading buffer for mixing with samples before running on gel (available from biotechnology supply companies). UV transiluminator, for viewing gels, and camera for taking photographs of gels. Assorted laboratory equipment, such as microfuge tubes, pipet tips, etc. Ice. Marker DNA, for determining molecular weights.

Each person in the scenarios will be a different type of DNA. You can decide ahead of time which, if any, of the suspects will match the DNA found at the crime scene. By varying the matches in each scenario, student groups can be exposed to different possibilities and can compare and contrast their results. You will place the various DNA samples in tubes labeled accordingly. THE CRIMES THE GREENLEAF MURDER Alan Greenleaf, a successful and wealthy businessman, was found shot to death in his bedroom. Nothing was stolen from the house, and there was no forced entry. Police believe he was murdered by either his wife, Karen Greenleaf, or his mistress, Lucinda Teal.

Mrs. Greenleaf had been served divorce papers just days before the murder. Due to a prenuptial agreement, Mrs. Greenleaf would be left to live a lifestyle less affluent than the one to which she was accustomed once the divorce was final. Mr. Greenleaf had been seeing Lucinda Teal for almost two years before his death. Although he was not divorcing Mrs. Greenleaf to marry Ms. Teal, Lucinda still had high hopes of becoming the next Mrs. Greenleaf. However, the romance was not going well lately, and Mr. Greenleaf and Ms. Teal had been seen in public, having a very loud argument, several hours before Mr. Greenleaf's death. Several pieces of evidence were found at the scene of the crime, including fingerprints from Mrs. Greenleaf and Ms. Teal. But since both women frequented the bedroom with Mr. Greenleaf, this evidence could not be used against either one. Several drops of blood were found on Mr. Greenleaf's body, possibly coming from the murderer during a struggle for the gun. DNA was obtained from this blood sample. DNA was also obtained from each of the suspects and from Mr. Greenleaf.

THE MOODY BLUES MURDER Moody Blues Waters, a jazz musician, was found murdered late one night in the club at which he was headlining. Moody was a talented musician, but he found it difficult to make ends meet. He began dealing drugs to make some extra cash. He often carried large sums of money as well as drugs wherever he went. When Moody was found, there were no drugs or money on him. The police suspect that Moody was killed by his drug supplier, Eric Solis. Eric is a rather paranoid soul and may have concluded that Moody was stealing from him. Mr. Solis is not a patient man and would rather "shoot first and ask questions later." Eric was also not very fond of jazz and complained that the music hurt his ears. Normally, the police would have considered Eric Solis their primary suspect. However, several people, coming back from a late night of partying in the jazz district, remember seeing a man running from the club just about the time of the murder. The man was picked up by police several blocks away. His name is Wild Jim Watson, a small time thief with a long criminal history. Police speculate Moody may have caught him in the club after hours, and Wild Jim may have panicked and killed Moody. Wild Jim was carrying over $1000 when the police picked him up.

The only evidence found at the club were some blood stains near Moody's body. Preliminary testing showed that Moody had a different blood type than the blood found at the scene. Therefore, police believe the stains were from the murderer. DNA was isolated from the blood stain, and samples of DNA were obtained from Moody, from Eric Solis, and from Wild Jim Watson. THE ORANGE JUICE MURDER Joey Piedmont had an orange juice delivery route for nearly twenty years. Six days a week, starting at 5 AM, he would deliver orange juice to the restaurants and vending machines in his delivery area. He made a very good living. One morning he was found dead, lying next to his delivery truck in a back alley. He had been stabbed and beaten and all of his money was gone. The police had a difficult time finding any leads. However, they began to suspect two boys from a local gang may have committed the murder. Rico Smarty and Johnny Christo had been implicated in several violent crimes but there was never enough evidence to convict either of them. When the police found Rico and Johnny several hours after the murder, they were both drinking bottles of orange juice. When Joey Piedmont's body was found, he was holding a broken Tropicana bottle. It appeared he had used the broken bottle as a weapon against his attacker. There was dried blood on the jagged edge of the broken glass, and police speculate that Joey may have cut the murderer in a struggle. Technicians were able to purify DNA from the dried blood stains, and DNA samples were obtained from Joey Piedmont, Rico Smarty, and Johnny Christo.

THE PROCEDURE ANALYSIS OF THE MURDERS

You will create a DNA fingerprint for the victim (V) and for each of the suspects (S1, S2, and S3).

1. It is necessary to first prepare the agarose gel in which you will analyze the DNA. Seal the ends of the casting tray with tape and place the comb in the designated area. Obtain a solution of hot liquid agar. Please wear gloves for these next steps, as the substance ethidium bromide is very toxic. Add 3 uL of ethidium bromide solution to your gel. Pour the agar into the casting tray and set it aside on your bench to cool. 2. Next, you will cut the DNA of each sample with a restriction enzyme. This will break the DNA into small pieces that can be compared among samples. Keep all materials on ice until instructed otherwise. You will need four tubes:

In In In In

tube tube tube tube

#1, #2, #3, #4,

place place place place

5 uL of the victim's (V) DNA 5uL of suspect 1's (S1) DNA 5 uL of suspect 2's (S2) DNA 5 uL of suspect 3's (S3) DNA

Then place 4 uL of buffer in each tube, and then 1 uL of restriction enzyme in each tube. Centrifuge the tubes to mix the solutions. 3. Place the tubes in a water bath at 370 degrees Celsius. Incubate the reactions for 30-60 min. After incubation, remove these samples from the water bath. Add 1 uL of loading dye to each sample. 4. Wear gloves when handling your gel. Prepare your solidified gel by removing the masking tape and comb. Place the gel in the gel chamber and add enough TBE buffer to just cover the gel. 5. Load your samples in the gel and electrophorese at 120 volts for approximately forty minutes. After the electrophoresis run, the DNA will be viewed using a UV light source. Be sure to wear protective eye and facewear to prevent exposure to the UV light. Photograph the gel. 6. View the pattern of bands created by the DNA fragments. Does the pattern made by the DNA found at the crime scene match the pattern made by either of the suspects? Can you conclude who the murderer is?