Video Tutor Sessions: DNA Profiling

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1 Video Tutor Sessions: DNA Profiling Narration 0:00 Hey there. I m Eric Simon, and welcome to another Video Tutor Session. In this videocast, I m going to walk you through the process of creating a DNA profile. Understanding how a DNA profile is made is helpful for a few reasons. First, it will familiarize you with several DNA technology procedures. Even more significantly, it will relate to the very important topic of DNA structure. Sound good? Then let s get started! 0:29 DNA profiling has rapidly transformed the field of forensics, the scientific analysis of evidence from crime scenes and other legal investigations. A quick note before we get started. You may have noticed that I am using the term DNA profiling rather than the term DNA fingerprinting. When the technology first came into use in the 1980s, the term DNA fingerprinting was widely used. But forensic scientists prefer the term DNA profiling because it more accurately reflects the information obtained. There is a fundamental difference between how an ordinary fingerprint and a DNA profile can be used: An ordinary fingerprint is only useful if you have another fingerprint to compare it to. A DNA profile, on the other hand, can provide some information in and of itself. For example, you can tell if a DNA profile came from a male or a female. And you can compare it to other known DNA profiles to identify family relationships. Your fingerprints, on the other hand, bear no discernable relationship to the fingerprints of your parents or any other relative. Thus, the term DNA profile is a more accurate description of how the information can be used. 1:48 To make a DNA profile, you start with samples of biological material. This can be blood, saliva, or other DNA-containing body fluids collected from a crime scene, from suspects, or even from old evidence. These samples can be miniscule. For example, a key piece of evidence proving that Ted Kaczynski was the Unabomber was a tiny bit of saliva recovered from the back of a postage stamp affixed to a package he sent. Because biological samples can be so minute, a key step in DNA profiling is to amplify the sample. DNA amplification means to take a very small sample of DNA, perhaps even just a few molecules, and precisely copy it many times to produce a larger sample that is suitable for testing.

2 2:28 To amplify a sample of DNA, researchers use a method called PCR. PCR stands for polymerase chain reaction. A polymerase is an enzyme (here s a good hint: you can usually recognize the names of enzymes because they almost always end with -ase ). Polymerase, you can therefore guess, is an enzyme involved in making polymers. In this case, polymerase refers to an enzyme that makes polymers of DNA nucleotides. In other words, polymerase constructs polynucleotides, or DNA strands. A chain reaction is a process that triggers itself, repeating over and over. So polymerase chain reaction refers to a process whereby a sample of DNA is used to create a bigger sample of DNA, which is used to make an even bigger sample, and so on in a chain reaction, until a sample large enough for testing has been created. 3:44 Conceptually, PCR is fairly simple. DNA is subjected to successive rounds of heating and cooling. When the DNA double helix molecule is heated, it causes the two strands of the double helix to separate. Once separated, DNA polymerase will build the missing strands along each half using nucleotides that are in solution with the DNA. When the mixture is cooled, you ll have double-stranded DNA containing the newly synthesized DNA strands. You have therefore doubled the amount of DNA. This completes one cycle of amplification. You then repeat the process: heat, synthesize, cool. Every time you go around that cycle you double the amount of DNA. 4:34 This is a PCR machine that I borrowed from my college. To amplify some DNA, I load this little test tube with the DNA to be copied, some DNA polymerase enzyme, a supply of DNA nucleotide monomers, and some short DNA strands called primers that allow the reaction to start, and a few other chemicals. I then load this tube into the PCR machine, program it using this keypad, and let it run. The machine heats and cools the DNA according to the program that I ve entered. Each cycle takes about five minutes and doubles the amount of DNA. So in just a few hours, you can go from a single DNA molecule to millions of identical copies, enough for testing. The polymerase enzyme is extremely accurate, so you can be assured that all of the DNA molecules that have been created contain the identical DNA sequence. 5:38 There is an important detail about PCR that I still need to discuss. When a forensic scientist uses PCR to create a DNA profile, he or she doesn t amplify the entire DNA sample. A PCR reaction is only accurate on stretches of a few hundred or a few thousand nucleotides, so you wouldn t be able to copy the entire genome even if you wanted to. Instead, DNA profiling depends on amplifying certain fairly small regions of the genome. The spots that are amplified to create a DNA profile are called short tandem repeats, or STRs.

3 An STR is a site along a chromosome known to contain consecutive copies of a short DNA sequence. For example, there is a site along your chromosome number 7 that contains between 6 and 15 copies of the sequence GATA. How many times the GATA sequence is repeated in a row varies from person to person. I might have 8 GATAs in a row, while you might have 12. A standard DNA profile consists of 13 such STR sites scattered throughout the genome. All of the standard STR sites contain a 4-nucleotide segment that is repeated between 3 and 51 times and exists in at least ten different variations within the human populations. Taken together, these 13 sites provide a unique DNA profile that is accurate enough to make a positive match to just one human that has ever lived. (For the purposes of this discussion, we ll exclude the possibility of identical twins.) 7:22 So, a standard DNA profile involves using specific primers to amplify these 13 STR sites via PCR, starting from a sample of evidence. Once the DNA is amplified, how can you make the results visible? To do that, we need to discuss another important and very common DNA technology technique: gel electrophoresis. 7:49 Gel electrophoresis is a method that sorts molecules of DNA (or other macromolecules) based primarily on their size. The key to understanding gel electrophoresis is to remember the structure of DNA. The monomer of DNA is a nucleotide, which consists of 3 parts: a sugar, a phosphate, and a base. The phosphate, which I ve represented here as a yellow ball, has a negative charge. A DNA molecule is therefore going to contain a universal coating of negative charge running along its outside, one negative charge for each nucleotide in the molecule. 8:26 Have you ever made Jell-O? To do that, you boil some water, add a packet of flavored gelatin powder, mix it up, pour it in a container, and let it cool. Making a gel for gel electrohporesis is very similar. This powder is called agarose. It s a seaweed extract containing a polysaccharide that, in waters, forms a dense, sticky network. Agarose has several culinary uses, such as a thickener in soups. In our case, we are going to add it to water and microwave it. (Normally I would carefully weigh this out measured. I m just giving you an idea here). Once it is dissolved, I can pour the hot agarose solution into this square-shaped plastic mold. I m going to add this little plastic comb to make holes in order to make holes the gel. I ll put this in there Now, I ll set this aside and let it cool, which takes about 20 minutes. I ve got one here that has already cooled. Notice that it is kind of stiff, like stiff Jell-O, or a soft sponge. If we were to examine this gel under a microscope, we would see a very dense

4 thicket of agarose fibers running all through it. Picture a very dense jungle with vines running every which way. 10:00 Now that I ve created a gel, I can use it to help display the amplified DNA. I start by putting the gel into an electrophoresis chamber. Then I add a liquid buffer that helps conduct electricity through the chamber. Next, I ll remove the comb. Notice that this leaves indentations, called wells, into which I can load the DNA solution. This tube contains the DNA that I amplified via PCR. I ll use this pipette to carefully load the DNA into the wells. In this one gel, I might put DNA from a crime scene in one well, DNA from a suspect in another well, and DNA from a second suspect in yet another well. Once all the DNA is loaded, I connect the chamber to a power supply. Notice that I ve connected the negative pole of the electricity at the top, near the DNA samples, and the positive pole at the bottom. I then turn on the power. If you look carefully, you can see small bubbles rising up from the wires that are conducting the electricity. And now we wait. 11:33 What s going to happen to the DNA inside the gel? Remember that DNA has a negative charge. So which way is it going to move? In nature, opposite electrical charges attract, so the DNA will move its way down through the gel toward the positive pole. And here is the key to gel electrophoresis: the bigger a molecule of DNA is, the harder a time it will have moving its way through the gel. Imagine that I enter a very crowded party. I wish to get to the opposite corner of the room. Standing next to me is my small son. Which one of us will get across the room more quickly? Well, My son will, because he can weave and dart between people much more easily than a big guy like me. Similarly, small pieces of DNA will move through a gel much more quickly than large pieces can. Thus, if we turn off the electricity after a certain amount of time, smaller pieces of DNA will be near the bottom and the larger pieces will be near the top. We thus will have sorted the DNA molecules by size. 12:42 This animation shows a hypothetical DNA gel. At the top, imagine we load a mixture of DNA molecules. Over time, as the gel runs, you can see the molecules separating by size. Smaller molecules will end up near the bottom with the bigger molecules staying near the top. 13:02 Okay, I ve run this gel for a few hours. Notice that, unlike the gel in the animation, you can t actually see the DNA within this gel. The DNA must be in the middle of the gel somewhere, but we can t see it. To know what happened, we need to make the DNA visible. One way to do that is to stain

5 the DNA using a dye. Here, I ll add this dye. This is a common dye called methylene blue. It will make the DNA appear as blue bands. If we let the gel sit in this dye for a while and then rinse it off, the dye sticks to the DNA within the gel, making the DNA visible. I ran this gel last night and then stained it to make the DNA visible. You can see it here in this picture. The bands near the bottom are relatively small lengths of DNA, while these bands near the top are longer. Each of these bands is a set of identical DNA fragments of a particular length. In the laboratory, you can also use a chemical that makes the DNA glow under UV light. This makes the DNA even more visible. 14:12 Now that we ve used PCR to amplify a sample of DNA evidence, run it out on a gel to separate the DNA molecules by size, and then stained it to make it visible, we can complete our DNA profile. I m going to explain using a hypothetical example. Imagine that some DNA is collected from a crime scene. This figure shows two STR sites and the repeats within them. Notice that in the first STR site, the sequence AGAT is repeated 7 times, and in the second STR site, the sequence GATA is repeated 8 times. Now imagine that we have two suspects. We obtain samples of DNA from those suspects. How do we tell if either suspect s DNA was left at the crime scene? 14:58 This figure summarizes the steps that we have described in creating a DNA profile. Step 1 is to collect and isolate DNA from the crime scene and from our two suspects. Step 2 is to amplify the DNA using PCR. We won t amplify all of it, just a selected number of STR sites that are known to differ from person to person. Third, we run the amplified DNA on a gel. Each of these bands represents DNA collected from a different STR site. What does it show? Consider the topmost row of bands. These represent particularly long stretches of DNA. Apparently, all 3 DNA samples are identical at this STR site. Can you tell which suspect s DNA matches the crime scene? Clearly, Suspect 1, the middle lane, has some STR sites that are different from the crime scene DNA. But suspect 2, in the right lane, matches the crime scene DNA at every STR site shown. From this, we can exclude suspect 1 from the crime scene and confirm that suspect 2 left DNA at the crime scene. That doesn t necessarily mean that suspect 2 committed any crime, but it does offer proof that the suspect was there and left DNA at the scene. 16:22 This example highlights one of the primary advantages of DNA profiling as a forensics tool: it is equally useful for proving innocence as well as guilt. Indeed, hundreds of wrongly convicted people have been exonerated through DNA evidence.

6 16:38 I hope this Video Tutor Session has given you a good understanding of the techniques involved in creating a DNA profile. Remember that the entire process depends on a particular physical property of DNA: the evenly spaced negative charge of the phosphate groups along the polynucleotide chain. Perhaps you ll get the chance to create a DNA profile in your laboratory course. It s fun and easier than you might imagine. I m Eric Simon, and I encourage you to pay attention during those TV crime shows to see what you recognize.