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PCR - DNA Fingerprinting
M.Ed., Stanford University
Winner of multiple teaching awards
Patrick has been teaching AP Biology for 14 years and is the winner of multiple teaching awards.
Because DNA is unique to an individual, we can use DNA fingerprinting to match genetic information with the person it came from. First, we use the polymerase chain reaction (PCR) technique to copy a tiny fragment of DNA so that there is enough to use in gel electrophoresis. Gel electrophoresis uses gel and electricity to separate DNA fragments based on size, creating a distinct pattern that represents an individuals genetic information.
In shows like CSI, Miami, New York or wherever they often throw up the term DNA fingerprinting. One of the most common methods of DNA fingerprinting is something called PCR and what it's all about is using PCR and Gel Electrophoresis to examine DNA that's what they mean by DNA fingerprinting it's not really somebody's finger print. Now PCR stands for Polymerase Chain Reaction which is a process for copying DNA and what it does is that it uses a special heat stable DNA Polymerase to copy a specific gene that you're interested in.
Now Gel Electrophoresis is this idea of using the fact that DNA is negatively charged to take your copy DNA put it into the agarose gel or some other materials and then you use electricity to drive that charged DNA through the gel and because that gel acts like an obstacle force it separates up the DNA fragments based on their size. Let's take a closer look at this YouTube video that shows the process known as PCR and we're inside of a test-tube filled with DNA from suspect if we're in CSI but all we're interested in, is this one particular section of DNA called the target sequence highlighted in green. Now this is going to take advantage of some of the steps involved in DNA replication the process of copying DNA.
Now normally with DNA replication we have to open up the helix, well to open that up in your cells you use an enzyme. In this test tube we're going to heat it up to 95 degrees Celsius which will separate the two sides, because that is almost boiling temperature. Now we cool it a little bit and allow a premade thing called a primer that tells which gene we're interested in copying to come in and so by cooling to right around 60 odd degrees or so that allows the primer to bind to our target sequence. Now the orange little things is that enzyme that can survive these high temperatures. And these green guys with sticks on them, those are the nucleotides the raw material for building our DNA copy. So the enzyme does what it's supposed to do, it finds the primer and says okay and it starts copying, and it keeps going.
And if you give it enough time it'll finish copying the entire molecule going this way and that one will copy going that way. Remember the two strands of DNA are anti-parallel, they go on opposite directions. But we only give it maybe 2 minutes at most and so at that point we then let it stop and we're at the end of cycle one. And so now that we're done with cycle one we can begin cycle two and it's the exact same thing, we heat it up to 95 degrees Celsius which is enough to separate our old, original template strands and our newly made copies. We cool it to 60 degrees Celsius that's cool enough for the primers that still are floating around in the test tube to bind to the beginning and end portions of our gene of interest. Then we go to the right temperature for the enzyme, the DNA polymerase it finds the primer and goes okay and it starts to copy and that's the end of cycle two.
At this point we have 4 copies now each of our copies contains information that's not part of our DNA but at the beginning of cycle 3 when we heat it up notice there's a couple of short little segments that are only the length of our gene of interest. We cool it, primer stick, the tag-primers comes along and binds it, it's called tag-primers that's short for thermokineses which is just the name of the creature came from but now we have a couple of our target molecules made. So we're ready to begin I believe this is cycle 4, so again we're going to heat it up, that' the end of cycle 3 so we heat it up for cycle 4, we separate our strands, we cool it enough for the primers to come on in, they bind to the beginning and end portions of our DNA gene tag polymerase does it's copying job and again we've made a number of copies of just the size that we want. Now original we had more of these longer ones but now we're starting to get more and more of the shorter ones.
As we begin cycle 5 we do the exact same thing over and over that's why it's called a chain reaction, each time we're doubling the number of our copies. And we just run it through, and this is such a simple process, this is one of the reasons why this when it was first invented people were going wow how did they think of this and there's a lot of a pack full of stories about how the guy actually did think of that, but he is niow a very rich man because everybody does this process. Now you can see we've got 22 molecules and that's after only 5 cycles. Each cycle takes maybe 90 seconds to couple of minutes, so you this 30 times and that takes you maybe 90 minutes and at the end of it you've got a large number, billions of copies of your target.
Now you can't see an individual molecule but you can see billions of molecules. Now how are we going to visualize this? How are we going to see how big that is? That's where the Gel Electrophoresis comes in. So we'll stop the YouTube and we'll go to a PowerPoint slide and let's imagine we've done a DNA fingerprint of 3 people and we're looking to see, we're not using this to identify who they are like you would see a side, but we're doing this trying to figure out what genes do they have? Let's suppose we've found a gene that if you have a longer version of it, you're more likely to get a particular cancer. If you have a short version of it, you're less likely to get a particular cancer.
Well we have patient 1, patient 2 and patient 3, now in this fourth row here what we have is pre made DNA so that we can use it like a ruler and what we do is we loaded our DNA samples into these holes here called the wells. We turn on the current, this end is negatively charged, this end is positively charged DNA has a negative charge to it so it is repelled by the negative side and goes zoom towards the positive end. And little guys one thousand base pairs long move a lot faster than the big 10,000 base pair of long pieces of DNA. Now this person here, we only see one band, this person here we see one band, this person we see two. Why is that? Oh yeah everybody has two copies of every gene, this person has two copies of the long version. Their homozygous for this particular condition.
This person here is homozygous for not, for the shorter version i.e. not having the cancer. This person here is heterozygous, so this person has a medium chance, this person has a low chance, this person with a double dose of the longer more, greater chance of having cancer gene. This person is at greater risk, and this is one of the uses of DNA fingerprinting is to assess your genetic risks so that you can make wise, intelligent decisions about what you're going to do in your environment. If this was you I would not smoke and watch your diet and do everything else you can to keep this genetic prediction from becoming a genetic reality.
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