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RFLP - 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. The restriction fragment length polymorphism technique (RFLP) "cuts" out genes which are likely to be differentiating factors using restriction enzymes. Are separated by size using gel electrophoresis. The pattern formed will be particularly unique because there is more variability in the genes examined.
When I first started college that's when DNA fingerprinting started entering the popular knowledge base and people were talking of this DNA technology that could be used to identify people and was considered almost a magical thing. In fact a friend of mine who is an English major he was very confident that he knew exactly what DNA fingerprinting was. I remember him explaining to somebody else it was if you left a fingerprint a Scientist could pick up a bit of your fingerprint put it into a pitridish and grow an entire new copy of you and then look at your clone's face and identify you that way.
Unfortunately that's not how it works, it's a little bit more complicated and a little bit less complicated more on the less complicated side. There's two big kinds of DNA fingerprinting one is called PCR, the one I'm going to talk about right now is called the RFLP that's a really long, sorry there's a really short version of the actual name which is Restriction Fragment Length Polymorphism but the basic idea of DNA fingerprinting no matter if it's RFLP or PCR is that you're examining DNA using in this case RFLP and a measurement tool called Gel Electrophoresis to do that DNA examination. So if we take a look at this Restriction Fragment Length Polymorphism this gigantic title what does that mean? Well there's something about fragments and the lengths, now a restriction fragment is something that is created when a special kind of cutting enzyme called a restriction enzyme cuts the DNA poly is a root word that many of you know it means many, morph means shape or form and so a polymorphism means that you see different sizes or shapes in the length of people's DNA fragments. So the basic idea of RFLP is that you're using those restriction enzymes, specialized enzymes that cut DNA at highly specific sequences to cut up DNA into different lengths.
Each person because our DNA is different from one individual to the next will wind up creating a different pattern of fragment lengths. And then you use this process called Gel Electrophoresis. What's that? Well Gel Electrophoresis uses a kind of Gel typically agarose which looks kind of like jello without any food diet added to it and you run an electric current through it. Now DNA has a negative charge so it's forced from the negative side towards the positive side by this electric field and the smaller pieces of DNA can wine their way through the gel a lot faster than the larger pieces. Let's take a look at this PowerPoint slide here and we can get a better idea hopefully of how this RFLP restriction fragment length polymorphism works.
Let's suppose we have sample one and sample two, these are two different sources of DNA. Now I have a restriction enzyme that looks for the sequence GAATC and so it goes along this DNA and goes GAATC and cuts it right there. It keeps going doesn't find that sequence again. In sample one going on the other strand remember DNA strands are anti parallel they move in opposite directions a restriction enzyme comes along this strand goes GATTC and cuts it there. What that winds up giving me is this fragment here which is shortish and this fragment here which is longish. Now sample two comes from somebody else with a different sequence at that particular site instead of GAATC they have GTATC and the restriction enzyme goes along and says GTA never mind and ignores it and similarly GAT and ignores it.
Alright so sample two's DNA winds up not being cut so it's very long, we take our sample one DNA put it into a hole or well in the gel take sample's two DNA puts it I there and then assuming that we have a large enough sample because you cannot see one DNA molecule you need several million DNA molecules all together in order to be able to actually see it with naked eye. So we put our sample of DNA from person number one into this well, we put our sample of DNA from person number two in that well then we set up our electric currents so this side of the gel is negatively charged repelling the DNA this side here is positively charged attracting the DNA and the DNA strand will start to move through.
Now because we have in sample two's well a whole bunch of identical DNA molecules that are large, they have a hard time lumbering through this obstacle course. Whereas in sample one, we have a sample of two different kinds of DNA fragments, the short guys who zipped, and this larger guys who lumber through not quite as fast as speedy Gonzales down here so kind of to there. Then we turn off the current and then we can add special stains that make the DNA visible and then we can see our pattern. And you could use this in back in the 80s and 90s that were using this kind of patterns to identify suspects. It has also been used a lot in doing some of the initial mapping work on the human genon project and in standard research. Also during genetic engineering when you're having to cut DNA this is a very simple and quick way to determine whether or not you're successful.
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