here we are at part two of this chapter and we will be exploring two techniques which are used um well they're used quite commonly in a lot of different arenas in biology but we will be talking about here in the context of criminology and their purpose is in particular this context for what's known as dna profiling dna profiling is being able to look at various individuals dna and use the unique properties of our dna to help identify an individual of some kind based upon their dna for whatever reason and then we'll talk about some of those reasons here in a minute but the first technique that we're going to talk about is the polymerase chain reaction and this is a very commonly used technique and it's used to amplify dna and what we mean by amplified dna is you can take literally one molecule of dna and you can in a very short amount of time make a very large amount of that dna so it's basically just making many many copies of dna and um this is exactly what is done on many uh coveted tests they've heard pcr before how many pcr tests that have there been conducted for covet and basically what they'll do is the way that it works is that the virus that causes kova 19 is sars kobe 2 and that has a nucleic acid sequence to it and so if that sequence that very very small sequence is found anywhere uh in your saliva or your nasal mucosa or anywhere in your body it will be present in a sample that you take of your body whether it be a spit sample or a nasal swab and even if just one copy of that specific sequence that is carried by the viruses found in your body pcr will make many many many many millions of copies of it and then it makes it much easier to detect if in fact that sequence of of rna is in your body or not and if that sequence of rna is in your body then it's likely the case that it's in there because um you at some point had a virus or currently have a virus that's replicating inside of your body okay so how is this done it sounds a little like witchcraft but i assure you it's not um it relies on a couple of things there must be some sort of dna template or could be an rna template that's eventually converted but there has to be nucleic acid and specifically the region of nucleic acid that is of interest so in the context of a cova-19 test the region of nucleic acid that would be present would be a portion of the viral genome that's packaged up inside the virus okay then what all that's required is you have to add nucleotides which are these nucleotides are all the building blocks that will link together to form dna you have to put primers on there because we didn't really talk a whole lot about this but dna replication always begins when a primer and then from there the polymerase can bind and start replicating okay so these are some of the three ingredients you need primers you need the polymerase and you need the nucleotides the specific polymerase here is called attack polymerase because it comes from a specific type of prokaryote where the ability for this to turn on and off is temperature dependent and it can also withstand high temperatures okay so here's how it's done the first cycle looks something like this basically the first step is that the the nucleic acid would be denatured so in other words this has to be split um into two so these bonds that keep the two strands if it is double stranded that the the bonds that keep the two strands together have to be broken so that this can split apart and then that will allow the primers to get in here the part is a temperature so the temperature will go way up um i forget what the exact number is but it's like 95 degrees or something like that celsius okay then the temperature is brought down to 60 degrees uh or so and because the primers are smaller than the big chunks of dna they're able to quick get in here and bind faster than the original stands can then come back together and rejoin with one another and as soon as the primers get on there then it's gonna keep these it should keep these uh two pieces of nucleic acid from coming back together again so you open up the the double strand you quick put some primers on and then that should keep everything open the annealing is done by uh dropping the temperature down so the first the temperatures of the whole mixture here is brought up to 95 and that's brought down i think it's to like 60 the actual number is not important but i think it's something like that okay and then uh what's gonna happen is that the polymerase will bind to any region where there's a primer polymerase always has to bind to a place where there's a an rna primer and then it just goes through and it literally just replicates the dna just this in the same way that it normally would in our cells when they're undergoing the s phase it's we're just making that happen here for this little small chunk okay so now we've ended up we've started with one fragment of dna and then we've ended up with two and then we can just repeat the whole cycle again so the second cycle again you'll you'll open these up the primers will bind and the polymerase will then replicate it so now we ended up with uh four because we got two from each of the um the ones that were there at the end of the first cycle and then after this the third cycle each of those makes two copies and so we can just keep going many many many cycles oftentimes in the past when i've done this it's usually around somewhere between 20 and 30 cycles so if you were ever interested to know how many uh copies of dna that would be there'd literally just be two to what either 20 or whatever number of cycles were completed and that ends up being a huge huge huge amount of of copies so it in a very and this happens quite rapidly in just a very quick amount of time one one copy can be made into many in like quick like less than 20 minutes in fact i actually just had to get a copic test for my job they now require that i get one in order to stay employed there so um it was pretty cool they had like this little machine you put like a big white chip into it and then and then there's the nasal swab and you literally just stuck the nasal swab into the chip the microchip uh thing and then within like 20 minutes the result was given so i think that what that machine was that little chip that i put the swab in it was a thermal cycler or it was it was the machine that does this reaction and it was heating it up and um you know it's basically the nasal swab probably went into a solution that had uh all of these primers and nucleotides and polymerase there and it literally just heated up and cooled it down heated up cooled down heated up cooled down a whole bunch of times so that within about 20 minutes there is a whole bunch of of these that would have been made had the original uh nucleic acid sequence from the virus but in my nasal cavity in my in my my not okay and then uh yeah so it's just like a little in-house little machine that does all this i think is how it must work and then it's much more easy to determine if there's uh the viral genome present in my body based upon how many copies of this would have been made it can it can be more readily detected okay another technique that's very commonly used and actually will also employ the polymerase chain reaction is gel electrophoresis gel electrophoresis is what allows us to create a dna fingerprint or in other words if because all of our dna is unique if we chop it up at certain spots it will create different size fragments and then we can actually pull those fragments through a porous gel and separate out all of the different fragments based upon their size and so because we all have different size fragments that are made there will be different uh bands of dna that show up on a particular gel and then you can compare the different types of dna so how this is done it utilizes what are known as restriction sites and also restriction enzymes so a restriction enzyme is a is a protein that cuts dna and it cuts dna always at a very specific spot which is called a restriction site and there are lots of different restriction enzymes and each has a sequence of bases that it will cut at so you can kind of alter which restriction enzymes you want to use based upon which restriction sites you want to cut but because our dna is all different the place where our restriction sites end up for any particular restriction enzyme varies for example it might be in my dna there's a restriction site right here for a particular restriction enzyme and then if that was the case i mean if it truly cut it right here that would result in this little piece so i'd have a little piece of dna here and then i'd have this really really really long piece like this so this as i if i tried to separate this out on a porous by dragging it through a porous gel then um i would get two different bands one that's composed of the little fragments and one that's composed of the big fragments the big fragments would take longer to get pulled through the gel so they would be closer to the origin and the shorter fragments could sneak through that gel that porous gel a lot faster and then they would migrate further in the same amount of time otherwise like if we like on your dna if your restriction site came here then if we cut that then it would lead to two fragments that are more similar in size and so there'd be two different bands and ultimately there's actually a lot of different restriction sites in different places so it makes many different uh sized fragments of dna that can get pulled through a gel the way that a gel is made for electrophoresis is um we use a cafe which is illustrated here so this is just like a piece of plastic and then you'll basically tape off the two ends here and here and we're actually abs so you'll you'll get to take a crack at it and then they put a comb in there and then they pour what's known as agarose into the casting tray agarose is like it's made out of agar which is like a molecule that's derived from seaweed and it's in uh hot temperatures it's all dissolved and it's a liquid but then as it cools it solidifies and the molecule of the the molecular structures in agar or agarose are such that when it cools it creates pores so it allows stuff to actually molecules to actually get moved through and then again the premise is that it's the smaller molecules that will move faster and then the larger molecules they move much more slowly because it's harder for them to wiggle through all those microscopic pores okay now what happens then is that uh once the gel is oops sorry once the gel is made so once this agar solidifies they take the comb out and it will leave little wells in the gel and then it's into those wells that dna will be dispensed we use a tool called a micro pipet which is basically an instrument that allows us to administer a very very very small amount of volume usually in this case it's about 20 microliters which is a very small amount of volume okay so what we're doing what we're seeing here is they're actually loading dna into those wells and the dna is purple because it's dyed with a loading dye and that allows us to basically see which of these wells has dna in it the loading dye also makes the dna heavy so it sinks down to the bottom of those wells that are made once all the dna is loaded up and keep in mind at this point the dna that is added is dna that likely has undergone the polymerase chain reaction so there's a lot of whatever particular dna we're interested in in evaluating and it's also been digested so it's been cut with restriction enzymes already okay so this in here is amplified dna that has been cut with restriction enzymes so already the fragments of dna that are going to be present are uh present okay and then we load this gel into something that looks similar to this this is called a electrophoresis chamber electrophoresis because what it does is actually there's two electrodes there's the um the positive and the negative electrode i can't ever remember which one is which i think this is a negative and this is positive and what happens is that electricity flows across this way through the fluid there's actually fluid in here into which the we'll put the gel and you can actually see there's dna dispensed in these wells and as the electrical current passes through the gel it will pull the dna across this happens because dna is negatively charged because of the fact it has that sugar phosphate background well phosphate has negative charges on it so all in all there's a slight negative charge on molecules of dna well when we apply a current across here the negatively charged dna is attracted to the positive field of the electrophoresis chamber and so it drags for lack of a better word the dna fragments through the gel and then those that are the smallest will migrate faster and those that are slowest or biggest will migrate slow basically separates them all out based upon their size so it looks something like this the the bigger they are in terms of base pairs the closer they will be um the and then the the smaller that they are the greater the distance they will uh migrate so in other words you might start off with putting some samples into the wells and then um usually also when this is completed we put a marker in one of the wells the marker is a sample of dna that's been cut a certain way so that we actually know the size of the dna fragments that will congregate to form all the different bands after we uh run the gel so let's say we run the gel we turn the electrophoresis chamber on put an electrical current through it it starts dragging all the dna fragments through then you should start to see um once it's all done a separation of the dna into the various uh sizes that it is and then the marker will tell us um for any you know dna sample that we run let's say we run it and there's a sample of dna fragments here there's a sample of dna fragments here here and here simple after we cut it with the restriction enzyme and ran it through the gel and made four groups of dna that all varied in the size of the fragments in those groups we can then figure out the relative size of the fragments in each of these groups by comparing it to the latter is what it's sometimes known as or the marker so in this case if i was wondering okay i see a band of dna here on the gel this means that in this region of the gel a whole bunch of chunks of dna are here and i'm wondering how big the size of those dna fragments is i can just come over across and we see that it lines up with this particular band so anything that's in this region likely would be something that is of a size of approximately 600 base pairs in length or similarly if i want to know how big the dna fragments are in this chunk i can come across saturn i can see it's somewhere in between 300 and 200 base pairs so maybe it's about 250 base pairs is the size of each of the dna chunks in this group okay so pretty cool and this again allows us to uh do all sorts of stuff so for example here's a a picture of a gel that has been run here we see the marker here we see all the bands and it's they've labeled them based upon the size in kilobases so like 4 300 kilobases or 3 500 kilobases so they've labeled the size of each of the dna chunks that are in these bands and then they've also um put some sort of fluorescent dye on them of some kind and shining some sort of light on them so they glow i don't know exactly what they put on them or what kind of light they use to make it glow like this but here you can see like let's say this is dna just for the sake of example here the dna that was in this well was dna that was found and isolated from uh a murder weapon at a crime scene or something okay and then there was four suspects or there's five suspects so we put the suspect's dna in here we treated it with the in the pcr reaction to amplify it and then we put it in these wells we you know cut it with the same restriction enzymes that we cut the dna that was found at the crime scene that was also amplified and then we run it all out you can see that this suspect right here yielded dna bands that fall on the same location as the dna that was found in the crime scene so this would suggest that this individual here has the same dna as the dna that was isolated at the crime scene now it doesn't necessarily mean the individual is guilty it just means that uh the dna that was found at the crime scene or on the murder weapon or what have you is the same dna as the individual whether it was them that actually did the crime it's harder to determine but something like gel electrophoresis can tell us at least if this particular individual was at the scene of the crime or if their dna was truly found on a weapon of some kind or something like that there are a lot of different applications for this um that go above and beyond just solving crimes uh these can be used for paternity and maternity tests so you could run the dna of mom dad and baby and see uh if baby's dna more closely matches mom's dna or dad's dna if there's a match there um that would help to confirm uh if a child really did in fact belong to a parent or not or if there was another potential thing that happened okay um as we said earlier there's uh it can help to id crime and forensics for organ donation to see what donors might have a genome that's most similar to somebody that needs to be receiving an organ they could use this test they could use this to diagnose inherited disorders so you could take the blood of somebody that has for example hemophilia and amplify their dna of the from their genome and run it and then and then if somebody else had that same mutation it would likely be the case that there was a matching band pattern on the gel depending on how it was cut and treated and run okay um test for pathogens and foods so you can take food and you know grab the dna and amplify it and see if it matches some sort of pathogen that has a known banding pattern for some particular protocol that was used to digest the dna and sort it out okay you can determine if something's genetically modified or not so and then also comparative biology basically seeing how similar the genomes are between different um animals that maybe live now or didn't live now or are excinct or whatnot it's powerful because one only needs a very very small sample of dna to do this test because pcr allows you to take one simple small molecule of dna and making gazillions of copies of it and then once it's amplified it's really easy to you know cut it and run it and separate it out and see how it looks and compares to other things either case uh it's a pretty simple test to do we're going to do it in lab and this is something i did a lot when i worked at the u of m and a lot of different research groups used this technique for a variety of reasons so very interesting little piece there of biotechnology that we can use and interesting to talk about it here in the context of of criminology this concludes our discussion of the use of biology and criminology