Bozeman science: Molecular biology overview

it's mr. Andersen and in this podcast I'm going to talk about all of molecular biology before I get there I want to talk about a story close to home in the 1970s some researchers went to Yellowstone Park there in the geyser basin they pulled these bacteria out called thermus aquaticus and from that they pulled out an enzyme called tak polymerase that means thermus aquaticus polymerase or the DNA polymerase found in this bacteria cool thing about it is that it can handle really high temperatures and survive and the whole genetic engineering molecular biology is built upon this enzyme so we're talking about trillions of dollars how much of that goes back into Yellowstone Park none and so they've changed their policy on that but it's great way to kind of frame this idea of molecular biology and how important it is when I was thinking of how I wanted to explain this in a way that made sense I always kind of fall back on an analogy and so basically what I want to talk about is how we've gone over the last 30 or 40 years to the point where we now know all the DNA inside a human and so basically think about it this way imagine if you look at these magazines right here there's a whole bunch of information that's found inside here there's a whole bunch of text inside here and basically if I could take all of the text that I want and make something out of it like a really cool ransom note then I would be manipulating that text and so I want to kind of use this as an analogy for what we've done in molecular biology and so what was the first thing that was ever done well the first ransom note ever in the terms of molecular biology looked something like this and you might say that's not very impressive basically what is it it's like cutting out letters from a magazine but turning it upside down so you can't see the letters and so what was the first thing that we were able to do is we were able to cut the DNA and we do that using in a restriction enzyme but you should think of a restriction enzyme like a scissors it's able to cut the DNA how do we paste DNA well basically to paste DNA the glue stick equivalent of that is hydrogen bond DNA will just come back together again once it's been cut because of the hydrogen bonds what's the next ransom note well it would look something like this basically it was the first recombinant DNA we took DNA from a frog if I remember right and a bacteria and they were able to combine those two using this hydrogen bonding but again not very impressive ransom note next thing that happened was something like this so you can see in here that what we're doing is we're actually ordering these little fragments cut out of a magazine from small to large but again you can't see what the letters are so that's called gel electrophoresis and then we developed something called PCR polymerase chain reaction where we made a copy of that and so if this is that original in PCR we make a duplicate of that and we make an exact duplicate so every duplicate that we make from that is going to be exactly the same and that's what PCR does makes copies of DNA that's exactly the same ok what's the next advancement well the advent of the marker so marker is basically going to be a little section of DNA that marks that specific DNA so if you read that there's they found the marker or the genetic marker for breast cancer that means that there's a piece of DNA that's inherited by all people who have this genetically based on breast cancer and then finally we get to where we are today so right now we've used DNA sequencers to figure out all the letters inside our DNA but it's hieroglyphics in other words you can't really read what it means we know what all the letters are but we don't know where the genes or what the genes express in other words what proteins they make and so when we look at this ransom note this is the future this is where we want to get but we're still quite a ways out and so basically going back to the big things in this talk that you'll need to remember first one is the scissors the role of the scissors will be played by restriction enzymes the role of the glue will be hydrogen bonds that are found within the DNA the ruler or the measuring device is something called gel electrophoresis to copy that we use the polymerase chain reaction and then to actually read it we use something called a DNA sequencer or a gene sequencer it's going to look at all of the letters even though we might know what know what they do we can find them right now so let's start with the scissors and that's a restriction enzyme where the restriction enzyme come from well basically they come from bacteria because bacteria locked in this million year war with viruses and the viruses you can see inject their DNA into the bacterial DNA and they make more viruses and so how do you fight back well basically what they do is they methylate their DNA first of all what does methylate mean you basically add a methyl group to all of their DNA from the bacteria to protect it and then they're going to secrete these or create these restriction enzymes and what the enzymes do is they chop up or cut DNA since all of the bacterial DNA is protected by these methyl groups what it's really going to chop up is all this foreign DNA and so all of that viral DNA is broken apart and then the bacteria after its done that can return to its specific shape so let me show you how restriction enzyme work so if this is a section of DNA a real common restriction enzyme is something called eco r1 it comes from the word ecoli this is a restriction enzyme one and basically what it'll do is it'll scan the DNA until it finds this specific sequence and it's literally going to cut it in half and so if I were to find that where is that going to be up here well you can see we have a GAA T so it's going to cut right like that through here it's going to go all the way to the end and just going to cut it in half like that and so if we take a look at that basically what will it do it will break that into two fragments if we take another one Oh what did you see that can be right back together again so what brings it back together again they're going to be little hydrogen bonds here between those two ends and in fact we call this a sticky end and this is sticky end because they're going to be hydrogen bonds form between the two and so once the enzyme gone it comes right back together again if we look at another one this is the hind III enzyme basically it's going to cut between these two A's and so it cut it right here all the way down here and it's going to cut it into two fragments like that if we apply that enzyme that was found in this specific type of bacteria if it goes away then it comes right back together again what if we add both of those enzymes what's it going to do well it's going to cut it into two and two points order to restriction sites and then we're going to have three fragments that come from that so those are restriction enzymes what do we use them for in molecular biology basically to cut DNA and then to glue it back together again all you need is hydrogen bonds and so the first recombinant DNA that of a frog and a bacteria how did they get together basically they cut them both with the same enzyme they had the same sticky ends and then they're able to come back together again what's the next one that we have to do so what we've done is showing you how to cut that's a restriction enzyme how to glue that's hydrogen bond now we have to figure out how to separate the DNA according to its length and so to do that let me talk about pachinko machines pachinko machines are essentially these vertical machines they're really popular in Japan you put a ball up at the top you usually flick the ball up at the top then that ball is going to bounce down and if it eventually goes where you want it to go if it get eventually goes into a little cup down here at the bottom then you might get more of those balls back but that case it wouldn't so imagine a game of pachinko where we had one ball like this that's bouncing its way down and then we had instead of another ball we had like eight balls that are attached together so something like that so what would happen to them if we drop them in a pachinko machine does it make sense that they're sometimes going to go slower they're going to wrap around it's going to take them way longer to get down to the bottom and so the single pachinko ball would already be at the bottom where this big chain of pachinko balls isn't even made it very far and so how does that apply in DNA well think of DNA instead of pachinko so basically if we were to take a small fragment of DNA it's going to work its way farther down through that pachinko machine sooner than one of these a big fragments and again we don't use pachinko machines but we do use gel electrophoresis and so how does this work basically inside you have a gel the gel is kind of the consistency of jell-o it's going to have little wells on one side where you insert the DNA and what's pulling the DNA well in a pachinko machine it's going to be the gravity but here you can see that there is a red cable that means there's a positive charge on this end so there's a positive electrode all the way across there's going to be a negative electrode you can see it right back here on this side so basically the DNA is going to migrate across that gel DNA has a negative charge that's going to be pulled towards the positive end and so if we look at this this is kind of that if you've ever heard of like a DNA fingerprint what's going on well basically the DNA is going to be pulled in this direction we could say this since it's been on for a while that this fragment right here is going to be bigger than this fragment right because this small fragment has made its way farther from these original wells where it was up here and lots of times you'll run a ladder as well a ladder is a bunch of DNA with known distances or known quantities and so you can read across and see how big is that how many how many nucleotide pairs do we have when we do this in class we use a guy called ethidium bromide and basically it'll die the DNA you can put it under a blacklight and you can see where the DNA is it's normally clear you can't see it okay what's the next thing we have to do remember after now that we've sorted according to its length now we have to make copies of it and to do that we use the polymerase chain reaction or PCR this was invented by Kary Mullis he was riding his motorcycle one day and came up with this idea and so to do that we need a few extra things in our PCR machine one thing we need is a primer primer is going to be a little section of DNA that'll grab onto the DNA it'll allow that polymerase to drive down the DNA now where's tech polymerase from remember it's that bacteria because in the PCR we're going to heat this up really hot what else do we need we need nucleotide so we need new letters and so let's check this out basically we put it into PCR machine which looks kind of like a photocopier you put your DNA in the top and then it's going to quickly heat it and cool it and heat it and cool it and so as it heats this it's going to unzip that DNA in the middle it's going to break all those hydrogen bonds what's the next thing that's added well the next thing that will be added is going to be the primer the primer is going to bond to the complementary sides of the DNA now we create the primer because it's going to target a specific gene that we want what happens next well once the primer is in place then tack polymerase is going to grab on now this looks familiar if you know anything about DNA replication how does that work well we unzip our DNA it's helicase that does that we put a primer down and then it's going to be DNA polymerase inside us that makes copies of our DNA but in a PCR machine it stack polymerase and the reason why is tak polymerase can can withstand these really hot temperatures okay watch carefully what happens next well basically as the tack polymerase runs in either direction it's going to add complementary letters to either side and so basically what do we have we had one strand of DNA now we have two so the PCR machine will cycle it'll again heat up the DNA so it unzips those hydrogen bonds we're going to add primer on either side tak polymerase is going to grab on and as it races down we're going to produce complementary sides and again heat it up we're going to unzip the sides the primer is added on tak polymerase on and now we have if you look at it we've now got 8 strands of DNA and so this is just in a few minutes we've gone from 12 to 24 28 and now we just get 16 you get that exponential growth so we can make a whole bunch of DNA very very quickly so we're almost to the end what's the last thing and our analogy of the ransom note is really reading what those letters are and to do that we use a DNA sequencing DNA sequencer sometimes it's hard to explain basically if you were to Google the Sanger method you'd find some videos it might help a little more but basically it's like a PCR machine so if you look down here we got the primer we got the TAC polymerase we got our letters but then we have these 30 or these four special letters they're called die deoxynucleotides and so they're just like adenine cytosine thymine and guanine but they have a specific color and so what happens you'll heat it up primer will be added tak polymerase will be added and then we're going to start adding those letters and so if I were to go across if there's a tea here there'd be an a here and a tee and a G and a/c and we'll say that's a tea and the tea SI jeje SI ay ay ok so usually it's just going to make copies of it but occasionally as it makes those copies instead of putting a regular a in it'll put one of these we're days in there and what that we're day is going to do is it's going to be dyed it's going to have a specific color and it's also going to stop the sequence and so basically you can't add new letters and so a lot of the time you'll get a regular run-of-the-mill copied DNA strand but occasionally you'll get these fragments that just have an a at the front and they have a specific color which is going to be green maybe we run it again the next time and the a seems normal but the next one is a tea that's weird and so that's going to stop it at that point but it's going to give it a color of red or maybe another time we're going to get an A and a tee and a G but then we're going to put a weird see on there and that's going to stop it the sea is going to have this blue color and so basically what you can do is then run gel electrophoresis all these fragments are going run through a gel electrophoresis machine or running a gel and basically what they're going to do is they're going to separate according to the fragment length well what's the smallest fragment which went the farthest that's going to be this little fragment of a with the weird a at the beginning and so what does that tell us well it tells us that the first letter is going to be an A and so a computer can go through and read all these colors and it can read the sequence of the DNA can figure out the letters in our DNA and so the human genome project that was the job of that to sequence all of the DNA in a human this is these are the DNA sequencing machines but what I want you to remember is that we've sequenced all of the DNA in a typical human and as you grow up they'll be able to sequence all the DNA specifically inside you but at this point we're kind of right here we've got the DNA we know the order that it's in but we don't know what proteins those make or how those proteins interact and so the next project after the Human Genome Project is the human proteome project and so that's a lot I know um but I hope that's helpful