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hello and welcome to another online lecture from the university of washington's synthetic biology course so today we're going to talk about primer design to start off imagine you have a plasmid of DNA and as we know a plasmid is just a circular loop of DNA that is common in bacteria also imagine that we are particularly interested in this segment of DNA denoted in red well how would we go about isolating that segment of DNA from the other thousands of base pairs in the entire plasmid well we're going to need to do something called primer design and use a tool known as PCR if you are unfamiliar with PC R go ahead and scroll down to the description section where a hyperlink is posted that will direct you to a video that will explain PCR further so assuming that you are familiar with PCR we'll continue forward what I've drawn down here is a zoomed in view of the region of DNA that we are actually interested in and as you remember each of these letters dictates a different base that is available in DNA so we have G's C's A's and T's and as you might remember G's bond with C's and A's bond with T's and G's actually bond with C's with three hydrogen bonds and a is bond with T's with two hydrogen bonds so a GC combination is actually a stronger bond than an eighty combination that will be important later on as I explained something so in order to isolate the region of DNA that we are actually interested in we need to design primers that will dictate that region we do that by designing one forward primer and one reverse primer I will explain this further in the expanded view of our segments of DNA below so as you know DNA is double-stranded and written classically in the 5 prime to 3 prime direction so that means that the complementary strand or this strand down here below is written in 3 prime or 2 5 prime which means that it is actually written backwards to standard convention so if I was going to design a primer that worked in the forward direction as shown above this forward direction here here I'll mark it for you as forward this one is forward and this one is reverse so if I was going to design my forward primer I would need it to be active on the 3 prime end and what that means is that DNA polymerase can actually bind on to a 3 prime end of DNA and synthesize more DNA from that end the 5 prime end however is in an inactive state that DNA polymerase is unable to bind to that means that all synthesis of DNA needs to originate from a 3 prime end and move in the 3 prime direction so if I was going to design a forward primer it would need to bind on to this complementary strand or this bottom strand of DNA and that and that will become clear in a moment so if I was going to design a primer I need it first to be about 20 bases long so filling in the complementary bases of DNA I have G C a a t g c a see t a see G a T t C G a okay so I have 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 and 20 so this is an interesting dilemma another aspect of primer design is that you want it to anneal very strongly especially on the ends and even more importantly on the 5-prime end so just as a reminder this is a segment of DNA and here I'm just drawing the the phosphate backbone and this is 5 prime and this is the 3 prime end 3 but as I mentioned previously a's and T's only have 2 bonds and GS and C's have 3 hydrogen bonds between them that means that this T at the end is a weaker bond than if I had moved this to 21 bases long for my primer so what I would actually do if I were the one designing this primer I would move this one more forward and make this a 21 base long primer and incorporate this C here and what that does is it helps anchor down this position for when DNA polymerase actually binds in so I'm going to draw a DNA polymerase molecule and it's going to attach here DNA polymerase I'll label that for you this is D a polymerase oh and it's actually going to attach to the three prime end and continue synthesizing DNA in this direction so that makes sense so we have our forward primer down below and it's going to synthesize DNA in that direction so that part is done and if you'll notice our forward primer is actually the exact same sequence as our top strand of DNA look we have G C a a T G C a CT and so on and so forth and it's the exact same as our top strand so when writing DNA you can literally just take the first 20 bases or 21 if that's what you need in order to incorporate a G or C at the end and use that as your forward primer so forward primer is done and mark this as forward now designing the reverse primer is a little bit more complicated so in the same manner what we're going to do is write out the complementary base pairs to this sequence for 20 bases long so here we have G and you'll notice this is the same as this bottom strand down here but we have to change something in order to get it into standard notation so we have G a t g TC a T G a G a t c g a a t c g and luckily our at our three prime end in this situation actually ends on a G or C so we can keep this one at 20 bases long and as a reminder this is the five prime end and this is the active three prime end so another DNA polymerase could bind here and synthesize DNA in this direction go ahead and draw that for you just to make it a little bit more of a visual process and this is also DNA polymerase D P and this will synthesize DNA in that direction so now we have designed our reverse primer right well actually this is where things get a little bit more confusing we have to do something called the reverse complement of this strand of DNA in order to actually design our primer because if we were to send into the company that actually synthesizes our primers for us gcta AG and so on this direction we would actually get something that was v prime on this end and three prime on this end which would not anneal to this upper strand so in order for our primer to actually function in the way that we have designed it to we have to take something called the reverse complement so up above here I have these bases and what I essentially have to do is write it in Reverse so I have G I'm going to write G a t g t c a t g a G a t c g a a t c g and so this is our reverse complement of this segment of DNA and as you can see the g's line up a it's just this flipped around 180 degrees so this is now the 5 prime end and this is the 3 prime so this would be something that we could actually send into a company to synthesize for us whereas the other version would not work and so this is our reverse primer so another thing that you should consider while you're constructing your primers is the total GC content and what that is is the percentage of your primer that is composed of GS and C's and as I mentioned before a G and a C is a stronger bond than an A bonded to a T that means that a 20 base segment primer that is a low percentage of GC bonds will actually have a lower annealing temperature than a primer that has a higher GC content what that means when I say annealing is that the primer physically bonds to our gene or gene segment of interest and this temperature annealing this low-temperature annealing of G and C means that when the temperatures increased in PCR this primer physically cannot bind on it does not have enough energy in its bonds to bind on to our gene of interest which means that PCR most likely will not work so bear in mind that the total GC content when designing your primers and try to get it around 50% if possible another thing to think about is that primers come in a price range depending on their size so up here I have designed a standard 20 base primer that's because primers under 60 base pairs are within a certain price category anything above 60 base pairs of a primer the synthesis actually becomes less and less reliable so that the company has to physically as an manually purify your primer for you which increases the price significantly so try to design primers that are below 60 base pairs including any regions that are known as sticky ends those should be covered in another video that will further denote them but just really quickly in the time that I have left I will give you an example of an application in which we would design primers with sticky ends so imagine I have another the same circular plasmid of DNA and I have designed these primers in order to isolate this region here and I'm actually interested in inserting it into this other plasmid that has these openings and this is actually a double-stranded segment of DNA so I'm going to draw it as double-stranded you can notice that I've drawn these overlaps here these single-stranded sticky ends on this segment so I'm assuming that I've already come in and use a restriction enzyme which is covered in another one of our videos if you are interested in what that is to actually cut the DNA and leave these ends open so I've designed my primers and they move in the forward and reverse directions so I have my forward and reverse primers and these are 20 base pairs long I could also design what are called sticky ends on the primers that do not line up and are not complementary to my and my original plasmid so this segment will not bind to this original plasmid instead it actually lines up with the sequence over here these sticky ends on my my plasma that I've used my restriction enzyme on that I want to insert this gene into so a standard ratio for primer design is 20 base pairs on your gene of interest and then 40 base pairs for the plasmid that you are going to physically insert the gene of interest in to and what that does is it keeps it ideally below your 60 base pair limit that I mentioned previously so just drawing that in on the primers up here as you can see I have drawn this sticky end coming off so DNA polymerase will synthesize DNA in this direction creating the rest of this strand ink that will incorporate the entire sequence of interest but it will also end up having this sticky end on it that can be inserted into the new gene or into the new plasmid well that's about it if you are interested more in how to insert genes into plasmids go ahead and see our video on Gibson assembly