Sanger Sequencing of DNA

in biochemistry it's very useful to be able to determine what the sequence of nucleotides in some DNA molecule because if we know what the sequence of nucleotides is that will give us information about how gene expression takes place and what types of proteins are produced now what's the process by which we can actually sequence our DNA molecule well the process is known as Sanger dye deoxy method or simply Sanger DNA sequencing now before we discuss the steps of this process let's discuss an important molecule used in this process and let's see why it is actually used so the molecule is this molecule here it's called two prime three prime dye deoxy nucleoside triphosphate or simply D G and T P now this molecule is almost identical to a normal DC nucleoside triphosphate the only difference is the sugar component contains a three prime carbon dan does not contain a hydroxyl group so remember in a normal deoxy nucleoside triphosphate the presence of the hydroxyl group on the 3 prime carbon allows DNA polymerase to actually form a phosphodiester bond with the next nucleotide in line so in the process of DNA synthesis when we're replicating a DNA strand the DNA polymerase needs this hydroxyl group to be present on the 3 prime carbon to actually form the phosphodiester bond and if that hydroxyl group is not present as in this case it will not be able to form that phosphodiester bond and so DNA replication would essentially stop and so what this DD ntp molecule is used for in this method is to basically stop the process of DNA replication and we'll see why that's important towards the end of this lecture so let's move on to these four steps so in step one of the Sanger DNA sequencing we have two action obtain that DNA molecule that we want to sequence so let's suppose we have a double-stranded DNA molecule as shown on the board now the second step of this process will involve DNA replication and remember DNA replication only takes place if the two strands of DNA have separated so in step one what we essentially want to do is we want to denature the double helix structure of the DNA we want to separate the two strands of DNA and the way that we're going to separate them is by adding sodium hydroxide so remember a base or an acid if we mix the DNA in either a basic or acidic solution in this case a basic the base will essentially ionized the bases of our DNA molecule and that will disrupt and break the hydrogen bonds and so if we take the double strand DNA molecule and we add sodium hydroxide we produce these two individual strands of DNA now one of these single strands of DNA can actually be chosen for the sequencing process now it doesn't matter which one of these DNA strands we choose because if we choose this one for example then once we determine the sequence of this DNA strand we can easily determine what the sequence of the other strand is because these two strands are complementary they have complementary base pairing so the G bases with our C and the a basis with our T's so once we know this sequence we know what the other sequence is simply by the base pairing process so let's choose this single-stranded DNA molecule we isolated we place it into our beaker that contains only this molecule here and then we move on to step number two and in step number two we want to basically replicate this DNA molecule and so what that means is we need three different things we need a DNA primer we need DNA polymerase and we need the building blocks the four types of normal deoxy nucleoside triphosphates so in step two the solution containing the single strand of DNA this one here is mixed with number one or a a labeled radioactively-labeled DNA primers so we need the DNA primer to basically for that DNA polymerase to actually work because remember the DNA polymerase will only initiate replication if the primer is present and what that means is we have to know what this initial sequence is on that DNA molecule because to build the DNA primer we have to know what the complimentary sequence is to this group here so if this is a CG then we have to build a primer that contains a sequence T G C and so we can build that in a laboratory and we also radioactively-labeled that DNA primer because that will basically allow us to pinpoint exactly where that molecule is when we undergo the process of gel electrophoresis so we add a label DNA primer that is complementary to the three end of that single stranded DNA molecule that we want to sequence so this is the three end of the DNA molecule and remember DNA polymerase reads from three to five and it builds from five to three and that's why this is the end that we actually want to build the DNA primer for so we add the DNA polymerase and we also add the four types of deoxynucleotide triphosphates so we add datp dgtp dctp and TTP and finally the important component in the Sanger dioxyde method is a tiny amount of one of the four types of D G and T P molecules remember we have four different types that can exist and that's because we have four different types of bases so this base could be adenine it could be guanine it could be cytosine or it can be a sign mean and so we have four different types of DG ntp molecules and in step two we have to add a tiny amount about 1% with respect to the other nucleoside triphosphates of a specific dioxin nucleoside triphosphate so we don't add all four types we only add one time now why is that important well let's see what that actually does by examining the following diagram so we essentially take this DNA molecule the single strand and we mix it with these four components so we have the radioactively labeled DNA primer that is complementary to the 3n we have the four types of deoxynucleotide triphosphates these four ones shown here we have the DNA polymerase and we have a very small amount so let's say about 1% of the DD ATP so that's the specific Gd and TP that we're going to choose for this specific experiment for that specific beaker now what will begin to happen well what will happen is the complementary DNA primer will hybridize itself to this section here as shown in the following diagram so this is our DNA primer it will form these base pairs as shown in the following diagram so T base pairs with a G base pairs with C and C base pairs with G and then the DNA polymerase will bind onto the primer and it will use the hydroxyl group on this cytosine to basically form that first phosphodiester bond and so it will take a diamond it will take this molecule here to basically form the first base then it will move on on to the second base which is a T and that will basically form an A now we have two types of a's that we can use one of them is the normal deoxyadenosine triphosphate the other one is a dye see adenosine triphosphate and the dye deoxyadenosine triphosphate does not contain a hydroxyl group on the third prime carbon and what that means is if that DNA polymerase actually uses the DG ATP to place this base it will not be able to continue that DNA replication process because that molecule does not have the hydroxyl group that is needed to produce the phosphodiester bond with the next base and so if this a comes from the DD ATP this process will end and this will be the molecule that we will synthesize and this is why we have fragment number one molecule number one now because we only have one percent of this DD ATP this DNA polymerase will only sometimes use the GG ATP usually it's going to use the normal datp molecule and if it uses the normal datp that contains the hydroxyl then it will continue synthesizing those phosphodiester bonds and so we can produce fragment number two so if this was normal then it will continue so the DNA polymerase would add the thymine then the guanine then the cytosine then the thymine and now it comes to a tea so that means there's once again the possibility for an A and the a can either come from this normal d ATP or the abnormal DG ATP that lacks the hydroxyl and if it's this group here then once again we will stop the synthesis and this fragment will be produced now if it wasn't that molecule then we would add the next in so if the a was normal the normal datp it would produce the next nucleotide in line and so the next one is also an a because this is a T so once again this is an A and now we have a possibility between this world this one and let's suppose it's once again the G a teepee and so it will stop at once again after this because it lacks that hydroxyl and so at the end in our mixture in beaker number one after we conduct step number two with the DGA TP these are the other three fragments that will be present inside our beaker number one now we take that beaker number one and we place it into SDS page so as DS polyacrylamide gel electrophoresis so this is our setup and we have four different wells now well number one we label as the DD ATP because this is step 2 where we use the GG ATP we take the mixture and we place it into well number 1 Lane number 1 and we allow these two separate based on their masses remember in gel electrophoresis the smaller our molecule is the farther down it will move along that gel so if this is molecule 1 2 & 3 this band will be for molecule 1 this band will be for molecule 2 and this band the highest up will be the largest molecule molecule 3 now we continue the same process three more times and the second time around we use a different DG and TP the third time around we use yet another GG and TP and the final fourth time we use the final GG and TP so let's suppose the second time around instead of using the DGA TP we used DD GTP and so instead of having the fragments where we stopped on the A's we're going to have the fragments where we stopped on the G's and so because we only have to see so one this see doesn't count because it's part of the primer so we have one see and we have a second see so the complementary would have a G here and a G here so we only form two fragments and this Lane would contain two bands because we only contain two fragments with different sizes then we repeat the process instead of using this one we use DD CTP so C that means we have to count up to G's here not including this one because it's part of the primer so we have one G we have two Gs and that means we're going to have two complementary C's so we're going to have two different fragments once again one two and finally if we use this one we have to look for our adenine so we uh we have to look for the adenine so we have one two three and four we should have four fragments and that's exactly what we get in this particular case so basically in step three we take step two and we repeat that same step three different times with the other three D G and T Peas and in step four once the four reactions are completed we run gel electrophoresis each reaction mixture is placed into a lane so Lane one lane to lane three lane four and the results are then transferred onto a polymer sheet and then we use x-ray order radiography to basically determine exactly where those radioactively-labeled fragments actually were and so this is the diagram that we get now how can we use this to actually determine what the sequence of that initial DNA molecule is well we know what the first three nucleotides are because that's the primer so we have T G and C so the question is what are these remaining nucleotides here well let's try to use the following set up to beit's determine what the sequence is so we know that the farther down along our page the the smaller our fragment is and the closer the nucleotide sequence is to the beginning and the fragment all the way at the bottom basically describes this right over here so that means the first nucleotide following the primer is the one lowest at the bottom so this and and according to this graph according to this on set up the lowest one at the bottom corresponds to a T so this one is a team the next one is we have an a the next one is so let's make a little bit bigger we have a t we have an A then we have a tee next we have a G next we have a C next we have a tee then we have two A's 1 & 2 then we have a tee then we have a G and then we have a seat and so this is the C

Channel: AK LECTURES

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Keywords: Sanger sequencing, Sanger dideoxy method, Sanger DNA sequencing, sequencing DNA molecules, sequencing DNA, biochemistry, DNA sequencing, dideoxynucleoside triphosphate, ddNTP

Id: IAf_6TZ6BYc

Length: 16min 6sec (966 seconds)

Published: Tue Feb 17 2015