Sanger Sequencing

by Georgina Cornwall, PhD

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    00:00 Before we move in to how sequencing works.

    00:04 I need to introduce you to one more player in the game. That is the dideoxynucleotide.

    00:11 You will recall that the three prime OH handle is really key for DNA to continue synthesis.

    00:20 If there is no three prime OH handle on that ribose sugar, then DNA polymerase cannot add new nucleotides to the chain. Dideoxy, DNA deoxyribonucleic acid is missing the three prime OH handle to grab onto and add new nucleotides. When we sequence DNA, we are going to be replicating DNA in vitro. We are unzipping it, asking for it to replicate and continually making more strands in order to see what the sequence is. Termination of replication occurs every time a dideoxynucleotide shows up. Now dideoxynucleotides have been made for all of the nucleotides.

    01:14 We can have As,Ts, Cs, and Gs. Let us take a closer look at what happens when we have each of these nucleotides in a vial and we are asking replication to happen.

    01:29 First of all, the ingredients for replication that are pretty key are going to be our template strand of DNA. We better have some DNA polymerase. We probably need to have some primers. We need normal nucleotides for certain and a few dideoxynucleotides. In the case of DNA sequencing, we are going to have four seperate vials. This is the enzymatic method where we have one vial that has all of the regular ingredients and dideoxy-As and another one that has dideoxy-Gs and then another one that has dideoxy-Cs and then another one that has dideoxy-Ts. There are four different reactions going on in which we were asking for multiple rounds of synthesis of DNA. In the container that contains just dideoxy-C for example, we will see that every so often DNA replication will terminate, but it will terminate when one of these dideoxynucleotides is put down. For example, here you have three different lengths at which synthesis stopped at the first G. Synthesis stopped at the second G so on and so forth. You got to keep in mind that there are hundreds of different fragments, thousands probably of different fragments of variant lengths depending on how far DNA polymerase went along before picking up a dideoxynucleotide, which had no three prime OH handle, so termination of replication happened. In the tube that has dideoxy-Cs, we see the same thing happen, replication happens, thousands of copies were made. Some of them stop early. Some of them stop later and then we will look at dideoxy-A. We have the same thing going on in that vial and in the fourth container, we have the same thing going, but now they all end in Ts. Now we can use these fragment lengths and the endings and guess what, to figure out the lengths of the fragments, we are going to do gel electrophoresis. Each of the different reactions or vials are put into a different lane in a gel electrophoresis and we will let the process go, run our current across the gel. The DNA fragments move towards the positive pole because they are negatively charged. Now we can read our gel and it is important to think about what is going on. Each lane has fragments that end in G, C, A, and T respectively and because of the position in the gel, we can read the fragments. Now the shortest fragments go the farthest and the longest fragments go the least far.

    04:17 The ones that are closest to the wells are going to be at the three prime end of the DNA strand.

    04:24 We can read the sequence like this. The first one we know is in the T lane and so it must be a T. The second one in the sequence as we have read down the gel is AG and we so can see that it's a G because of its position, there is lots of them in there and so the next one in the line is a G. Following that, we can see the next one down the line is the C so on and so forth. We can put the A in position and we can read the entire gel till we get to the end and we have a sequence. Now you can only so many or so long of a sequence in one gel. Clearly we cannot have a gel that goes all the way around the world. We have to chop it into pieces, which is why we had that chopping into pieces part.

    05:14 This whole technique, the enzymatic method is pretty laborious and used to take a long time for us to get these small sets of sequence and then overlap the fragments and align them and figure out what the whole genome sequence is, but things became automated. That was really a pivotal point. We could do automated DNA sequencing and in this case, it works in just the same way. We have DNA replication occurring and we have dideoxynucleotides, but they are just done in one vial. We mix it all together. We have got one reaction. We have got dideoxy-A, dideoxy-G, dideoxy-C, dideoxy-T. And we have all of the other regular nucleotides.

    05:59 Synthesis is going to occur as normal. Most of the time DNA polymerase is going to pick up the right nucleotides and then every once in a while you will pick up a dideoxy, no three prime OH handle, termination of synthesis. So we have varying fragment lengths. Exactly the same philosophy going on in this automated sequencing. How it comes automated then is that we can label those dideoxynucleotides with a glowing or fluorescing label. And we have them in four different colors to match the four different letters. And the core part now is that we can run electrophoresis in a capillary tube, a very small tube and as the nucleotide fragments move across the gel, there is a laser that essentially reads them at the end of the capillary tube as they fall off the end of the gel and then a computer puts together that sequence and spits out precisely what that DNA sequence is. A lot less laborious, much quicker and this is one of the ways that allowed us to sequence the human genome much more rapidly than we would have if we were using the enzymatic method being much slower.

    07:20 So automated still using enzymes, but it is an automated method. That is the way that we sequence genomes these days.

    About the Lecture

    The lecture Sanger Sequencing by Georgina Cornwall, PhD is from the course Genomics.

    Included Quiz Questions

    1. Dideoxynucleotides
    2. Regular DNA nucleotides
    3. Template strand
    4. DNA polymerase
    5. Taq Polymerase
    1. …the incorporation of a chain terminating dideoxynucleotides by DNA polymerase during in vitro DNA synthesis.
    2. …the incorporation of a chain initiating dideoxynucleotides by DNA polymerase during in vitro DNA synthesis.
    3. …the incorporation of a chain terminating deoxynucleotides by DNA polymerase during in vitro DNA synthesis.
    4. …the incorporation of a chain initiating deoxynucleotides by DNA polymerase during in vitro DNA synthesis.
    5. …the incorporation of a chain initiating dideoxy-ribonucleotides by DNA polymerase during in vitro DNA synthesis.
    1. …the lack of 3’-OH group on the ribose moiety of the dideoxynucleotide.
    2. …the lack of 5’-OH group on the ribose moiety of the dideoxynucleotide.
    3. …the lack of 2’-OH group on the ribose moiety of the dideoxynucleotide.
    4. …the lack of 1’-OH group on the ribose moiety of the dideoxynucleotide.
    5. …the lack of 4’-OH group on the ribose moiety of the dideoxynucleotide.
    1. Capillary gel electrophoresis
    2. Chromatogram
    3. Fluorescent dye-labeled ddNTPs
    4. X-ray film
    5. Autoradiograph

    Author of lecture Sanger Sequencing

     Georgina Cornwall, PhD

    Georgina Cornwall, PhD

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    really good
    By Cerys C. on 28. September 2020 for Sanger Sequencing

    simply explained so well understood, very well done. lots of info but concise