Dideoxy Sequencing: Videos & Practice Problems

Dideoxy sequencing, also known as Sanger sequencing, is a DNA sequencing method that utilizes dideoxynucleotides (ddNTPs) as chain terminators. The process involves five key components: unknown template DNA, DNA polymerase, DNA primers, deoxyribonucleotides (dNTPs), and ddNTPs. The chain termination PCR generates DNA fragments of varying sizes, which are then separated by gel electrophoresis. The resulting gel allows for the determination of the DNA sequence by reading the fragments from bottom to top, revealing the complementary sequence through base pairing rules.

Dideoxy Sequencing

In this video, we're going to begin our lesson on dideoxy sequencing. Dideoxy sequencing is a specific DNA sequencing method or technique that uses ddNTPs or dideoxynucleotides as elongation terminators in order to help determine the sequence of the DNA. Now, dideoxy sequencing was actually first discovered way back in 1977 by a scientist named Frederick Sanger, and for that reason, sometimes it is commonly referred to as just Sanger Sequencing. This was the first method of DNA sequencing that uses these dideoxynucleotides. We'll be able to talk more about this dideoxy sequencing and Sanger sequencing as we continue to move forward in our course. So, I'll see you all in our next video.

Components of Dideoxy Sequencing

This video, we're going to focus on the specific components that are needed for dideoxy sequencing, but we're not going to get into the actual process of dideoxy sequencing until we get to another video later on in our course. In this video, we're only focusing on the specific components. And so the components that are needed in dideoxy sequencing reactions include the following 5 components that we have labeled down below a through e. And of course, these labels a through e that you see here correspond with the labels a through e that you see down below in our image. And so the very first component that is needed in a specific dideoxy sequencing reaction is, of course, the unknown template DNA of interest whose sequence we do not know, which is why we want to conduct dideoxy sequencing. And so over here, what we have is our template DNA, and you can see this is the DNA here. And we do not know the sequence of the DNA, which is why we want to conduct this sequencing to figure out what is the sequence of this DNA. So, that is the first component. Then, the second component that we are going to need is actually a DNA polymerase, which you might recall is an enzyme that polymerizes or builds DNA. And so DNA polymerase is the main enzyme that's needed for DNA replication. And so, what you'll see here down below is the second component that we need is the DNA polymerase. And of course, this is the components of dideoxy sequencing here. Okay?

Now, the third component that is needed is going to be DNA primers. And these DNA primers are going to anneal to the template strand. And so recall that DNA primers were used in the specific technique that we talked about in our previous lesson videos called polymerase chain reaction or PCR. And so, what we'll see moving forward is that dideoxy sequencing is actually going to use a special type of PCR, and we'll see that moving forward. Now the, DNA primers you can see down below right here, and there will need to be, of course, 2 DNA primers.

Now, the 4th component that is going to be needed are all 4 DNA, deoxyribonucleotides. And so these are going to be the normal, deoxyribonucleotides that are used in DNA, during normal cellular DNA replication. And so, these deoxyribonucleotides include DATP, DTTP, DGTP, and DCTP. So this is basically the adenine, thymine, guanine, and cytosine. And then, you can see these deoxyribonucleotides down below here. And again, these are the normal DNA nucleotides.

And then of course, for dideoxy sequencing where the, the 5th and final component that we're going to need is a small amount of a single dideoxyribonucleotide. And so this is the special type of nucleotide. The DDATPs, DD TTPs, DDGTPs, and DDCTPs. And so in a particular dideoxy sequencing reaction within a single test tube, we would only want to use just one single, nucleotide. And so we would have to separate these reactions using different test tubes, to use different dideoxynucleotides And we'll talk more about this as we move forward in our course. And so recall that these DDNTPs, these dideoxyribonucleotides, they are going to terminate the DNA synthesis due to the presence of a 3 prime hydrogen, atom or group. And so, what you'll notice is down below right here, you can see that these are the dideoxyribo, DNA nucleotides. And so these are the special chain terminating, DDNTPs, dideoxynucleotides. And so these are really the 5 major components that are needed for dideoxy sequencing and these are the components that you will see mentioned as we move forward and talk more about the specifics of the process of dideoxy sequencing.

And so for now, this here concludes this lesson and, we'll be able to talk more about this as we move forward.

Which of the following is NOT required for the reactions in dideoxy sequencing?

• A. Deoxyribonucleotides.
• B. RNA polymerase.
• C. DNA primers.
• D. Template DNA.
• E. Dideoxyribonucleotides.

Answer: (B)

Chain-Termination PCR

In this video, we're going to continue to talk about dideoxy sequencing as we discuss the chain termination PCR steps or the chain termination polymerase chain reaction steps. Recall from our previous lesson videos that we already discussed polymerase chain reaction or PCR. Be sure to check out those older videos on PCR, polymerase chain reaction, before you continue here. Also recall from our previous lesson videos that DNA synthesis reaction is actually terminated when a dideoxynucleotide or a ddNTP is added to the 3' end of the growing DNA strand. The use of these ddNTPs to terminate the chain is really what chain termination PCR relies on.

In the first two steps of dideoxy sequencing, it requires setting up a chain termination PCR which is just a PCR reaction that's going to include small amounts of ddNTPs. In this first step, we're going to need to set up 4 separate reactions in 4 separate test tubes. Notice in our image on the left-hand side, you can see that we've got these 4 different test tubes where we're setting up 4 different reactions. Each of these separate reactions will contain all of the components needed for a normal PCR and also a small amount of a different ddNTP, which distinguishes one tube from another. In one test tube, it has all of the components for a normal PCR, but it also includes the ddNTP for cytosine, providing chain termination at all of the cytosine nucleotides upon amplification of the DNA. In another test tube, it has all of the normal components but includes the ddNTP for thymine, so chain termination in this tube will occur at all of the thymine nucleotides. Another test tube will contain all of the components for a normal PCR and a small amount of the ddNTP for adenine, and similarly, the last test tube will have all the components for a normal PCR but with the ddNTP for guanine. These four test tubes differ from each other in the small amount of the different ddNTP added, leading to chain termination at specific nucleotides: C's in the first, T's in the second, A's in the third, and G's in the fourth.

At the top here is mystery DNA, the specific DNA sequence that we want to sequence and determine. Dideoxy sequencing can help us determine the sequence of this mystery DNA, and we have to set up chain termination PCR for this. The mystery DNA will serve as the template DNA for amplification during this PCR and will go into all 4 of these test tubes. Then, in step number 2, we conduct the actual PCR reaction, the chain termination PCR reaction. DNA synthesis will produce a bunch of fragments of DNA because of the ddNTPs which will terminate the DNA synthesis reaction and create fragments that terminate at the specific nucleotides indicated in each tube.

The DNA synthesis produces fragments of DNA that will be complementary to the unknown target, the mystery DNA. Below in our image, upon conducting the actual chain termination PCR in step number 2, the DNA is replicated. You can see the products, the PCR products, and notice that there are various sized fragments of DNA generated. Some fragments have only one nucleotide, others more, and there's a different colored background at the end of each chain that represents the ddNTP being incorporated and terminating the chain at that nucleotide. By arranging these PCR products based off the size of the fragment from 5' to 3' end, the dideoxynucleotides at the chain's end allow for generating these different-sized fragments. Analyzing these PCR products is something we're going to talk about in our next lesson video. Analysis of these PCR products can reveal the sequence of the mystery DNA. This concludes our introduction to the chain termination PCR, and we'll be able to get some practice applying this and discuss exactly how these PCR products can be analyzed to reveal the sequence of the DNA in our next video. See you all there.

Determining the DNA Sequence from a Gel

So after chain termination PCR, the next steps in dideoxy sequencing involve determining the DNA sequence from a gel. And so, in the final two steps of dideoxy sequencing, the DNA sequence is finally going to be determined. In step number 3, which is a continuation of the chain termination PCR step from our previous lesson video, the fragments from all four chain termination PCR reactions are going to be separated by size using gel electrophoresis. If we take a look at our image below, notice on the far left-hand side what we have are the products of our chain termination PCR. These products are going to be different-sized fragments. These different-sized PCR products or different-sized fragments can be separated via gel electrophoresis. Recall from our previous lesson videos that gel electrophoresis loads each of the different samples toward the top of the gel in specific wells and then separates the fragments within each lane based on their size.

In step number 4, what we need to do is determine the sequence. The sequence of the DNA can be determined either manually, using the gel from gel electrophoresis, or the sequence can also be determined automatically using a computer on what is known as a chromatogram, which is this plot that you see over here on the right. We are going to focus on determining the sequence using the gel. The gel that you see below can actually be read backwards from bottom to top, and you read the gel across all lanes to reveal the complementary DNA sequence from 5' to 3'. I'll show you what I mean by this in the image below. In the example, it says to determine the mystery DNA sequence by analyzing the gel electrophoresis results from dideoxy sequencing. Notice over here in this gel, again, we have each of these lanes containing a different chain termination PCR reaction from the previous. That means that each of them is going to be ending with a different nucleotide. The ones with 'C' here end with the nucleotide 'C,' the ones with 'T' end with the nucleotide 'T,' and so on. The 'A's end with 'A's, and the 'G's are going to end with 'G's. The shortest fragments represent the fragments closest to the 5' end of the PCR product. To reveal the sequence from 5' to 3', we need to start at the bottom. You read the gel backwards and notice that the band at the very bottom, closest to the bottom, is highlighted here in lane 'T.' That means that this first nucleotide is going to be a 'T,' and we can go ahead and put that here, in this position, as the first nucleotide. Then reading the gel backwards, the next one closest to the bottom is the yellow one, representing a 'G' nucleotide, which is going to be the next nucleotide. Then the next one at the bottom here is an 'A,' so we would put an 'A' here. Then we have a 'C' and another 'C,' so we get two back-to-back 'C's.' Then, we have a 'T,' an 'A,' and last but not least, we have a 'G' in the final position toward the 3' end. You can see that the sequence has been revealed by reading the gel backwards, from bottom to top starting at these positions and working in this direction, revealing it from 5' to 3' end.

Next, we need to realize that now that we've revealed the complementary DNA sequence, which is the sequence of the PCR products, to reveal the mystery DNA sequence, we need to remember that the complementary DNA sequence is going to be complementary to the mystery DNA sequence. So, we would use our complementary base pairing rules to figure out the sequence of the mystery DNA. 'T's always base pair with 'A's on the opposite strand, so we have an 'A' here. 'G' is always base paired with 'C's, 'A's always base pair with 'T's, 'C's with 'G's, 'C's with 'G's, 'T's with 'A's, 'A's with 'T's, and 'G's with 'C's. What you see here is the mystery DNA sequence from 3' to 5' since recall that DNA strands are gonna be anti-parallel with respect to one another when they are complementary base pairing. Here we've revealed the sequence of the mystery DNA. You can see how dideoxysequencing and analyzing the gel backwards can be used to reveal the sequence.

Once again, if the gel is not going to be analyzed manually, another way to analyze the DNA sequence is using a computer, which can generate a chromatogram, a plot that looks something like this, and the chromatogram is also going to reveal the sequence. This concludes our brief lesson on how to determine the DNA sequence from the gel, using dideoxy sequencing. We'll be able to get some practice applying these concepts as we move forward in our course. I'll see you all in our next video.

According to the gel below, which of the following is the correct sequence on the unknown DNA molecule?

• A. 5 '- GCGATGCCAT - 3'
• B. 5' - ATGGCATCGC - 3'
• C. 5' - TACCGTAGCG - 3'
• D. 5' - CGCTACGGTA - 3'
• E. None of the above are the correct sequence.

Answer: (C)

Dideoxy sequencing is also known as chain termination sequencing because:

• A. The dideoxy nucleotide prevents further synthesis of DNA due to the lack of a free 5' carbon.
• B. The dideoxy nucleotide prevents further synthesis of DNA due to the lack of a free 3' OH.
• C. The dideoxy nucleotide prevents further synthesis of DNA due to the lack of a nitrogen-containing base.
• D. Chain termination is the same as sequencing by synthesis.
• E. None of the above are correct.

Answer: (B)

The final step in a Sanger DNA sequencing reaction is to run the DNA fragments on a gel. What purpose does this serve?

• A. It adds ddNTP to the end of each DNA fragment.
• B. It changes the length of the DNA fragments.
• C. It separates DNA fragments based on their sequence.
• D. It separates DNA fragments generated during the sequencing reaction based on one nucleotide differences in their size.

Answer: (D)

Here’s what students ask on this topic:

What is dideoxy sequencing and how does it work?

Dideoxy sequencing, also known as Sanger sequencing, is a DNA sequencing method that uses dideoxynucleotides (ddNTPs) as chain terminators. The process begins with the preparation of a reaction mixture containing the template DNA, DNA polymerase, DNA primers, normal deoxyribonucleotides (dATP, dTTP, dGTP, dCTP), and a small amount of one type of ddNTP. During DNA synthesis, the incorporation of a ddNTP terminates the chain because ddNTPs lack a 3' hydroxyl group necessary for forming a phosphodiester bond with the next nucleotide. This results in a series of DNA fragments of varying lengths. These fragments are then separated by size using gel electrophoresis, and the DNA sequence is determined by analyzing the pattern of terminated fragments.

What are the key components needed for dideoxy sequencing?

The key components needed for dideoxy sequencing include:

• Template DNA: The unknown DNA sequence to be determined.
• DNA Polymerase: An enzyme that synthesizes new DNA strands.
• DNA Primers: Short DNA sequences that anneal to the template DNA and initiate synthesis.
• Deoxyribonucleotides (dNTPs): The normal nucleotides (dATP, dTTP, dGTP, dCTP) used in DNA synthesis.
• Dideoxynucleotides (ddNTPs): Modified nucleotides that terminate DNA synthesis when incorporated.

These components work together in a chain termination PCR to produce DNA fragments that can be analyzed to determine the DNA sequence.

How are DNA fragments separated in dideoxy sequencing?

In dideoxy sequencing, DNA fragments are separated by size using a technique called gel electrophoresis. After the chain termination PCR, the resulting DNA fragments of varying lengths are loaded into wells at the top of a gel matrix. An electric current is applied, causing the negatively charged DNA fragments to migrate towards the positive electrode. Smaller fragments move faster and travel further through the gel, while larger fragments move more slowly. This separation by size allows for the determination of the DNA sequence by analyzing the pattern of fragments on the gel.

What is the role of dideoxynucleotides (ddNTPs) in Sanger sequencing?

Dideoxynucleotides (ddNTPs) play a crucial role in Sanger sequencing by acting as chain terminators. Unlike normal deoxyribonucleotides (dNTPs), ddNTPs lack a 3' hydroxyl group, which is necessary for forming a phosphodiester bond with the next nucleotide. When a ddNTP is incorporated into a growing DNA strand during synthesis, it prevents the addition of any further nucleotides, effectively terminating the chain. This results in a series of DNA fragments of varying lengths, each ending with a ddNTP. By analyzing these terminated fragments, the DNA sequence can be determined.

How is the DNA sequence determined from a gel in dideoxy sequencing?

After performing gel electrophoresis to separate the DNA fragments by size, the DNA sequence is determined by reading the gel from bottom to top. Each lane of the gel corresponds to a different ddNTP (A, T, G, or C). The position of the bands in each lane indicates where the chain terminated at that specific nucleotide. By reading the sequence of bands across all lanes, the complementary DNA sequence can be reconstructed from 5' to 3'. This sequence is then used to determine the original template DNA sequence by applying complementary base pairing rules.