DNA Sequencing 1 - Online Tutor, Practice Problems & Exam Prep

DNA polymerase synthesizes DNA using an RNA primer, a template strand, and deoxynucleotide triphosphates, relying on the energy from breaking phosphate bonds. It cannot initiate synthesis but elongates existing strands. Dideoxy DNA sequencing employs dideoxynucleotide triphosphates, which lack a 3' hydroxyl group, halting synthesis and allowing for the determination of DNA sequences through length analysis of truncated strands. This method involves running four reactions with different dideoxynucleotides, followed by gel electrophoresis to identify the sequence based on strand lengths.

DNA Sequencing

You might remember from biology that DNA polymerase actually requires a few things in order to synthesize DNA, including an RNA primer, a template strand, and deoxynucleotide triphosphates. It's going to use that template strand to know the correct order of bases to put together because the template strand is going to have a base sequence that's complementary to the sequence that DNA polymerase wants to synthesize. And it's going to use the deoxynucleotide triphosphates as for the polymer, and remember, it's going to be using the energy from breaking those phosphate bonds in order to actually drive this reaction. Now, it actually has to use an RNA primer because DNA polymerase can't start synthesizing a new strand of DNA on its own. It can only elongate a strand that already exists. So it's going to, or in our cells rather, we lay down a small stretch of RNA on the template strand and then DNA polymerase comes and attaches and lengthens that. Of course, it creates a strand of DNA and later the RNA is removed but you don't really need to worry about all that. You just need to understand the basics of how DNA polymerase works. You can see the reaction carried out by DNA polymerase summarized in this equation right here:

primer of length n + deoxynucleotide triphosphate → primer of length n + 1 + pyrophosphate + H +

And as byproducts from that reaction, we get pyrophosphate and a hydrogen ion. These byproducts are actually going to become important later when we talk about some other DNA sequencing methods. But for now, we're going to talk about the original DNA sequencing method and that is dideoxy DNA sequencing. Dideoxy DNA sequencing is a method that relies on these dideoxynucleotide triphosphates. Normally a deoxynucleotide triphosphate is missing an OH group on the 2' carbon. In dideoxynucleotide triphosphates, they're actually missing not only the 2' hydroxyl but also a 3' hydroxyl group. Now, this is very important because during the course of DNA synthesis, we have that 3' hydroxyl, which is the attachment point for the next nucleotide. In the course of the reaction, we add our new nucleotide onto that 3' hydroxyl, creating a bond there, and then our byproducts are a hydrogen ion and pyrophosphate. If our OH group is missing on that 3' hydroxyl, we don't have anything to add a new nucleotide onto. So, when a dideoxynucleotide triphosphate is added to a nucleic acid, it's going to halt synthesis, causing the strand of DNA to stop at that particular nucleotide.

How is this actually used for sequencing? Well, in a clever way. It is a seemingly low-tech method of sequencing DNA, but there's very sophisticated thinking behind it. You run four DNA synthesis reactions. In each, you use some deoxynucleotide triphosphates at a millimolar concentration and a very small micromolar concentration of dideoxynucleotide triphosphates. So you have mostly the normal ones that DNA polymerase wants to use for synthesis and just a small amount of these dideoxynucleotides to halt synthesis whenever they're added. This results in a small chance of adding a dideoxynucleotide triphosphate, meaning that in the big vat where this DNA synthesis is carried out, only a small portion of the strands of DNA you synthesize are going to be truncated. You do this reaction four times because in each, you only want to add one type of dideoxynucleotide triphosphate—Thymine in reaction 1, Cytosine in reaction 2, Adenine in reaction 3, and Guanine in reaction 4. You have some way of identifying which is which. You separate these reactions so that you only have one type of dideoxynucleotide triphosphate you're using, making it easier to parse out each one. Basically, what you end up with are strands of different lengths from each DNA synthesis reaction. You separate them using something like gel electrophoresis to determine the order of lengths and from that, you can actually determine the sequence. So you see the primer, how long the primer is, and then you see a strand that came from the reaction with dideoxy G, right? Like number 4 here. That strand is one nucleotide longer than the primer, so you know that that's going to be a G. And then from that same batch of DNA, you have one that's 2 nucleotides longer than the primer, and so on. In this way, you're actually able to determine the sequence of the DNA. It's a roundabout way of determining the sequence if you think about it, which is why I say it's not the most high-tech method, but there's sophisticated thinking behind this.

Alright. So let's actually turn the page and talk about some specific ways in which this is used.

Here’s what students ask on this topic:

What are the requirements for DNA polymerase to synthesize DNA?

DNA polymerase requires several components to synthesize DNA: an RNA primer, a template strand, and deoxynucleotide triphosphates (dNTPs). The RNA primer provides a starting point for DNA synthesis, as DNA polymerase cannot initiate synthesis on its own. The template strand serves as a guide, with its base sequence being complementary to the new DNA strand. The dNTPs are the building blocks that DNA polymerase uses to elongate the new strand, and the energy for this process comes from breaking the phosphate bonds in the dNTPs.

How does dideoxy DNA sequencing work?

Dideoxy DNA sequencing, also known as Sanger sequencing, involves using dideoxynucleotide triphosphates (ddNTPs) that lack a 3' hydroxyl group. This absence halts DNA synthesis when incorporated into the growing DNA strand. The process involves four separate reactions, each with a different ddNTP (A, T, C, or G). The resulting DNA fragments of varying lengths are then separated by gel electrophoresis. By analyzing the lengths of these fragments, the DNA sequence can be determined, as each fragment ends with a specific ddNTP, indicating the position of that nucleotide in the sequence.

Why is an RNA primer necessary for DNA polymerase?

An RNA primer is necessary for DNA polymerase because the enzyme cannot start synthesizing a new DNA strand from scratch. It can only add nucleotides to an existing strand. The RNA primer provides a short, pre-existing strand with a free 3' hydroxyl group, which DNA polymerase can then elongate by adding deoxynucleotide triphosphates (dNTPs). This primer is later removed and replaced with DNA.

What role do dideoxynucleotide triphosphates play in DNA sequencing?

Dideoxynucleotide triphosphates (ddNTPs) play a crucial role in DNA sequencing by terminating DNA synthesis. Unlike regular deoxynucleotide triphosphates (dNTPs), ddNTPs lack a 3' hydroxyl group, preventing the addition of further nucleotides. In dideoxy DNA sequencing, small amounts of ddNTPs are added to four separate reactions, each containing a different ddNTP. When a ddNTP is incorporated into the growing DNA strand, it halts synthesis, creating fragments of varying lengths. These fragments are then analyzed to determine the DNA sequence.

How is gel electrophoresis used in dideoxy DNA sequencing?

In dideoxy DNA sequencing, gel electrophoresis is used to separate DNA fragments based on their length. After the four sequencing reactions (each with a different dideoxynucleotide triphosphate) are completed, the resulting DNA fragments are loaded onto a gel. An electric current is applied, causing the fragments to migrate through the gel matrix. Shorter fragments move faster and travel further than longer ones. By comparing the positions of the fragments from each reaction, the DNA sequence can be determined, as each fragment ends with a specific nucleotide.