The ability to determine the DNA sequence of an individual is a powerful tool for paternity questions and criminal investigations, among other uses. This lesson will describe one laboratory method that can be used to sequence DNA.
Sanger Sequencing Method
Reporter: Professor Pear, I think our readers are going to enjoy learning how forensic scientists can use DNA to distinguish between suspects like you did in the case of the Spiral Staircase Killer.Professor Pear: Actually, there are a number of different ways a scientist can use DNA to identify a person. DNA sequence analysis has gotten more and more sophisticated over time.Reporter: Um, why don’t you just tell me about a basic method for distinguishing between two people?Professor Pear: Well, I guess I can tell you about the method that Fredrick Sanger developed for DNA sequencing.
The Sanger method allows scientists to determine the DNA sequence of a sample. There are now more sophisticated ways to analyze forensic samples, but understanding how basic sequencing works will be a good starting point for your readers.
Professor Pear: Sanger sequencing is really quite ingenious. It’s a laboratory procedure that determines DNA sequence through the use of dideoxynucleotides. That’s why it’s also sometimes referred to as dideoxy sequencing.Reporter: Uh, Professor, what is a dideoxynucleotide?Professor Pear: Oops. Silly me.
A dideoxynucleotide is a nucleotide that is missing the 3′-hydroxyl group of its sugar. It’s basically a special kind of nucleotide that scientists use in the Sanger sequencing method. Let’s revisit dideoxynucleotides after I tell you a little about the sequencing procedure.
Dideoxynucleotides Are Chain Terminators
Professor Pear: But before we discuss the sequencing procedure, let’s consider a simple example.Imagine you and I are playing a word game. The object of the game is for you to guess the sentence I have written based on a series of clues I provide to you. I will tell you a letter and the location of that letter in the sentence.
For instance, if I tell you the third letter from the left of the sentence is e, the first letter from the left is t, and the second letter from the left is h, you could tell me that the first three letters of this sentence are the.Note that it doesn’t matter what order I give you each clue as long as each clue provides you with a letter and position within the sentence. If I provide a clue for each character in the sentence, you can assemble a complete sentence, right?Reporter: That makes sense, but what does it have to do with DNA sequencing?Professor Pear: That’s conceptually how Sanger sequencing works. We need two pieces of information to apply the strategy of our sentence game to DNA sequencing.
First, we need to know the identity of the nucleotide. Is it a G, A, T, or C? Second, we need to know the location of the nucleotide.
For instance, is it the first, sixth, or tenth nucleotide of the sequence?Dideoxynucleotides are the answer to both of these questions.Recall the structure of DNA. Nucleotides in a DNA molecule are held together by phosphodiester bonds. These phosphodiester bonds can form because the phosphate on the 5′ end of a new nucleotide can react with the hydroxyl, or -OH group, at the 3′ end of the growing DNA molecule.A dideoxynucleotide lacks this 3′-hydroxyl group.
That means that polymerase can’t add another nucleotide once a dideoxynucleotide has been added to a nucleotide chain.
Chain Termination DNA Sequencing
Professor Pear: Scientists can use a mixture of regular nucleotides (abbreviated dNTP) and dideoxynucleotides (ddNTP) to sequence DNA. Let’s start with a mixture of some ddGTPs, but mostly the four regular dNTPs: template DNA, buffer, one DNA primer, and DNA polymerase.Reporter: Wait.
Those ingredients sound familiar. Aren’t those basically the same ingredients you use in PCR?Professor Pear: Yes! This DNA sequencing procedure is similar to PCR, the laboratory procedure used to create copies of DNA. However, instead of a pair of DNA primers like PCR, Sanger sequencing uses only one primer.
You see, the primer provides a starting reference point.Note in our word game if you didn’t know which side of the sentence your clue was using as the reference point, it quickly becomes a very difficult game. Thus, using a single primer provides a clear reference point for our sequence data.
Most of the time, DNA polymerase adds dNTPs to the primer as it forms a new DNA molecule. However, whenever it happens to add one of the rarer ddGTPs, no further nucleotides can be added because of the structure of the dideoxynucleotides. At this point, we know that the last nucleotide in this sequence is a G because we only added ddGTPs to this tube.
Many, many of these reactions are taking place simultaneously in the tube, but the point at which a ddGTP is incorporated is completely random in each case. Therefore, all of these reactions are going to produce different-length DNA molecules ending in a ddGTP.
Reporter: Okay. I think I understand how to produce those fragments, but it seems like it would take forever to sort through all that data.Professor Pear: Oh, good point. All that data probably does seem a little overwhelming. But, remember that gel electrophoresis is a good way to distinguish between DNA molecules of different sizes. Let’s see what kind of results we’d get if we performed four different reactions with each of the possible ddNTPs and then analyzed that data on a gel.
Interpreting Sanger Sequencing Data
Professor Pear: Here’s an example of that data in a sequencing gel.
It may look like information overload, but let’s take it one step at a time.
Recall that shorter DNA fragments travel farther in a given amount of time than longer DNA fragments during gel electrophoresis. Therefore, if we start at the bottom of our gel and ‘read’ upward, we can ‘read’ our DNA sequence with the sequencing primer as the starting reference point.The band located lowest on the gel is an A.The next smallest band is a T.The third band is a G, and so on and so forth.In this way, we can easily determine the sequence of the DNA segment we were analyzing.Reporter: Wow.
That analysis was much less complicated than I thought it would be.Professor Pear: As I mentioned before, this is a very simple version of analyzing DNA.Nowadays, forensic analysis uses more sophisticated techniques and technology that are faster and more sensitive than this basic example. However, hopefully this example helped you understand the basic principles of how scientists can determine the DNA sequence of an individual. And, hopefully this explanation has given you a sense of how powerful forensic evidence can be to a criminal case such as this one.
By testing fourteen different locations in the human genome, we were confident that Mr. Teal’s, and not Colonel Custard’s, DNA was found at the scene of the crime. This helped get the case against Colonel Custard dropped and led the DA to consider charging Mr. Teal with killing Mr. Bones in the spiral staircase with the lead pipe.
Reporter: Professor, thank you for your explanation. That was very enlightening. Let me just double-check that my notes are correct.Sanger sequencing is a laboratory procedure that determines DNA sequence through the use of dideoxynucleotides as sequence terminators.A dideoxynucleotide is a nucleotide that is missing the 3′-hydroxyl group of its sugar.
It is used in Sanger sequencing as a chain terminator.When a dideoxynucleotide is incorporated into the growing DNA chain, it prevents any further nucleotides from being added. By performing this reaction with each of the four different dideoxynucleotides, DNA products of different lengths are produced.
These sequencing products can be ordered by size using gel electrophoresis. And, using the primer as a starting point, the DNA sequence is read from the bottom of the gel upward.
You will be able to explain the importance of DNA sequencing and how the Sanger method works after watching this video.