Introduction to DNA Sequencing - Online Tutor, Practice Problems & Exam Prep

DNA sequencing is the process of determining the complete sequence of nucleotides in a DNA molecule, which can identify organisms, such as bacteria. Special nucleotides called dideoxynucleotides (ddNTPs) are crucial in this process, as they terminate DNA synthesis by blocking phosphodiester bond formation due to their unique structure. This characteristic is essential for techniques like dideoxy sequencing, where the incorporation of ddNTPs halts elongation, allowing for accurate sequencing of DNA strands.

Introduction to DNA Sequencing

In this video, we're going to begin our introduction to DNA sequencing. DNA sequencing refers to the specific process of determining the complete sequence of nucleotides, or the order of nucleotides, in a DNA molecule. DNA sequencing can be used to sequence either a small fragment of DNA or the entire genome of an organism, all of the DNA within the organism. The genome of an organism is unique to that specific type of organism. In our example below, we show how DNA sequencing can be used to sequence the entire genome of a bacterial species to help identify that bacterial species. Notice in this silly little cartoon down here, we have a little bacteria over here saying, "Hey, you don't know me, you don't know what type of bacteria I am," and the scientist is saying, "Yes, I do, because I've sequenced your DNA." Sequencing the DNA of an organism can help to identify that organism.

It turns out that DNA sequencing uses very special nucleotides called dideoxynucleotides. At least one type of DNA sequencing uses these special nucleotides called dideoxynucleotides. We're going to introduce dideoxynucleotides in our next lesson video. Dideoxynucleotides are commonly used in DNA sequencing techniques due to their unique features, which we'll talk about in our next lesson video. But for now, this concludes our brief introduction to DNA sequencing, the process of determining the complete sequence or the order of nucleotides in a molecule. We'll be able to get some practice applying these concepts and learn more about DNA sequencing as we move forward in our course. So I'll see you all in our next video.

Dideoxy Nucleotides

In our last lesson video, we mentioned that dideoxynucleotides are special nucleotides that are commonly used in DNA sequencing techniques. And so here in this video, we're going to talk more about these dideoxynucleotides. It's helpful to recall from way back in our previous lesson videos when we first introduced DNA that phosphodiester bonds are the types of bonds that form between the 3' hydroxyl group (OH group) and the 5' phosphate group of two nucleotides during DNA synthesis.

Now, these dideoxynucleotides are sometimes referred to as DDNTPs. Dideoxynucleotides, again, are very special nucleotides that differ from the usual normal DNA nucleotides that a cell might use. They actually contain a 3' hydrogen atom instead of containing the 3' hydroxyl group like what the normal DNA nucleotides have. By replacing this 3' hydroxyl group with a 3' hydrogen atom, this actually blocks the covalent formation of the phosphodiester bond. Dideoxynucleotides will block the formation of a phosphodiester bond. During the elongation step of DNA synthesis, if a DDNTP is incorporated, then the elongation will come to a stop.

This is going to be very important once we get to the specifics of the DNA sequencing technique as we move forward. The elongation is going to be terminated by the incorporation of the ddNTP at the 3' end of the new DNA strand. In this example, we're looking at how dideoxyribonucleotides can terminate elongation during DNA synthesis.

In the image at the top here in the greenish color, what we have is the regular DNA nucleotide, which is the deoxyribonucleotide. This is the normal nucleotide as we covered it when we first introduced DNA. At the bottom here is the special DNA nucleotide that we're calling the dideoxynucleotide ribonucleotide. Notice that it has the hydrogen atom at the 3' position instead of having the hydroxyl group at the 3' position. Over here, we have the normal deoxyribonucleotides which can form phosphodiester bonds, highlighted here between two different nucleotides.

It's important to note that these dideoxyribonucleotides cannot form phosphodiester bonds, as indicated by the red X. This is going to have important implications during the elongation phase of DNA synthesis. With the normal nucleotides, DNA synthesis will be able to proceed as normal. However, with these dideoxyribonucleotides, when there's a hydrogen atom at the 3' end instead of a hydroxyl group, DNA replication is going to come to a stop. It will be terminated, as mentioned above.

These dideoxynucleotides are going to help terminate the elongation of DNA synthesis. This will be important as we move forward and talk about dideoxy sequencing. This here concludes our brief introduction to dideoxynucleotides. As we move forward, we'll be able to talk about dideoxy sequencing. I'll see you all in our next video.

Here’s what students ask on this topic:

What is DNA sequencing and why is it important?

DNA sequencing is the process of determining the complete sequence of nucleotides in a DNA molecule. This technique is crucial because it allows scientists to identify organisms, understand genetic variations, and study evolutionary relationships. For example, sequencing the genome of a bacterial species can help identify the species and understand its characteristics. DNA sequencing is also essential in medical research, forensics, and biotechnology, as it provides detailed genetic information that can be used for diagnostics, treatment development, and understanding genetic disorders.

How do dideoxynucleotides (ddNTPs) function in DNA sequencing?

Dideoxynucleotides (ddNTPs) are special nucleotides used in DNA sequencing techniques. Unlike normal nucleotides, ddNTPs have a hydrogen atom at the 3' position instead of a hydroxyl group. This structural difference prevents the formation of phosphodiester bonds, which are necessary for DNA strand elongation. When a ddNTP is incorporated into a growing DNA strand, it terminates the elongation process. This termination is crucial for methods like dideoxy sequencing, where the incorporation of ddNTPs at various points allows for the determination of the DNA sequence by analyzing the lengths of terminated fragments.

What are the steps involved in the dideoxy sequencing method?

The dideoxy sequencing method, also known as Sanger sequencing, involves several key steps: 1) DNA denaturation to separate the strands, 2) Primer annealing to provide a starting point for DNA synthesis, 3) DNA synthesis using a mixture of normal nucleotides and ddNTPs, 4) Termination of DNA synthesis when a ddNTP is incorporated, and 5) Gel electrophoresis to separate the terminated fragments by size. By analyzing the pattern of terminated fragments, the sequence of the original DNA strand can be determined.

What are phosphodiester bonds and their role in DNA synthesis?

Phosphodiester bonds are covalent bonds that form between the 3' hydroxyl group of one nucleotide and the 5' phosphate group of another nucleotide during DNA synthesis. These bonds create the backbone of the DNA molecule, linking nucleotides together in a chain. In normal DNA synthesis, the formation of phosphodiester bonds allows the DNA strand to elongate. However, in the presence of dideoxynucleotides (ddNTPs), which lack the 3' hydroxyl group, the formation of these bonds is blocked, leading to the termination of DNA strand elongation. This property is exploited in DNA sequencing techniques to determine the nucleotide sequence.

How does DNA sequencing help in identifying bacterial species?

DNA sequencing helps in identifying bacterial species by determining the unique sequence of nucleotides in their genomes. Each bacterial species has a distinct genetic makeup, and by sequencing its DNA, scientists can compare the obtained sequence with known sequences in databases. This comparison allows for the accurate identification of the bacterial species. DNA sequencing is particularly useful in microbiology for diagnosing infections, studying bacterial diversity, and tracking the spread of bacterial pathogens.