DNA Sequencing 2 - Online Tutor, Practice Problems & Exam Prep

Sanger sequencing, or dideoxy sequencing, utilizes radio-labeled dideoxynucleotide triphosphates to identify DNA sequences by truncating strands during synthesis. Gel electrophoresis separates these fragments, allowing for sequence reading from smallest to largest. This method evolved to fluorescent labeling for efficiency. Pyrosequencing detects light from ATP production linked to nucleotide addition, while Ion Torrent sequencing measures pH changes from proton release. Both methods enhance DNA sequencing accuracy and efficiency, crucial for genetic analysis and research.

DNA Sequencing 2

Referred to as Sanger sequencing, this is basically dideoxy sequencing, where you use radio-labeled dideoxynucleotide triphosphates. Radio-labeled means they have some sort of radioactive tag on them so that you can identify them. As we discussed, you use a much smaller concentration of the dideoxynucleotides compared to the deoxynucleotides, so that only a small amount of the strands are truncated or shortened during synthesis. Of course, you only use one type of dideoxy which determines which base is present when you find a shortened strand because if there's only one type of dideoxynucleotide that you used in the synthesis, anytime you find a shortened strand, you know that the last nucleotide on that strand is going to be whichever one you were using in that particular reaction. You then use gel electrophoresis to separate the fragments of different lengths. You can see that right here. This is our gel, and you can see our fragments from our reaction with G are found here, and our fragments from reaction C are along this path, A along this path, and T along this path. If you were to try to read this code, you'd see that the actual code here is something like 'ATGCTTCG'. I'm going to stop right there. This 'G' is corresponding to this mark right here. So all I'm doing is reading down the gel more or less to determine the code. Of course, I know this to be the order because the smaller strands will travel further in the gel, and this arrow going up this way shows us the direction of travel. The strands down on this end right here are going to be the smallest, and the largest will be at the opposite end. So, you just read from smallest to largest, and that's your sequence. Eventually, these radio-labeled dideoxynucleotide triphosphates were replaced with fluorescent ones. The reason is that it's easier in the lab to scan for the light emitted from fluorescent molecules as opposed to working with radioactive substances. Also, you usually have to use some pretty nasty chemicals when working with these radioactive substances. So, it's more efficient, and you don't have to use these nasty chemicals. That's why there was this transition to using fluorescent ones. But basically, the exact same concept except instead of using gel electrophoresis, you use a capillary gel column. It's like electrophoresis gel except you're running it in a column and you're eluting out the various strands. As the strands come out, with the smaller ones coming out first and the bigger ones later, you have a photodetector that scans and determines what frequency or wavelength of fluorescence each strand is emitting. Based on the color that they're emitting, it's going to determine which labeled dideoxynucleotide triphosphate you have present. So, much in the same way we went through the gel, if we were using a spectrophotometer, we'd read as the strands come out: green, red, black, blue, and we'd know that green means A, red means T, black means G, and blue means C. Next, let me explain more about pyrosequencing and ion torrent sequencing.

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What is Sanger sequencing and how does it work?

Sanger sequencing, also known as dideoxy sequencing, is a method used to determine the nucleotide sequence of DNA. It involves the incorporation of radio-labeled dideoxynucleotide triphosphates (ddNTPs) into a DNA strand during synthesis. These ddNTPs lack a 3' hydroxyl group, causing chain termination when incorporated. The resulting DNA fragments of varying lengths are then separated by gel electrophoresis. By reading the sequence from the smallest to the largest fragment, the DNA sequence can be determined. This method has evolved to use fluorescent labels instead of radioactive ones for safety and efficiency, and capillary gel columns instead of traditional gels for separation.

What are the advantages of using fluorescent labels over radioactive labels in DNA sequencing?

Fluorescent labels offer several advantages over radioactive labels in DNA sequencing. Firstly, they are safer to handle as they do not involve exposure to harmful radiation. Secondly, fluorescent labels are easier to detect and quantify using automated systems, which increases the efficiency and accuracy of the sequencing process. Additionally, the use of fluorescent labels eliminates the need for hazardous chemicals required to work with radioactive substances. This transition to fluorescence has made DNA sequencing more accessible and streamlined in laboratory settings.

How does pyrosequencing detect nucleotide addition in DNA sequencing?

Pyrosequencing detects nucleotide addition by monitoring the release of pyrophosphate (PPi) during DNA synthesis. In this method, DNA polymerase adds nucleotides to a growing DNA strand, releasing PPi. The enzyme sulfurylase converts PPi to ATP, which then activates luciferase to produce light. The presence of light indicates that a nucleotide has been successfully added. By sequentially adding different nucleotides and detecting light emissions, the DNA sequence can be determined. This method is highly efficient and allows for real-time monitoring of DNA synthesis.

What is Ion Torrent sequencing and how does it differ from pyrosequencing?

Ion Torrent sequencing is a method that detects nucleotide addition by measuring changes in pH rather than light emissions. When a nucleotide is incorporated into a DNA strand, a proton (H⁺) is released, causing a change in pH. Ion Torrent technology measures these pH changes to determine the DNA sequence. Unlike pyrosequencing, which relies on light detection from ATP production, Ion Torrent sequencing uses semiconductor technology to detect pH changes. This method is fast, cost-effective, and scalable, making it suitable for high-throughput DNA sequencing applications.

What are the key differences between Sanger sequencing and next-generation sequencing (NGS) methods like pyrosequencing and Ion Torrent sequencing?

Sanger sequencing and next-generation sequencing (NGS) methods like pyrosequencing and Ion Torrent sequencing differ in several ways. Sanger sequencing uses chain-terminating ddNTPs and gel electrophoresis to determine DNA sequences, making it suitable for smaller-scale projects. In contrast, NGS methods are designed for high-throughput sequencing, allowing for the simultaneous sequencing of millions of DNA fragments. Pyrosequencing detects light emissions from ATP production, while Ion Torrent sequencing measures pH changes from proton release. NGS methods are faster, more cost-effective, and capable of handling larger and more complex sequencing projects compared to Sanger sequencing.