Chain Termination Method

Definition

The chain termination method, also known as the Sanger sequencing method, is a DNA sequencing technique that allows for the determination of the precise order of nucleotides (A, T, C, and G) within a DNA molecule. This method relies on the controlled termination of DNA synthesis to generate a set of DNA fragments of varying lengths, which can then be analyzed to decipher the DNA sequence.

5 Must Know Facts For Your Next Test

1. The chain termination method involves the controlled incorporation of chain-terminating dideoxynucleotides (ddNTPs) during DNA synthesis, which lack a 3' hydroxyl group and prevent the addition of further nucleotides.
2. The DNA sample is divided into four separate reactions, each containing a different fluorescently labeled dideoxynucleotide (ddATP, ddCTP, ddGTP, or ddTTP).
3. The DNA fragments of varying lengths, each terminated by a specific dideoxynucleotide, are then separated by size using gel electrophoresis.
4. The separated DNA fragments are detected by their fluorescent labels, and the sequence of the DNA is determined by the order in which the fluorescent bands appear on the gel.
5. The chain termination method is widely used in automated DNA sequencing machines, where the fluorescent signals are detected and analyzed by a computer to generate the final DNA sequence.

Review Questions

• Explain the role of dideoxynucleotides in the chain termination method of DNA sequencing.
  • In the chain termination method, dideoxynucleotides (ddNTPs) play a crucial role. These modified nucleotides lack a 3' hydroxyl group, which is necessary for the addition of the next nucleotide during DNA synthesis. When a ddNTP is incorporated into the growing DNA chain, it prevents the addition of further nucleotides, causing the chain to terminate. This controlled termination of DNA synthesis at different positions along the template DNA molecule allows for the generation of DNA fragments of varying lengths, which can then be separated and analyzed to determine the sequence of the original DNA sample.
• Describe how the separation and detection of the terminated DNA fragments is achieved in the chain termination method.
  • The chain termination method utilizes gel electrophoresis to separate the DNA fragments of varying lengths. Each of the four separate reactions (containing ddATP, ddCTP, ddGTP, or ddTTP) produces a set of DNA fragments terminated by a specific dideoxynucleotide. These fragments are then loaded onto a polyacrylamide gel and subjected to an electric field, which causes the DNA fragments to migrate through the gel at different rates based on their size. As the fragments move through the gel, they are detected by their fluorescent labels, which are attached to the dideoxynucleotides. The order in which the fluorescent bands appear on the gel corresponds to the sequence of the original DNA sample, allowing for the determination of the nucleotide sequence.
• Evaluate the significance of the chain termination method in the context of DNA sequencing and its impact on the field of genomics.
  • The chain termination method, also known as the Sanger sequencing method, has been a revolutionary technique in the field of DNA sequencing and has had a profound impact on the advancement of genomics. This method allowed for the efficient and accurate determination of the nucleotide sequence of DNA molecules, which was a crucial step in the completion of the Human Genome Project and the subsequent rapid growth of genomics research. The chain termination method's ability to generate long, high-quality DNA sequences has enabled the detailed analysis of genomes, the identification of genetic variations, and the understanding of the genetic basis of diseases. Furthermore, the development of automated DNA sequencing machines based on the chain termination method has significantly increased the speed and throughput of DNA sequencing, making it a widely adopted technique in various fields, including molecular biology, genetics, and biotechnology. The impact of the chain termination method on the advancement of genomics and our understanding of the genetic code cannot be overstated.

Related terms

Dideoxynucleotides: Modified nucleotides that lack a 3' hydroxyl group, preventing the addition of further nucleotides and causing chain termination during DNA synthesis.

Gel Electrophoresis: A technique used to separate DNA fragments based on their size, allowing the determination of the DNA sequence in the chain termination method.

Fluorescent Dye Labeling: The process of attaching fluorescent dyes to the dideoxynucleotides, enabling the detection and identification of the terminated DNA fragments during the sequencing process.