Chain termination method

Definition

The chain termination method is a technique used in DNA sequencing that involves the incorporation of labeled dideoxynucleotides into a growing DNA strand, leading to the termination of the strand at specific bases. This method allows for the determination of the sequence of nucleotides in a DNA molecule by analyzing the lengths of the terminated fragments produced during the synthesis process. By using different labeled dideoxynucleotides, researchers can identify the order of nucleotides in the original DNA template.

5 Must Know Facts For Your Next Test

1. The chain termination method was developed by Frederick Sanger in the late 1970s and revolutionized DNA sequencing techniques.
2. In this method, each dideoxynucleotide is labeled with a different fluorescent dye, allowing for the simultaneous detection of all four bases in a single reaction.
3. The resulting fragments from the chain termination process are separated by size using gel electrophoresis or capillary electrophoresis, providing a clear readout of the DNA sequence.
4. The accuracy of the chain termination method makes it a reliable technique for sequencing small to medium-sized DNA fragments, although newer methods have since been developed for longer sequences.
5. This technique played a pivotal role in significant projects like the Human Genome Project, facilitating the mapping and understanding of human DNA.

Review Questions

• How does the incorporation of dideoxynucleotides affect the process of DNA synthesis in the chain termination method?
  • The incorporation of dideoxynucleotides into a growing DNA strand halts further synthesis because these nucleotides lack a hydroxyl group at the 3' carbon. When a dideoxynucleotide is added, it prevents additional nucleotides from being added to the chain, resulting in shorter, terminated fragments. This selective termination allows researchers to determine where each base was incorporated, ultimately revealing the sequence of nucleotides in the original DNA template.
• Discuss how gel electrophoresis is utilized in conjunction with the chain termination method to analyze DNA sequences.
  • Gel electrophoresis is used to separate the terminated DNA fragments generated by the chain termination method based on their size. After adding dideoxynucleotides during synthesis, each fragment varies in length depending on where termination occurred. When these fragments are loaded into a gel and subjected to an electric field, they migrate at different rates, allowing for separation. The smallest fragments move fastest and appear first, creating a pattern that can be analyzed to deduce the sequence of bases present in the original DNA molecule.
• Evaluate the impact of the chain termination method on modern biotechnology and its significance in genomic research.
  • The chain termination method significantly impacted modern biotechnology by providing a foundational technique for sequencing DNA. Its development led to advancements in genomic research, enabling scientists to map entire genomes, including complex organisms like humans. The accuracy and efficiency of Sanger sequencing facilitated critical breakthroughs in genetics, medicine, and evolutionary biology. Although newer high-throughput sequencing technologies have emerged, the principles behind chain termination continue to influence methodologies used today, demonstrating its lasting importance in the field.

Related terms

Dideoxynucleotide: A type of nucleotide that lacks a hydroxyl group at the 3' carbon position, which prevents further extension of the DNA strand once incorporated.

Sanger Sequencing: A DNA sequencing method developed by Frederick Sanger that uses the chain termination method to determine the nucleotide sequence of DNA.

DNA Polymerase: An enzyme responsible for synthesizing new DNA strands by adding nucleotides to a growing chain, crucial in both DNA replication and sequencing.