Chain termination

Definition

Chain termination refers to a process in DNA sequencing where the addition of nucleotides is halted, resulting in fragments of different lengths that represent the sequence of nucleotides. This technique is crucial in first-generation sequencing methods, such as Sanger sequencing, allowing for the accurate determination of DNA sequences by incorporating modified nucleotides that prevent further elongation of the DNA strand.

5 Must Know Facts For Your Next Test

1. Chain termination uses dideoxynucleotides, which are critical for stopping the elongation of the growing DNA strand.
2. In Sanger sequencing, each of the four dideoxynucleotides is labeled with a distinct fluorescent dye, allowing for the identification of each fragment during analysis.
3. The resulting DNA fragments from chain termination are analyzed using capillary electrophoresis, which sorts them based on size to read the sequence.
4. Chain termination effectively generates a mixture of DNA fragments that end at different lengths, representing all possible sequences derived from the original template.
5. This method was revolutionary for its time and set the standard for early DNA sequencing efforts, paving the way for modern genomics.

Review Questions

• How does chain termination contribute to the overall process of Sanger sequencing?
  • Chain termination is essential in Sanger sequencing because it allows for the generation of DNA fragments that are terminated at specific nucleotide positions. By incorporating dideoxynucleotides during replication, each fragment ends at a point that corresponds to the original template's sequence. This creates a collection of fragments that can be separated and analyzed to determine the exact order of nucleotides in the original DNA strand.
• What role do dideoxynucleotides play in chain termination, and how do they differ from regular nucleotides?
  • Dideoxynucleotides (ddNTPs) are key players in chain termination because they lack a 3'-OH group essential for forming phosphodiester bonds. When a ddNTP is incorporated into a growing DNA strand during replication, it prevents any further nucleotides from being added. In contrast, regular nucleotides contain a 3'-OH group that allows for continuous elongation. This difference is what enables the precise stopping of DNA synthesis at specific points, forming fragments necessary for sequencing.
• Evaluate the impact of chain termination methods on advancements in genomic research and technology.
  • The introduction of chain termination methods significantly advanced genomic research by providing accurate and efficient ways to sequence DNA. Sanger sequencing, based on this principle, was one of the first methods to enable researchers to decode entire genomes, leading to breakthroughs such as the Human Genome Project. The ability to determine genetic sequences has transformed our understanding of genetics, evolution, and disease, laying the groundwork for subsequent developments in high-throughput sequencing technologies and personalized medicine.

Related terms

Sanger Sequencing: A method of DNA sequencing that relies on chain termination to create fragments of varying lengths, enabling the determination of the sequence of nucleotides.

Dideoxynucleotide (ddNTP): A modified nucleotide used in chain termination that lacks a 3'-OH group, preventing further extension of the DNA strand when incorporated.

Electrophoresis: A laboratory technique used to separate DNA fragments based on their size, commonly employed after chain termination to visualize the resulting sequences.