Which of the following components terminates the chain in a sequencing reaction?
A. Dideoxynucleotides
B. Klenow polymerase
C. DNA polymerase Ill
D. Deoxynucleotides

Role of Dideoxynucleotides in Chain Termination During Sequencing

Sequencing reactions, especially in Sanger sequencing, rely on the controlled termination of DNA synthesis to determine the nucleotide sequence. The component responsible for terminating the chain in sequencing reactions is dideoxynucleotides (ddNTPs). Understanding this mechanism is essential for mastering molecular biology techniques, especially for competitive exams like CSIR NET Life Science, IIT JAM, and GATE Biotechnology.

Correct Answer: (A) Dideoxynucleotides

The correct component that terminates the chain in a sequencing reaction is dideoxynucleotides (ddNTPs). These modified nucleotides lack a 3′-hydroxyl (-OH) group, which prevents the formation of a phosphodiester bond with the next nucleotide, thereby terminating DNA chain elongation.

Keyphrase: Dideoxynucleotides in DNA Sequencing

Dideoxynucleotides (ddNTPs) are critical for terminating DNA synthesis during sequencing reactions, especially in the Sanger method.

How Dideoxynucleotides Work in DNA Sequencing

Mechanism of Chain Termination

1. A mixture of normal deoxynucleotides (dNTPs) and fluorescently labeled dideoxynucleotides (ddNTPs) is added to a sequencing reaction.
2. DNA polymerase incorporates nucleotides into the growing DNA strand.
3. When a ddNTP is incorporated, it lacks the 3′-OH group, which is required for forming a phosphodiester bond.
4. The chain elongation stops at that point, producing fragments of varying lengths.
5. These fragments are separated by gel electrophoresis or capillary electrophoresis to determine the sequence.

Example Reaction:

• Template strand: 5′ – ATCGTACG – 3′
• Synthesized strand: 3′ – TAGCAT – 5′
• If ddATP is added, the strand would terminate at the position where ‘A’ is needed, generating fragments like:
  • 3′ – T
  • 3′ – TA
  • 3′ – TAG

Why Other Options Are Incorrect

Option B: Klenow Polymerase

• Klenow polymerase is a fragment of DNA polymerase I used in DNA synthesis but does not terminate the reaction.
• It lacks the ability to terminate chain elongation.

Option C: DNA Polymerase III

• DNA polymerase III is involved in DNA replication but not in sequencing reactions.
• It synthesizes new strands without chain termination.

Option D: Deoxynucleotides (dNTPs)

• Deoxynucleotides are the building blocks of DNA but do not cause termination.
• They allow continued chain elongation.

Importance of Dideoxynucleotides in Sanger Sequencing

High Sensitivity

• Dideoxynucleotides enable the identification of DNA sequences with single-nucleotide resolution.

Precision in Mutation Analysis

• Sequencing with ddNTPs helps identify point mutations and polymorphisms.

Cost-Effective and Accurate

• Sanger sequencing using ddNTPs remains a gold standard for sequencing small fragments of DNA.

Challenges in Using Dideoxynucleotides

Low Yield in Complex Genomes

• Large and repetitive sequences are difficult to sequence accurately.

Short Read Length

• The chain termination approach limits the length of DNA that can be sequenced.

High Cost for Large-Scale Projects

• Next-generation sequencing (NGS) is more suitable for whole-genome sequencing.

Applications of Dideoxynucleotide-Based Sequencing

Mutation Detection

• Identification of point mutations in genetic diseases.

Phylogenetic Studies

• Comparing DNA sequences to study evolutionary relationships.

Genetic Testing

• Diagnosing inherited disorders through gene sequencing.

Cancer Genomics

• Detecting somatic mutations and tumor-specific changes.

Future Developments in DNA Sequencing

1. Next-Generation Sequencing (NGS):

   • Overcomes limitations of Sanger sequencing with higher throughput.

2. CRISPR-Based DNA Analysis:

   • Precise gene targeting for improved sequence analysis.

3. Nanopore Sequencing:

   • Enables real-time and long-read sequencing.

Conclusion

Dideoxynucleotides (ddNTPs) are essential for chain termination in Sanger sequencing, allowing precise determination of DNA sequences. They work by preventing the formation of phosphodiester bonds, stopping DNA polymerization at specific sites. This method remains vital for genetic testing, mutation analysis, and research applications. For expert guidance on molecular biology techniques and competitive exam preparation, join Let’s Talk Academy — the leading institute for CSIR NET Life Science, IIT JAM, GATE Biotechnology, and DBT JRF.

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FAQs

Q1. Why do dideoxynucleotides terminate DNA synthesis?
Dideoxynucleotides lack a 3′-OH group, which is required for forming phosphodiester bonds.

Q2. What is the role of DNA polymerase in Sanger sequencing?
DNA polymerase incorporates nucleotides and ddNTPs during strand synthesis.

Q3. Why is Sanger sequencing still used despite NGS advancements?
Sanger sequencing provides high accuracy for small-scale sequencing projects.

This article was prepared with insights from Let’s Talk Academy, the top coaching institute for competitive life science exams