Which one of the following set of essential components are required for Sanger method of DNA sequencing in a required buffer containing MgC12 and Tris-HCl?
(1) DNA template, a primer, 4 deoxyribonucleotides, 4 labelled dideoxyribonucleotides, DNA polymerase
(2) DNA template, a primer, 4 labelled dideoxy ribonucleotides, DNA polymerase, DNA ligase
(3) DNA template, 4 deoxyribonucleotides, 4 labelled dideoxy ribonucleotides, DNA polymerase, DNA ligase
(4) DNA template, a primer, 4 labelled dideoxy ribonucleotides, DNA polymerase, telomerase

Essential Components for Sanger DNA Sequencing

Sanger DNA sequencing, also known as the chain termination method, is a widely used technique for determining the nucleotide sequence of DNA. Developed by Frederick Sanger in 1977, this method relies on the controlled incorporation of dideoxynucleotides (ddNTPs) during DNA synthesis, which results in the termination of the growing DNA strand at specific points. Understanding the essential components involved in Sanger sequencing is crucial for accurate DNA analysis.

✅ Correct Answer:

(1) DNA template, a primer, 4 deoxyribonucleotides, 4 labelled dideoxyribonucleotides, DNA polymerase

Components Required for Sanger DNA Sequencing

1. DNA Template

• The DNA template provides the sequence that needs to be determined.
• It serves as the starting point for DNA polymerase to initiate synthesis.

2. Primer

• A short single-stranded DNA sequence that binds to the complementary sequence on the template DNA.
• Provides a starting point for the DNA polymerase to begin adding nucleotides.

3. Deoxyribonucleotides (dNTPs)

• Four standard dNTPs:
  • dATP (adenine)
  • dTTP (thymine)
  • dCTP (cytosine)
  • dGTP (guanine)
• These are incorporated into the growing DNA strand during polymerization.

4. Dideoxyribonucleotides (ddNTPs)

• Four ddNTPs are required:
  • ddATP
  • ddTTP
  • ddCTP
  • ddGTP
• Labeled with fluorescent or radioactive tags for detection.
• Lack a 3′ hydroxyl (-OH) group, which prevents further elongation of the DNA strand when incorporated.

5. DNA Polymerase

• Taq polymerase or modified DNA polymerase is used for strand synthesis.
• Adds dNTPs to the growing DNA strand and incorporates ddNTPs at random positions, causing termination of the strand.

6. Buffer Containing MgCl₂ and Tris-HCl

• MgCl₂ – Acts as a cofactor for DNA polymerase, ensuring proper enzyme function.
• Tris-HCl – Maintains the optimal pH required for the reaction.

How Sanger Sequencing Works

1. Primer Binding: A single-stranded DNA template is mixed with a primer that binds to the complementary sequence.
2. Extension: DNA polymerase starts adding dNTPs to the 3′ end of the primer.
3. Chain Termination: When a ddNTP is incorporated, extension stops because ddNTPs lack a 3′ hydroxyl group.
4. Gel Electrophoresis: The terminated fragments are separated based on size using capillary electrophoresis.
5. Sequence Analysis: The sequence is read from the shortest to the longest fragment using a fluorescence or radioactive signal.

Why Other Options Are Incorrect

❌ (2) DNA template, a primer, 4 labelled dideoxy ribonucleotides, DNA polymerase, DNA ligase

• DNA ligase is NOT required for Sanger sequencing.
• DNA ligase joins DNA fragments during replication and repair but plays no role in chain termination sequencing.

❌ (3) DNA template, 4 deoxyribonucleotides, 4 labelled dideoxy ribonucleotides, DNA polymerase, DNA ligase

• Missing a primer – A primer is essential for initiating DNA synthesis.
• DNA ligase is not required for sequencing.

❌ (4) DNA template, a primer, 4 labelled dideoxy ribonucleotides, DNA polymerase, telomerase

• Telomerase is an enzyme that extends the ends of chromosomes but is not involved in DNA sequencing.

Summary of Correct and Incorrect Answers

Key Differences Between dNTPs and ddNTPs

Applications of Sanger DNA Sequencing

✔️ Mutation detection – Used to identify point mutations in genetic diseases.
✔️ Genome sequencing – First method used for human genome sequencing.
✔️ Phylogenetics – Used to determine evolutionary relationships.
✔️ Molecular cloning – Confirms the accuracy of inserted DNA sequences.

Advantages of Sanger Sequencing

✅ High accuracy (~99.9%)
✅ Suitable for sequencing short DNA fragments (~500–1000 bp)
✅ Effective for single-gene sequencing

Limitations of Sanger Sequencing

❌ Expensive for large-scale genome sequencing
❌ Inefficient for sequencing long DNA fragments
❌ Requires high-quality DNA samples

Why Understanding the Components is Important

1. Accurate Sequence Determination – Correct combination of reagents ensures accurate reading of DNA sequences.
2. Troubleshooting Experimental Errors – Understanding the role of each component helps identify and resolve errors.
3. Adaptation to New Techniques – Knowledge of Sanger sequencing helps transition to next-generation sequencing (NGS).

Conclusion

The essential components required for Sanger sequencing include a DNA template, a primer, 4 deoxyribonucleotides (dNTPs), 4 labelled dideoxyribonucleotides (ddNTPs), and DNA polymerase. Understanding the correct experimental setup is crucial for successful DNA sequencing. For more insights into DNA sequencing and molecular biology, visit Let’s Talk Academy – your trusted resource for CSIR NET Life Science preparation!

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