DNA sequencing by the Sanger method takes advantage of which property of DNA synthesis to generate a sequencing ladder?
A. Addition of nucleotides requires a free OH group on the 3′ of the DNA strand
B. DNA has free 5′ phosphoryl group
C. DNA polymerase has proofreading capabilities
D. U is a base only found in RNA

How Sanger Sequencing Works: Understanding the Role of 3′-OH in DNA Synthesis

Sanger sequencing, also known as dideoxy sequencing, is a widely used method for determining the nucleotide sequence of DNA. It takes advantage of the fundamental property of DNA synthesis, which relies on the presence of a free 3′-OH group for the addition of nucleotides. This method was developed by Frederick Sanger in 1977 and remains one of the most accurate and widely used DNA sequencing techniques.

Correct Answer:

👉 The correct answer is A. Addition of nucleotides requires a free OH group on the 3′ of the DNA strand

What is Sanger Sequencing?

Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) during DNA replication. The technique involves using a DNA template, a primer, DNA polymerase, and a mixture of deoxynucleotides (dNTPs) and dideoxynucleotides (ddNTPs).

How Does Sanger Sequencing Work?

1. Template Preparation

• A single-stranded DNA template is prepared.
• A short primer complementary to the template is added to provide a starting point for DNA polymerase.

2. DNA Polymerization

• DNA polymerase extends the primer by adding nucleotides (dNTPs) complementary to the template strand.
• The reaction mixture contains:
  • dATP, dTTP, dGTP, and dCTP – normal deoxynucleotides.
  • ddATP, ddTTP, ddGTP, and ddCTP – chain-terminating dideoxynucleotides.

3. Chain Termination

• When a dideoxynucleotide (ddNTP) is incorporated, DNA synthesis stops.
• ddNTPs lack the 3′-OH group necessary for forming a phosphodiester bond with the next nucleotide.

4. Gel Electrophoresis

• The resulting DNA fragments of different lengths are separated using polyacrylamide gel electrophoresis or capillary electrophoresis.
• The sequence is read by detecting the fluorescently labeled ddNTPs.

5. Sequence Determination

• The order of nucleotides is determined by the position of the terminated fragments on the gel.
• The shortest fragment represents the 5′ end, and the longest fragment represents the 3′ end.

Role of 3′-OH in DNA Synthesis

The addition of nucleotides during DNA synthesis relies on the presence of a free 3′-OH group on the growing DNA strand. DNA polymerase catalyzes the formation of a phosphodiester bond between the 3′-OH of the last nucleotide and the 5′-phosphate of the incoming nucleotide.

When a ddNTP is incorporated:

• ddNTPs lack the 3′-OH group, which prevents further extension of the DNA strand.
• This causes termination of DNA synthesis at specific nucleotides.
• This property is exploited in Sanger sequencing to create a series of DNA fragments that differ by one nucleotide.

Why Is the 3′-OH Group Critical in Sanger Sequencing?

 DNA polymerase requires a free 3′-OH for adding new nucleotides.
 Without a 3′-OH, no further extension can occur.
 The selective use of ddNTPs allows for the controlled termination of DNA synthesis, which is necessary for generating a sequencing ladder.

Explanation of Other Options

B. DNA has free 5′ phosphoryl group

• The 5′-phosphate group is involved in forming phosphodiester bonds but does not influence the termination of DNA synthesis.
• The 3′-OH is critical for polymerization, not the 5′ end.

C. DNA polymerase has proofreading capabilities

• DNA polymerase can proofread and correct mismatched nucleotides, but proofreading does not affect chain termination in Sanger sequencing.
• The absence of 3′-OH, not polymerase proofreading, causes termination.

D. U is a base only found in RNA

• Uracil (U) is found in RNA, not in DNA.
• Sanger sequencing is based on DNA synthesis, not RNA synthesis.

Applications of Sanger Sequencing

Human Genome Project: First complete genome sequencing used Sanger sequencing.
Genetic Diagnosis: Used to identify mutations associated with genetic disorders.
Molecular Biology: Determining the sequence of cloned genes and plasmids.
Cancer Research: Identifying mutations and single nucleotide polymorphisms (SNPs) in cancer genomes.

Advantages of Sanger Sequencing

High accuracy (up to 99.9%).
 Suitable for sequencing fragments up to 1000 base pairs.
 Low error rate due to proofreading ability of DNA polymerase.
 Ideal for small-scale projects and targeted sequencing.

Limitations of Sanger Sequencing

 Low throughput compared to Next-Generation Sequencing (NGS).
 Inefficient for sequencing large genomes.
 Labor-intensive and time-consuming.

Sanger Sequencing vs. Next-Generation Sequencing (NGS)

Challenges in Sanger Sequencing

• Poor sequencing quality near the ends of the template.
• High background noise from mixed templates.
• Difficult to sequence high GC content regions due to secondary structures.

Conclusion

Sanger sequencing remains a gold standard for DNA sequencing due to its high accuracy and simplicity. The process relies on the critical requirement of a free 3′-OH group for nucleotide addition, which allows selective termination using ddNTPs. Understanding this mechanism helps in diagnosing genetic disorders, characterizing mutations, and advancing molecular biology research.