Understanding DNA Sequencing Using Sanger’s Method – Explained with an Example

DNA sequencing is a critical technique in molecular biology that allows researchers to determine the exact order of nucleotide bases in a DNA strand. One of the most widely used methods for DNA sequencing is Sanger’s method (also known as the dideoxy chain termination method). This method relies on the selective incorporation of chain-terminating nucleotides during DNA replication. In this article, we will explain how to interpret Sanger sequencing results and why the correct DNA sequence in the given example is 5′ GAATC 3′.

Correct Answer:

The correct DNA sequence is 5′ GAATC 3′ (Option B).

What is Sanger Sequencing?

Sanger sequencing, developed by Frederick Sanger in 1977, is based on the use of:

• A single-stranded DNA template
• DNA polymerase enzyme
• Primers
• Normal deoxynucleotides (dNTPs)
• Chain-terminating dideoxynucleotides (ddNTPs)

How It Works:

1. Primer Binding:

   • A primer binds to the single-stranded DNA template.

2. DNA Synthesis:

   • DNA polymerase adds nucleotides (dNTPs) to synthesize the complementary strand.

3. Chain Termination:

   • When a dideoxynucleotide (ddNTP) is incorporated, the chain is terminated.
   • ddNTPs lack a 3′ hydroxyl group, preventing further extension.

4. Gel Electrophoresis:

   • The resulting DNA fragments are separated using gel electrophoresis.
   • Shorter fragments migrate faster, and longer fragments migrate slower.

5. Autoradiography:

   • The gel is exposed to X-ray film to produce an autoradiogram showing the bands.
   • Each band represents a terminated DNA fragment.

How to Interpret Sanger Sequencing Results

To determine the DNA sequence from a sequencing gel, follow these steps:

1. Read from Bottom to Top:

• The shortest fragments are at the bottom of the gel because they migrate faster.
• The sequence is read in the 5′ to 3′ direction from bottom to top.

2. Identify the Nucleotide Order:

• The gel contains four wells labeled G, A, T, and C corresponding to the four nucleotides (guanine, adenine, thymine, and cytosine).
• Each band in a specific well corresponds to the nucleotide at that position in the sequence.

3. Complementary Sequence:

• The sequence obtained from the gel is the complementary strand.
• To find the original template sequence, generate the complementary strand of the result.

Example and Explanation

Given Autoradiogram Well Order:

• The wells are arranged in the order GATE (Guanine, Adenine, Thymine, and Cytosine).
• The sequencing result from bottom to top reads: G – A – A – T – C

Resulting Complementary Strand:

• The sequence directly from the gel (bottom to top) = GAATC
• The complementary sequence (template strand) = 5′ GAATC 3′

Why Option B is Correct

1. Gel Reading Direction:

   • Sequencing gels are always read from bottom to top (5′ to 3′ direction).
   • The shortest fragments, representing the 5′ end, are at the bottom.

2. Complementary Sequence:

   • The sequence read from the gel is complementary to the template strand.
   • The correct final sequence is 5′ GAATC 3′.

3. Other Options Are Incorrect:
   | Option | Sequence | Reason |
   |——–|———-|——–|
   | A | 5′ CTAG 3′ | Incorrect order of bases |
   | C | 5′ CTAAG 3′ | Incorrect nucleotide combination |
   | D | 5′ AAITG 3′ | Incorrect base presence (I is not a valid base) |

Steps in Sanger Sequencing – Detailed Overview

1. Reaction Setup

• DNA template mixed with primers, DNA polymerase, and dNTPs.
• A small amount of ddNTPs is added to terminate extension at specific points.

2. DNA Synthesis and Termination

• DNA polymerase synthesizes the strand.
• When a ddNTP is incorporated, the strand stops elongating.
• Fragments of varying lengths are produced.

3. Gel Electrophoresis

• The mixture is loaded onto a polyacrylamide gel.
• Fragments separate based on size under an electric field.

4. Autoradiography

• Radioactively or fluorescently labeled fragments are detected.
• A sequence pattern appears as distinct bands.

Advantages of Sanger Sequencing

 High accuracy for short DNA sequences.
 Useful for confirming mutations or variations.
 Ideal for sequencing small plasmids and PCR products.
Directly interpretable output.

Limitations of Sanger Sequencing

 Inefficient for long sequences (>1000 bp).
 Time-consuming and labor-intensive.
 Lower throughput than next-generation sequencing (NGS).

Applications of Sanger Sequencing

1. Mutation Detection

• Sanger sequencing is widely used to identify point mutations.
• Useful in cancer research and genetic disorders.

2. Genotyping

• Used for precise identification of genetic variations.

3. Plasmid Verification

• Confirming the presence of an insert in recombinant plasmids.

4. Evolutionary Studies

• Comparing sequences from different species to study genetic relationships.

Importance of Correct Sequence Interpretation

Reading the sequencing gel correctly is crucial for accurate genetic analysis. A mistake in reading can lead to incorrect conclusions about mutations, gene functions, or protein coding sequences. The ability to correctly interpret sequencing gels enhances the accuracy of genetic research and clinical diagnosis.

Conclusion

Sanger sequencing remains a cornerstone of molecular biology, despite the rise of next-generation techniques. Correct interpretation of the autoradiogram is essential for determining the accurate DNA sequence. In this case, the sequence obtained from the gel, GAATC, represents the complementary strand, confirming that the correct DNA sequence is 5′ GAATC 3′. Mastering Sanger sequencing ensures accurate genetic analysis, essential for research and clinical applications.