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DNA sequencing determines the exact order of nucleotides—adenine (A), thymine (T), cytosine (C), and guanine (G)—in a DNA strand. Among various methods, the Sanger method, also known as the dideoxy chain termination method, remains one of the most influential. Developed in 1977 by Frederick Sanger, this technique laid the foundation for projects like the Human Genome Project and is still widely used today for small-scale sequencing and validation despite the rise of next-generation sequencing (NGS).

What is the Sanger Method?

The Sanger method uses modified nucleotides called dideoxynucleotides (ddNTPs) that lack a 3’-OH group. When incorporated into a growing DNA strand, they prevent further elongation, producing DNA fragments of varying lengths. By separating and analyzing these fragments, scientists can reconstruct the original DNA sequence.

In simple terms, while normal nucleotides extend the chain, ddNTPs act as “full stops,” helping to read the DNA sequence base by base.

Principle

The principle lies in controlled termination of DNA synthesis:

1. DNA polymerase extends the DNA strand with standard nucleotides (dNTPs).

2. Random incorporation of a ddNTP halts chain growth because no further nucleotide can attach.

3. Running four parallel reactions (A, T, C, G) generates fragments ending at different points for each base.

4. When separated and read, these fragments reveal the DNA sequence.

Steps of the Method

The Sanger dideoxy method follows clear laboratory steps:

1. Denaturation – Heat unzips double-stranded DNA into single strands.

2. Template Preparation – Multiple copies of the target DNA are generated for accuracy.

3. Primer Attachment – A short primer binds to the DNA, giving DNA polymerase a starting point.

4. Four Reaction Tubes – Each tube contains DNA polymerase, dNTPs, and one fluorescently labeled ddNTP (A, T, C, or G).

5. Chain Synthesis and Termination – DNA synthesis proceeds until a ddNTP is incorporated, producing fragments of varying lengths.

6. Denaturation of Fragments – Chains are separated into single strands.

7. Electrophoresis – Fragments are separated by size using gel or capillary electrophoresis. Fluorescent tags identify the terminal nucleotide, allowing the sequence to be read.

This workflow can be visualized in a Sanger sequencing flow chart: template → synthesis → chain termination → fragment separation → sequence reading.

Applications

The Sanger method remains relevant for:

• Clinical diagnostics (e.g., detecting BRCA1/2 mutations linked to breast cancer).

• Forensic science (DNA identification).

• Molecular biology (validating cloned DNA).

• Microbiology (identifying bacterial strains).

• Evolutionary biology (comparing DNA among organisms).

Advantages

• High accuracy with error rates as low as 0.001%.

• Long read length, up to 900 base pairs in one run.

• Cost-effective for small projects.

• Reliable for confirming NGS results.

Disadvantages

• Low throughput, unsuitable for large genomes.

• Expensive and time-consuming for large-scale sequencing.

• Labor-intensive compared to automated NGS systems.

Comparisons

• Sanger vs. Maxam-Gilbert: Sanger is safer (no toxic chemicals) and easier to use.

• Sanger vs. NGS: NGS is faster and cheaper for large genomes, but Sanger remains unmatched for accuracy in small fragments and validation.

Conclusion

The Sanger method of DNA sequencing continues to be a cornerstone of genetics. By using ddNTPs to terminate chain growth, it enables accurate, reliable sequencing of DNA fragments. While NGS dominates large-scale projects, Sanger remains indispensable in diagnostics, research, and validation due to its precision and reliability.

As Frederick Sanger’s work shows, progress in science relies on innovative techniques—and his method remains one of the most enduring in molecular biology.