Sanger Sequencing Method : Principle, Steps, and Applications

Sanger sequencing, also known as the chain termination method, is a widely used DNA sequencing technique developed by Frederick Sanger in 1977. Despite the rise of next-generation sequencing (NGS), Sanger sequencing remains a gold standard for sequencing small DNA fragments due to its high accuracy and reliability. This method plays a crucial role in clinical diagnostics, genetic research, and forensic science.

In this article, we’ll explore how Sanger sequencing works, its step-by-step process, applications, advantages, and a comparison with NGS, helping you understand why it’s still relevant in modern molecular biology. 🚀

2. What is Sanger Sequencing?

Sanger sequencing, also known as the chain termination method, is a DNA sequencing technique developed by Frederick Sanger in 1977. It was the first widely used method for determining the nucleotide sequence of DNA and played a crucial role in early genome sequencing projects, including the Human Genome Project.

Principle of Sanger Sequencing

The method is based on selective incorporation of chain-terminating nucleotides called dideoxynucleotides (ddNTPs) during DNA replication. These special nucleotides lack a hydroxyl (-OH) group at the 3’ carbon of the sugar molecule, preventing the addition of further nucleotides and causing termination of DNA synthesis at specific points. By analyzing multiple terminated DNA fragments, researchers can determine the original DNA sequence.

Key Features of Sanger Sequencing

• High Accuracy – Reliable for sequencing small DNA fragments (~800-1000 bp).
• Low Throughput – Suitable for targeted sequencing but not ideal for whole genomes.
• Gold Standard for Mutation Detection – Used in clinical diagnostics and research.
• Requires a DNA Template – A single-stranded DNA fragment is needed as the starting material.

Although next-generation sequencing (NGS) has largely replaced Sanger sequencing for large-scale projects, it remains an essential tool for small-scale sequencing tasks, such as mutation analysis, microbial identification, and validation of NGS results.

3. Sanger Sequencing Steps: How Does It Work?

Sanger sequencing follows a step-by-step process that involves DNA amplification, selective chain termination, fragment separation, and sequence analysis. Below is a detailed breakdown of each step:

1: DNA Template Preparation

• The DNA sample to be sequenced is extracted and purified.
• It must be single-stranded DNA (ssDNA), which serves as the template for replication.

2: PCR Amplification (Optional Step)

• In some cases, polymerase chain reaction (PCR) is used to amplify the target DNA before sequencing.
• This ensures there is enough DNA for sequencing.

3: Chain Termination via Dideoxynucleotides (ddNTPs)

The core of Sanger sequencing relies on a modified DNA replication process:

1. A reaction mixture is prepared containing:
   • Single-stranded DNA template (the DNA to be sequenced).
   • DNA polymerase (an enzyme that synthesizes new DNA strands).
   • Deoxynucleotides (dNTPs) (A, T, C, G) for normal DNA synthesis.
   • Dideoxynucleotides (ddNTPs) (A*, T*, C*, G*) labeled with fluorescent dyes.
2. DNA polymerase extends the strand until a ddNTP is randomly incorporated, causing chain termination.
3. This process generates DNA fragments of varying lengths, each ending at a different nucleotide.

4: Fragment Separation via Gel or Capillary Electrophoresis

• The DNA fragments are separated based on size using:
  • Gel electrophoresis (older method).
  • Capillary electrophoresis (modern automated method).
• Shorter fragments migrate faster than longer ones, allowing their order to be determined.

5: Detection and Data Analysis

• The fluorescently labeled ddNTPs are detected using a laser scanner.
• The signal is converted into an electropherogram, showing peaks that correspond to specific DNA bases (A, T, C, G).
• The final DNA sequence is read based on the order of these peaks.

Summary of Sanger Sequencing Workflow

• DNA extraction & preparation
• Optional PCR amplification
• Chain termination reaction with ddNTPs
• Separation of DNA fragments via electrophoresis
• Fluorescent detection and sequence reading

4. Applications of Sanger Sequencing

Sanger sequencing is widely used in genetic research, clinical diagnostics, forensics, and microbiology due to its high accuracy and ability to sequence small DNA fragments reliably. Below are some of its key applications:

1. Clinical Diagnostics and Genetic Testing

• Detecting genetic mutations linked to inherited diseases (e.g., cystic fibrosis, BRCA1/BRCA2 mutations in breast cancer).
• Confirming single nucleotide polymorphisms (SNPs) and small insertions/deletions (indels).
• Validating next-generation sequencing (NGS) results in clinical settings.

✅ Example: Used in diagnosing hereditary cancer syndromes and genetic disorders.

2. Cancer Research and Somatic Mutation Analysis

• Identifying mutations in oncogenes and tumor suppressor genes.
• Studying the genetic basis of cancer to develop targeted therapies.

✅ Example: Detecting TP53, KRAS, and FGFR3 mutations in various cancers.

3. Microbial and Viral Genome Sequencing

• Sequencing bacterial, viral, and fungal genomes for disease surveillance.
• Identifying drug-resistant pathogens in infections.
• Studying viral mutations (e.g., tracking SARS-CoV-2 variants).

✅ Example: Used for sequencing E. coli, Mycobacterium tuberculosis, and HIV.

4. Forensic Science and DNA Fingerprinting

• Analyzing DNA from crime scenes, paternity tests, and missing person cases.
• Identifying individuals based on unique DNA sequences.

✅ Example: Used in criminal investigations and paternity disputes.

5. Molecular Biology and Evolutionary Studies

• Sequencing genes and regulatory regions to understand gene function.
• Studying evolutionary relationships among species through phylogenetic analysis.

✅ Example: Used in comparative genomics to study human and primate evolution.

6. Biotechnology and Agricultural Genomics

• Genotyping plants and animals for genetic improvements.
• Developing genetically modified organisms (GMOs) with enhanced traits.

✅ Example: Used in crop improvement programs to study plant resistance genes.

Why Sanger Sequencing is Still Used in These Applications

• High accuracy for small DNA regions.
• Reliable for clinical and forensic applications.
• Gold standard for validating mutations and sequencing results.

5. Advantages and Disadvantages of Sanger Sequencing

Sanger sequencing is known for its high accuracy and reliability, making it a preferred method for sequencing small DNA fragments. However, it has some limitations compared to next-generation sequencing (NGS). The table below summarizes its key advantages and disadvantages.

6. Sanger Sequencing vs. Next-Generation Sequencing (NGS)

Both Sanger sequencing and next-generation sequencing (NGS) are widely used for DNA sequencing, but they differ significantly in terms of technology, throughput, cost, and applications. The table below provides a side-by-side comparison of these two sequencing methods.

Comparison Table: Sanger Sequencing vs. NGS

Which One Should You Use?

✅ Use Sanger Sequencing If:

• You need high accuracy for small DNA fragments.
• You are working on single-gene sequencing, mutation validation, or clinical diagnostics.
• You want a cost-effective solution for targeted sequencing.

🚀 Use NGS If:

• You need to sequence whole genomes, transcriptomes, or large genetic datasets.
• You require high-throughput sequencing with a large number of samples.
• You need to study complex genetic variations, such as structural variants or epigenetic modifications.

7. Cost and Availability of Sanger Sequencing

Sanger sequencing, although considered more traditional compared to next-generation sequencing (NGS), remains a reliable and widely used method for small-scale sequencing projects. Its cost and availability are significant factors when choosing this technique for specific applications. Below, we discuss the cost structure of Sanger sequencing and its availability in research and clinical settings.

Cost of Sanger Sequencing

The cost of Sanger sequencing depends on several factors, including the length of the DNA to be sequenced, the number of samples, and the sequencing service provider. In general, Sanger sequencing is more affordable for sequencing small DNA fragments, but it becomes expensive when used for large-scale projects.

Typical Cost Breakdown

• Per sample: The cost typically ranges between $5 to $10 per sequencing reaction for small fragments (~800–1000 bp).
• For larger DNA regions: If the DNA fragment is longer than 1000 base pairs, the cost may increase due to the additional sequencing reactions required.
• Discounts for bulk sequencing: Many sequencing service providers offer discounted rates for high-volume sequencing orders (e.g., 100+ samples).
• Additional Costs: In some cases, there may be extra costs for primer design, sample preparation, and data analysis (particularly when using automated data interpretation tools).

Cost Comparison with NGS

While Sanger sequencing is more affordable for small-scale sequencing, NGS becomes more cost-effective for larger projects. For example:

• NGS offers higher throughput at a lower cost per base when sequencing large genomes, but the overall initial setup and bioinformatics analysis can increase costs for NGS-based workflows.

Availability of Sanger Sequencing

Sanger sequencing is widely available in both academic research and clinical diagnostic labs. Many sequencing companies and core facilities provide sequencing services, making it accessible for researchers and clinicians worldwide.

Where to Access Sanger Sequencing

1. Academic and Research Institutions: Universities and research centers often have core facilities where Sanger sequencing services are available for internal researchers and external customers.
2. Commercial Sequencing Providers: Many companies, such as Macrogen, Eurofins Genomics, and Genewiz, offer sequencing as a service for both academic and commercial clients. These companies also provide automated data analysis tools to help interpret sequencing results.
3. Clinical Laboratories: Clinical labs use Sanger sequencing for diagnostic purposes, including mutation detection for genetic diseases and cancer, and genetic testing for inherited disorders.

Global Availability

chain termination method is available in most parts of the world, making it an accessible choice for researchers and clinicians in both developed and developing countries.

Factors Affecting Cost and Availability

• Sample preparation and shipping: Costs may increase for sample extraction, purification, or shipping to the sequencing facility.
• Sequencing service providers: Some regions may have limited access to sequencing facilities, leading to higher costs for sample shipping.
• Specialization: Some labs specialize in particular applications, like microbial genome sequencing or clinical diagnostics, which may affect pricing.

Conclusion

Sanger sequencing remains a gold standard for accurate and reliable DNA sequencing, particularly for small-scale projects like mutation detection, genetic testing, and targeted sequencing. While next-generation sequencing (NGS) has surpassed Sanger sequencing for large-scale projects due to its higher throughput and lower cost per base, Sanger sequencing continues to be a valuable tool in both clinical diagnostics and research. With its high accuracy and widely available services, it remains an essential method for precise DNA analysis in many scientific fields.

Frequently Asked Questions (FAQ)

1. What are the steps of Sanger sequencing?
chain termination method follows five key steps:

• DNA template preparation: Extract and purify the DNA to be sequenced.
• PCR amplification (optional): Amplify the target DNA region if necessary.
• Chain termination: Add dideoxynucleotides (ddNTPs) to stop DNA synthesis at specific points.
• Fragment separation: Separate the DNA fragments based on size using gel or capillary electrophoresis.
• Detection and analysis: Use fluorescent signals to detect the terminated fragments and determine the DNA sequence.

2. Why is Sanger sequencing better for small DNA fragments?
chain termination method is particularly well-suited for sequencing small DNA fragments (up to 1000 base pairs) due to its high accuracy and longer read lengths compared to other methods. The method ensures that each base is correctly identified in short, simple sequences, making it ideal for single-gene sequencing, mutation detection, and validation of results from other sequencing methods.

3. How does Sanger sequencing compare to next-generation sequencing?
While both Sanger sequencing and next-generation sequencing (NGS) are DNA sequencing methods, they differ in several aspects:

• Throughput: NGS can sequence millions of fragments simultaneously, making it ideal for large-scale projects, whereas Sanger sequencing is best for small-scale sequencing.
• Cost: Sanger sequencing is more cost-effective for small DNA regions, but NGS becomes cheaper for large-scale genome sequencing.
• Accuracy: Sanger sequencing provides high accuracy for short fragments, while NGS requires more advanced bioinformatics tools to ensure accurate results.
• Applications: Sanger sequencing is preferred for targeted sequencing, clinical diagnostics, and mutation validation, while NGS is better suited for whole-genome sequencing, metagenomics, and transcriptomics.