What the Most Common Applications of Sanger Sequencing?

Sanger sequencing, also known as the chain-termination method, is a DNA sequencing technique that relies on the incorporation of chain-terminating dideoxynucleotides (ddNTPs) during in vitro DNA replication. This process is guided by the activity of DNA polymerase, which synthesizes a complementary strand of DNA. Developed by Frederick Sanger and his team in 1977, this method revolutionized genetics and remains a cornerstone in DNA analysis.

How Sanger Sequencing Works

In order to determine which nucleotides are incorporated in the nucleotide chain, four ddNTPs often labeled with different fluorescent dye are utilized to terminate the reaction process of synthesis. Compared to dNTP, ddNTP has an oxygen atom, which is removed from a ribonucleotide. Following the synthesis process, the products’ reaction are usually loaded into four different lanes of a gel, based on different chain-terminating nucleotides and get subjected to the process of gel electrophoresis.

The core principle of Sanger sequencing involves terminating DNA synthesis at specific nucleotides. The process works as follows:

1. DNA Template Preparation: The target DNA to be sequenced is isolated and prepared.

2. Primer Binding: A short primer binds to the single-stranded DNA template, providing a starting point for DNA polymerase.

3. Chain Elongation with ddNTPs:

   • The reaction mixture contains the four standard deoxynucleotides (dATP, dTTP, dCTP, dGTP) and small amounts of fluorescently labeled ddNTPs.

   • Unlike dNTPs, ddNTPs lack a 3’-OH group, preventing further elongation when incorporated.

   • Each ddNTP is tagged with a different colored dye, enabling identification of the terminating base.

4. Separation by Size: The resulting mixture contains DNA fragments of varying lengths, each ending with a fluorescently labeled ddNTP.

5. Detection: Fragments are separated using capillary or gel electrophoresis, and the fluorescent signals are read by a laser detector to determine the DNA sequence.

Modern Sanger sequencing typically uses automated capillary electrophoresis, replacing traditional polyacrylamide gels, which allows high-throughput sequencing with greater accuracy and speed.

Ingredients Required for Sanger Sequencing

Sanger sequencing requires components similar to DNA replication or PCR:

• DNA template: The target DNA region to be sequenced.

• Primers: Short single-stranded DNA that binds to the template and initiates replication.

• DNA polymerase: The enzyme responsible for adding nucleotides.

• Deoxynucleotides (dNTPs): dATP, dTTP, dCTP, dGTP for normal strand elongation.

• Dideoxynucleotides (ddNTPs): Chain-terminating nucleotides labeled with fluorescent dyes (ddATP, ddTTP, ddCTP, ddGTP).

• Buffer and cofactors: Provide the optimal environment for DNA polymerase activity.

The technique involves making numerous copies of target DNA regions. Ingredients used in Sanger sequencing are the same as those required for replication of DNA in organisms or for PCR (polymerase chain reaction) that copies DNA in vitro. Some of these ingredients are not limited to the following:

• DNA templates to be sequenced
• Four nucleotides of DNA, including dGTP, dCTP, dTTP, and dATP
• Primers, which are short pieces of a single-strand DNA binds to DNA templates as well as serves as the starter for polymerase.
• Chain-terminating or dideoxy versions of nucleotides (ddGTP, ddCTP, ddTTP, and ddATP), all labeled with different dye colors.

Applications

Sanger sequencing remains highly valued due to its accuracy, reliability, and long-read capabilities. Its applications span research, clinical diagnostics, and forensic science.

The technique remains an accurate method of sequencing. It is widely used, especially in the clinical labs for various applications, including testing for specific familial sequence variants and diagnostic sequencing of genes. In addition, it can also be used to fill NGS data’s gaps and confirm variants that the NGS has identified. Other applications include the following:

1. Pharmacogenomics

Pharmacogenomics studies how genetic variations affect individual responses to drugs. Sanger sequencing identifies genetic variants influencing drug metabolism, efficacy, and risk of adverse reactions, enabling:

• Personalized treatment plans

• Reduced drug-related complications

• Optimized dosing strategies

2. Mutation Detection and Disease Diagnosis

Sanger sequencing is widely used to detect mutations responsible for genetic disorders, including:

• Sickle cell anemia

• Cystic fibrosis

• Hereditary cancers (BRCA1/2 mutations)

• Muscular dystrophies

Clinicians can pinpoint single nucleotide changes, insertions, deletions, and other genetic abnormalities with high accuracy, making it a gold standard for clinical diagnostics.

3. Genetic Diversity and Evolutionary Studies

Researchers use Sanger sequencing to study population genetics and evolutionary relationships. Applications include:

• Analyzing mitochondrial DNA for maternal lineage studies

• Sequencing chloroplast DNA in plants

• Investigating migration patterns and phylogenetic relationships

This approach helps scientists understand biodiversity, species evolution, and ecological interactions.

4. Forensic Analysis

In forensic science, Sanger sequencing plays a crucial role in DNA fingerprinting and identification:

• Sequencing short tandem repeats (STRs) or hypervariable regions of DNA

• Matching DNA samples from crime scenes to suspects

• Establishing family relationships in legal investigations

Its accuracy and reproducibility make it a trusted method in criminal investigations.

5. Filling Gaps in Next-Generation Sequencing (NGS)

Although NGS allows high-throughput sequencing, it sometimes produces ambiguous or incomplete regions. Sanger sequencing is often used to:

• Confirm variants identified by NGS

• Sequence difficult regions or repetitive sequences

• Validate clinical or research findings

This complementary role ensures data reliability and reduces errors in genomic studies.

6. Microbial and Viral Research

Sanger sequencing is also used in microbiology and virology to study:

• Bacterial strains and antibiotic resistance genes

• Viral genomes for outbreak tracking

• Genetic changes in pathogens over time

Such applications are critical in epidemiology and infectious disease research.

Advantages of Sanger Sequencing

• High Accuracy: >99.99% accuracy for single reads

• Long Read Lengths: Typically 500–1000 base pairs per read

• Established Protocols: Widely standardized and reproducible

• Versatile Applications: From clinical diagnostics to evolutionary research

Limitations of Sanger Sequencing

• Lower Throughput: Not ideal for whole-genome sequencing compared to NGS

• Cost per Base: More expensive for large-scale projects

• Time-Consuming: Manual steps can be labor-intensive without automation

Despite these limitations, it remains preferred for validation, clinical sequencing, and targeted studies.

Conclusion

Sanger sequencing remains a gold standard in DNA sequencing technology. Its principle of chain termination combined with the high specificity of fluorescently labeled ddNTPs allows for accurate and reliable sequencing. This method continues to support a wide range of applications:

• Detecting genetic mutations and variants

• Personalized medicine and pharmacogenomics

• Forensic investigations and criminal identification

• Genetic diversity studies in populations

• Complementing next-generation sequencing for validation

Even in the era of high-throughput NGS technologies, Sanger sequencing’s accuracy, long-read capacity, and versatility ensure it remains an indispensable tool in research, medicine, and forensic science.