If you are a health professional, you are probably familiar about Sanger DNA sequencing method. Call it old school or even ‘outdated’, but Sanger sequencing remains an important test in modern biology. Even though new DNA sequencing methods is faster and cheaper, DNA sequencing with Sanger method remains widely used in research, diagnostics, and validation of results.
To accommodate those who have no idea on how scientists decodes DNA, Sanger sequencing at its core determines the exact order of nucleotides (A, T, C, and G) in a piece of DNA.
Developed in 1977 by Frederick Sanger, it became the foundation of the Human Genome Project, which completion has set the stage for today’s high-throughput sequencing technologies.
This article will explain how Sanger sequencing works, its remaining applications, and the institutions that still use it. We will also explain why it remains valuable in our today’s science despite proliferation of new generation DNA sequencing (NGS) methods.
See Hyperdrive Bio article about New Generation DNA Sequencing.
What Is Sanger Sequencing?
Sanger sequencing is often called the “chain termination method” as it uses specially-modified DNA building blocks that copies a DNA sample and stops DNA replication at specific points.
The process starts with the sample DNA strand being replicated several times, and then divided into four containers; a DNA base (either C, A,G, or T) is added into each of the containers. After a heating and cooling process, it creates DNA fragments of different lengths.
Still into their separate containers, the fragments are mixed with a dye and loaded into a very thin layer of gel (now into four separate lanes), which is then exposed to an electric charge (capillary electrophoresis). The smaller fragments moves faster through the gel than longer ones, so the fragments became segregated through size. The electric charge is applied until the fragments reach the end of the gel.
The gel is dried and placed into a paper support. During the earlier days, x-ray visible dyes are used to make the bases appear, and then from the shortest to the longest.
Even though next-generation sequencing (NGS) can process millions of DNA fragments at once, Sanger sequencing is still considered the gold standard for short DNA reads. It is precise, relatively inexpensive for small projects, and provides clear results that are easy to interpret.
How Does Sanger Sequencing Work?
Note that there is an original and newer methods of Sanger sequencing that are faster and less labor intensive, but all follow the essential steps.
Below is the very simplified version of how Sanger sequencing is performed in labs:
Step-by-Step Process
- DNA Extraction – the DNA helix is isolated from a sample—this could be blood, tissue, or microbial cultures.
- PCR Amplification – the target region of DNA is copied many times using polymerase chain reaction (PCR). This ensures there is enough material for sequencing.Reaction Setup. A sequencing reaction is prepared with:
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Normal nucleotides (A, T, C, G)
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Special chain-terminating nucleotides called dideoxynucleotides (ddNTPs), each tagged with a fluorescent dye
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A DNA primer and DNA polymerase enzyme
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- Chain Termination – The DNA primer attaches to the DNA strand, then the DNA polymerase builds a new strand, it occasionally incorporates a ddNTP instead of a normal nucleotide. When this happens, the strand stops growing.
- Fragment Separation – The result is a collection of DNA fragments of different lengths, each ending with a fluorescent base. These fragments are separated by size using capillary electrophoresis.
- Data Reading – A detector now reads the fluorescent signals and translates them into a series of peaks, producing the final DNA sequence.
This workflow explains in simple terms how Sanger sequencing works: it creates fragments that stop at every possible position in the DNA, then reconstructs the full order of bases.
Applications of Sanger Sequencing
Even with the rise of NGS, Sanger sequencing still plays an important role in science and medicine because of its precision. Sanger sequencing has an accuracy of 99.9%, while NGS can do so from 99%-99.9%.
It is well suited for applications where accuracy matters more than volume.
Newer methods of Sanger sequencing are rather automated and can be done my machines, thus making it widely available.
Common uses include:
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Clinical Diagnostics
Sanger sequencing is used to detect mutations in specific genes linked to hereditary diseases, cancer, or rare disorders. For example, it is still used to confirm variants found by next-generation sequencing. -
Pathogen Identification
In microbiology labs, researchers sequence small genetic regions to identify bacteria, viruses, or fungi. This is common in infectious disease surveillance. -
Validating NGS Results
Because NGS can produce errors, Sanger sequencing often acts as a gold standard to double-check important findings. -
Forensic Science
Crime labs use Sanger sequencing for certain DNA analyses because of its reliability and straightforward interpretation. -
Basic Research
Scientists use it for cloning verification, small gene sequencing projects, and teaching purposes in academic labs.
Institutions That Still Perform Sanger Sequencing
Despite the availability of faster sequencing platforms, many major institutions continue to operate Sanger sequencing facilities. Some examples include:
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National Institutes of Health (NIH) — Core facilities at NIH still provide Sanger sequencing services for biomedical researchers.
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Centers for Disease Control and Prevention (CDC) — Sanger sequencing is used for pathogen genotyping and outbreak investigations.
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Cancer research labs — Many cancer research centers validate tumor mutation findings with Sanger sequencing.
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University labs — Nearly every major research university (e.g., Harvard, Stanford, Johns Hopkins, University of Michigan) maintains Sanger sequencing services in their genomics cores.
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Clinical testing labs — Companies that provide genetic testing, such as Labcorp and Quest Diagnostics, still rely on Sanger sequencing for confirmatory analysis.
Its ongoing use shows that while high-throughput methods dominate large-scale projects, Sanger sequencing is still used in medicine and biology.
Why Sanger Sequencing Still Matters
Sanger sequencing may not be as fast or scalable as modern sequencing, but it offers advantages that keep it relevant:
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High accuracy for short reads (typically 500–1000 base pairs)
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Cost-effectiveness for small projects
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Clear data that does not require extensive bioinformatics
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Acceptance by regulatory agencies as a confirmatory method
For healthcare companies and research institutions, tools like Voyager, Hyperdrive Bio’s database of healthcare sales leads, help connect with the labs and organizations that continue to rely on this trusted sequencing method.
Frequently Asked Questions (FAQ)
Q1: What is Sanger sequencing used for today?
Sanger sequencing is mainly used for clinical validation, pathogen identification, mutation detection, and small-scale DNA sequencing projects.
Q2: How does Sanger sequencing work in simple terms?
It works by creating DNA fragments that stop growing at specific points. Each fragment ends with a labeled base, and by analyzing all fragments, the DNA sequence can be read.
Q3: Is Sanger sequencing still used in hospitals?
Yes. Many hospital diagnostic labs still use Sanger sequencing to confirm genetic mutations and provide accurate results for patient care.
Q4: How accurate is Sanger sequencing compared to NGS?
Sanger sequencing has an accuracy rate of about 99.9% for short sequences, making it highly reliable for confirmatory testing.
Q5: Which institutions offer Sanger sequencing services?
Institutions such as NIH, CDC, major universities, and large clinical labs like Labcorp continue to provide Sanger sequencing services.
Sources
Sanger Sequencing Steps & Method. Sigma Aldrich.
https://www.sigmaaldrich.com/PH/en/technical-documents/protocol/genomics/sequencing/sanger-sequencing?srsltid=AfmBOoqq2QtpFw8bZ7AqAw0gDb4CWcUtogRryTHo5ymwVHoNaXcsaZDU
Sanger sequencing. NHS
https://www.genomicseducation.hee.nhs.uk/genotes/knowledge-hub/sanger-sequencing/
Sanger Sequencing. DNA Learning Center.
https://dnalc.cshl.edu/resources/3d/29-sanger-sequencing.html
