Sanger Sequencing: A Staple of Genetic Analysis

May 16, 2024

Sanger sequencing has served as the fundamental workhorse of sequencing for over 40 years, providing the backbone for monumental scientific achievements like the Human Genome Project. With its ability to read through DNA base-by-base, Sanger sequencing has become an essential tool for a wide range of genetic analysis applications. 

In this article, we will explore the Sanger sequencing methodology from its humble beginnings to its current standing as an indispensable staple technique. You will gain an appreciation for how this Nobel Prize-winning approach revolutionised genomics and molecular biology through its elegantly simple enzymatic process.

An Overview of Sanger Sequencing

Sanger sequencing, also known as the chain termination method, uses modified nucleotides to synthesise DNA strands. It relies on the incorporation of dideoxynucleotides (ddNTPs) during in vitro DNA replication to terminate DNA strand elongation.

The Process

The technique requires the use of DNA polymerase, a primer, deoxynucleotides (dNTPs), and ddNTPs – each labelled with a fluorescent dye. DNA polymerase initiates DNA synthesis from the primer, incorporating normal dNTPs. However, when a ddNTP is incorporated, it terminates DNA elongation due to the lack of a 3′ hydroxyl group. This results in DNA fragments of varying lengths.

Detection and Analysis

The DNA fragments are then separated by size using gel electrophoresis. A laser is used to excite the fluorescent dyes, allowing the sequence of the fragments to be read. The output is a series of peaks representing each nucleotide base. By compiling the sequences of many short fragments, the sequence of the entire DNA segment can be determined.

Applications and Limitations

Sanger sequencing is a foundational technique that enabled advancements like the Human Genome Project. However, its limitations in read length, scalability and cost spurred the development of next-generation sequencing methods. Although more advanced techniques now dominate, Sanger sequencing still has applications for targeted, smaller-scale sequencing and validating next-generation sequencing results.

Overall, Sanger sequencing has been crucial for the development of genomics. Despite its limitations, it remains an important tool for specific sequencing needs, and continues to provide a standard of accuracy for newer methods.

The Components of Sanger Sequencing

Sanger sequencing requires several crucial components to function. Firstly, it needs a DNA template, which is the DNA sample containing the target sequence that will be sequenced. The DNA template can be, plasmid DNA or PCR products.

DNA Polymerase

DNA polymerase is an enzyme that synthesises complementary DNA strands. In Sanger sequencing, DNA polymerase sequentially adds fluorescently labelled dideoxynucleotides (ddNTPs) to the growing DNA strand during DNA replication.


Primers are short, single-stranded DNA fragments that bind to the template DNA at a specific location. They provide a free 3’-OH group for DNA polymerase to begin DNA synthesis. Forward and reverse primers are used to sequence both strands of the DNA template.

dNTPs and ddNTPs

Deoxynucleoside triphosphates (dNTPs) are the building blocks for DNA synthesis. In Sanger sequencing, fluorescently labelled ddNTPs are used and compete with dNTPs to be incorporated into the growing DNA strand. Once a ddNTP is added, DNA synthesis terminates.

Sequencing Reaction

The sequencing reaction contains the DNA template, DNA polymerase, primers, dNTPs and fluorescently labelled ddNTPs. When the reaction proceeds, DNA polymerase synthesises a new DNA strand. During synthesis, ddNTPs are randomly incorporated, resulting in strands of varying lengths.

Gel Electrophoresis

Gel electrophoresis separates the DNA fragments by size. The fluorescently labelled DNA fragments are loaded into the gel, which is then subjected to an electric current. Shorter fragments move faster than longer ones, allowing the sequence to be read from the gel.

By understanding the components involved, one can gain better insight into how Sanger sequencing works to decipher the sequence of nucleotides in a DNA strand with high accuracy and resolution.

The Advantages of Using Sanger Sequencing

Sanger sequencing, also known as the dideoxy chain termination method, has been the workhorse of DNA sequencing for decades. This technique provides several benefits that have solidified its status as a staple of genetic analysis.


Compared to newer next-generation sequencing methods, Sanger sequencing is relatively inexpensive. It does not require the use of specialised equipment or reagents, making it accessible to most molecular biology laboratories. For smaller experiments, Sanger sequencing can achieve the desired results at a lower cost.

High Accuracy

Sanger sequencing is able to generate long, highly accurate reads of up to 1,000 base pairs. The dideoxy chain termination method has an extremely low error rate, allowing scientists to determine the exact sequence of bases with a high degree of confidence. This high level of accuracy and precision is essential for applications where single nucleotide changes can have major biological implications.

Targeted Analysis

Unlike next-generation sequencing techniques that provide a broad overview of all DNA or RNA in a sample, Sanger sequencing allows for the targeted analysis of specific genes or regions of interest. Researchers are able to selectively sequence predetermined areas of the genome using custom designed primers. This makes Sanger sequencing ideal for confirming the presence of known mutations or validating the results of genome-wide sequencing studies.

Wide Capabilities

Sanger sequencing can be used to determine the order of nucleotides in both DNA and RNA, and works for a variety of sample types including plasmids, PCR products, bacterial colonies, and genomic DNA. Its versatility and ability to provide detailed sequence information for any region of interest has secured Sanger sequencing an enduring and prominent role in molecular research. Although new technologies have increased throughput, Sanger sequencing remains an accessible, affordable, and reliable method for genetic analysis.

The Limitations of Sanger Sequencing

While Sanger sequencing has been instrumental in enabling significant discoveries in molecular biology, it does have some inherent limitations. 

Read Length

The maximum read length of 700 to 1000 base pairs means that Sanger sequencing cannot sequence longer DNA fragments efficiently. This makes de novo genome assembly challenging and requires the fragmentation of longer DNA strands.


Sanger sequencing has a limited ability to detect genetic variants and does not provide single-nucleotide resolution. It cannot easily detect insertions, deletions or structural variants in the genome. More advanced sequencing techniques are required for precision medicine applications that rely on the detection of rare or complex variants.


Sanger sequencing does not scale well to the high-throughput demands of modern genomics. It cannot produce the large volumes of data required for population-scale studies or personalised medicine. Next-generation sequencing methods are vastly more scalable, producing billions of reads per run.

While Sanger sequencing remains an important technique, especially for small-scale sequencing and validation, its limitations mean it cannot satisfy the demands of large-scale genomics research and biotechnology. More advanced high-throughput sequencing methods have now surpassed Sanger sequencing to become the dominant approach in molecular biology. However, Sanger sequencing continues to play a role as a benchmarking and validation tool due to its high accuracy and fidelity.

Applications of Sanger Sequencing

Sanger sequencing has enabled groundbreaking discoveries in genetics and molecular biology. Its applications span research, diagnostics, forensics, and more.

Genetic Research

Sanger sequencing has been instrumental in sequencing the first human genome and the genomes of other organisms. By revealing the precise order of nucleotides in DNA, it has enabled the discovery of genes and the mutations that cause diseases. Sanger sequencing continues to be used to study genetic variation and evolution.


Sanger sequencing is used to detect mutations that cause genetic diseases. It can identify single nucleotide polymorphisms (SNPs) and small insertions or deletions in patient samples to diagnose conditions like cystic fibrosis, sickle cell disease, and familial hypercholesterolemia. Sanger sequencing is also used in newborn screening programmes to detect treatable conditions.


In forensics, Sanger sequencing is used to analyse DNA from crime scenes and match it to suspects. By comparing sequences at multiple loci in the genome, investigators can identify individuals with a high degree of certainty. Sanger sequencing has been instrumental in exonerating wrongly convicted individuals and solving cold cases.

Other Applications

Sanger sequencing also has applications in virology, microbiology, and other fields. It is used to detect viral pathogens, study microbial evolution and epidemiology, authenticate cell lines, and more. As sequencing has become more affordable and higher throughput, Sanger sequencing remains useful for smaller-scale sequencing projects, validation, and niche applications that require a high degree of accuracy.

Recent Advances in Sanger Sequencing Technologies

Increased Automation and High Throughput

Recent improvements in Sanger sequencing have focused on increased automation and higher throughput.

Advanced robotic platforms and liquid handling systems now automate the sequencing workflow, including plasmid purification, reaction setup, and product analysis. These automated systems have enabled the parallel processing of up to 96 samples at a time, allowing Sanger sequencing to achieve throughputs of over 100,000 reads per day.

Faster Polymerases and Improved Cycle

Sequencing Chemistry

Faster thermostable DNA polymerases with higher fidelity and processivity have improved the speed and accuracy of Sanger sequencing. Newer polymerases can now extend up to 1,000 nucleotides per second with an error rate of just 1 in 100,000 bases. In addition, improvements in the chemistry of the sequencing cycles have optimised the efficiency of nucleotide incorporation and cleanup. These advancements have reduced the time required for a single sequencing reaction from 8-12 hours to just 2 hours.

Capillary Electrophoresis and High Resolution


The transition from slab gel to capillary electrophoresis in the 1990s significantly improved the resolution and accuracy of sequence readouts. Capillary electrophoresis uses narrow-bore capillaries instead of slab gels to separate DNA fragments by size. This allows for faster, higher resolution separations and detection. When coupled with laser-induced fluorescence detection, capillary electrophoresis can resolve DNA fragments that differ in length by only a single nucleotide. These technologies remain the gold standard for Sanger sequencing and enable reads up to 1,000 nucleotides in length with 99.999% base calling accuracy.

While next-generation sequencing is paving the way for higher throughput applications, Sanger sequencing remains an important tool for many laboratories. Ongoing improvements in automation, polymerases, and detection chemistries continue to enhance its capabilities, ensuring it will remain a staple of genetic analysis for years to come.

Alternatives to Sanger Sequencing

While Sanger sequencing has been the workhorse of DNA sequencing for decades, newer high-throughput sequencing technologies have emerged that offer advantages over the traditional Sanger method. These next-generation sequencing (NGS) technologies, also known as massively parallel sequencing, allow for faster and cheaper sequencing of large amounts of DNA.

Next-Generation Sequencing (NGS)

Next-Generation Sequencing (NGS) represents a quantum leap in DNA sequencing technology, revolutionising the field with its capacity for massively parallel sequencing. By concurrently sequencing millions of DNA fragments, NGS platforms such as Illumina enable rapid and cost-effective whole-genome sequencing, exome sequencing, transcriptome profiling, and metagenomic analysis. With its high throughput, NGS has become the method of choice for large-scale genomic studies, population genetics, cancer genomics, and personalized medicine, driving advancements in biological and medical research.

Third-Generation Sequencing

Third-Generation Sequencing platforms, exemplified by PacBio and Oxford Nanopore technologies, herald a new era of DNA sequencing characterised by long-read, single-molecule sequencing capabilities. Unlike short-read sequencing technologies, third-generation sequencing directly sequences DNA molecules in real-time, producing long reads that facilitate de novo genome assembly, detection of structural variants, and characterisation of complex genomic regions. Despite challenges related to error rates and throughput, third-generation sequencing holds immense promise for addressing previously intractable genomic complexities and unlocking new insights into genome structure and function.


Sanger Sequencing FAQs: Your Top Questions Answered

Sanger sequencing, also known as the dideoxy chain termination method, is a widely used technique for determining the order of nucleotides in DNA strands. If you have questions about how this staple of genetic analysis works, here are answers to some of the most frequently asked questions.

How does Sanger sequencing work?

Sanger sequencing employs DNA polymerase to synthesise DNA strands. DNA polymerase requires DNA primers to initiate synthesis and deoxynucleoside triphosphates (dNTPs) to extend the strands. By including dideoxynucleoside triphosphates (ddNTPs) in the reaction, DNA polymerase randomly incorporates ddNTPs, which lack a 3’ hydroxyl group, thus terminating elongation. This results in DNA fragments of varying lengths. The fragments are then separated by size via electrophoresis to determine the sequence.

What are the steps in the Sanger sequencing process?

The major steps in Sanger sequencing include:

  1. Isolating the DNA segment to be sequenced.
  2. Annealing DNA primers to the flanking regions of the target sequence.
  3. Adding DNA polymerase, dNTPs, and ddNTPs.
  4. DNA elongation and random ddNTP incorporation, resulting in termination of DNA synthesis.
  5. Separation of DNA fragments by size using gel electrophoresis.
  6. Detection of the fragments to determine the sequence.

What are the advantages and disadvantages of Sanger sequencing?

Advantages of Sanger sequencing include its low cost, high accuracy, and ability to produce long read lengths. Disadvantages include its low throughput and inability to sequence highly repetitive genomic regions.

How has Sanger sequencing impacted scientific research?

Sanger sequencing has enabled groundbreaking discoveries like the sequencing of the first human genome. It continues to aid research in fields such as medical genetics, evolutionary biology, and forensics. Though next-generation sequencing technologies have emerged, Sanger sequencing remains an important tool for validating results and sequencing challenging genomic regions.

Sanger sequencing has revolutionised life sciences research and clinical diagnostics. By understanding how this technique works and its applications, you can better appreciate its profound impact. Please let me know if you have any other questions!

Engage Bio Basic For Sanger Sequencing Services

With over a decade of expertise, Bio Basic stands as a premier provider of DNA sequencing services, delivering swift, reliable, and cost-effective solutions for research and development across the globe. Employing Sanger sequencing via capillary electrophoresis of fluorescent-labelled DNA fragments, we consistently achieve read lengths of up to 1000 base pairs, ensuring high-quality sequencing results. 

Committed to client satisfaction, we assign dedicated project specialists to each project, tailoring our approach to meet specific requirements and ensure successful project completion. Our dedication to excellence extends to offering complimentary services, including a one-time free re-sequencing option, allowing for further optimisation and improved results. 

Additionally, we provide free gel analysis for quality control of fragment lengths and offer access to over 100 free universal primers to enhance sequencing reactions. With a short turnaround time of within 24 hours upon sample receipt, we strive to exceed expectations and empower researchers in their quest for scientific discovery.