Sanger Sequencing for Specific and Small-Scale DNA Testing
Cost, time, and labor considerations still make the Sanger method a better choice amidst new sequencing technologies

Darwin’s postulation of “heritable traits,” followed by the discovery of DNA and its structure, led to our understanding of the essential role of genetic information in enabling biological functions, including the role of particular genes in disease. By sequencing DNA, i.e., finding out the precise order of nucleotides in genes, we can diagnose and better target certain diseases.
Although researchers do not completely understand the gene–function relationship yet, the advent of sequencing technologies have contributed to our ability to characterize the form and function of many genes and gene products, including for powerful clinical interventions.
A particularly effective technology is Sanger sequencing, and this article will provide an overview of Sanger sequencing, its applications, and its advantages and disadvantages compared to other sequencing technologies.
What is Sanger sequencing?
Sanger sequencing, also known as the chain-termination method, determines the DNA sequence of small, targeted regions of the genome. Developed in 1977, Frederick Sanger’s method remains a gold standard for high-accuracy, small-scale DNA sequencing.
How does Sanger sequencing work?
To perform Sanger sequencing, the patient’s DNA is used as a template for amplification through polymerase chain reaction (PCR). Along with normal nucleotides (dNTPs, including A, T, C, or G bases), this PCR reaction includes chain-terminating nucleotides (ddNTPs) that lack a 3'-OH group and are tagged with a unique fluorescent dye. Thus, when the growing DNA strand happens to incorporate a ddNTP, its elongation terminates, resulting in DNA fragments of various lengths ending with fluorescently-tagged ddNTPs.
Next, DNA fragments are sorted by size using a method called capillary electrophoresis, where the base at the end of each fragment is identified by a laser detector. The shortest fragments, corresponding to the beginning of the sequence, are detected first, followed by longer fragments.
Capillary electrophoresis is a laboratory technique used to separate molecules, such as DNA fragments, based on their size and charge, by passing them through a narrow capillary tube under an electric field. Smaller fragments move faster than larger ones, and the fragments are detected, often by fluorescence, to determine their sequence. |
The emitted fluorescence is captured and analyzed to create a chromatogram that reflects the sequence of bases in the DNA. By comparing this chromatogram with a reference sequence, genetic variations in the patient’s DNA can be identified.
How does Sanger Sequencing compare with other sequencing? What are its limitations or disadvantages?
The main difference between Sanger and other parallel sequencing methods is the volume of DNA they can sequence in a given run.
The Sanger method sequences a single DNA fragment at a time. In comparison, by definition, parallel sequencing methods such as next-generation sequencing (NGS) can simultaneously sequence millions of fragments per run. In other words, compared to other methods, Sanger sequencing has limited throughput, making it more expensive and less efficient for sequencing multiple samples or large genomes.
Sanger sequencing also has a lower detection sensitivity than some of the newer methods and is unlikely to detect novel or rare variants.
What are the advantages of Sanger sequencing?
Despite these limitations and the availability of newer sequencing methods like NGS, whole exome sequencing (WES), chromatin immunoprecipitation (ChIp), and more, Sanger sequencing continues to be the gold standard method for accurately detecting single nucleotide variants, or SNPs, and small insertions and deletions.
Key advantages of Sanger sequencing:
Sanger sequencing is flexible for testing specific familial variants anywhere in the genome (i.e., not limited to specific regions like the exome). This makes it ideal for situations where targeted testing is required.
Moreover, when it comes to data analysis, Sanger sequencing is significantly easier to interpret and less computationally intensive than alternatives like NGS, saving costs for computational resources and training, as well as time required for training and analyzing data and the need for advanced analytical expertise.
The Sanger method is also cost-effective for urgent testing of single samples and is more likely to be reimbursed by insurance.
This is particularly important when batching samples is not feasible, such as in prenatal or parental carrier testing during a pregnancy, according to data from the Norwegian newborn screening (NBS) program. The NBS program uses DNA extracted from dried blood spot (DBS) filter cards for genetic testing, with Sanger sequencing being “the most time- and cost-efficient method to use for monogenic disorders caused by variants in one single gene or a few genes only.”
Clinical applications of Sanger sequencing
These advantages make Sanger sequencing well suited to molecular diagnostic testing for several genetic diseases in the clinic, such as testing for cardiovascular inherited diseases.
In a recent webinar, Sumy Joseph, PhD, FACMG, of Cohesion Phenomics, described the case of a 52-year-old female presenting with symptoms, such as chest pain, heart palpitations, dizziness, and fatigue. Based on clinical and genetic tests, the individual was diagnosed with hypertrophic cardiomyopathy, a condition with thickened ventricular walls in the absence of other cardiac or systemic conditions.
Hypertrophic cardiomyopathy is caused by mutations in sarcomeric proteins, which are responsible for contraction, and is inherited in an autosomal dominant pattern.
To perform genetic testing, Joseph’s team used Sanger sequencing, revealing a mutation in myosin biotin protein. Their positive detection rate for variants was 45 percent, on par with the literature, but variants of uncertain significance (VUS) were detected at 11 percent, which is significantly lower than the 21 percent in literature.
“This shows that sequencing with Sanger, as a testing platform, using a defined number of genes that have a definitive association for the condition, is pretty robust in detecting clinically actionable variants,” Joseph concluded.
Overall, Sanger sequencing continues to be an efficient and effective method for identifying known genetic variants, making it a good initial method for genetic tests.