As the first mainstream technology for DNA sequencing, Sanger sequencing laid the foundations for next-generation sequencing (NGS) and contributed to major milestones in genomics like the Human Genome Project. Nearly fifty years after its introduction, Sanger remains a vital and widely used tool in molecular biology labs. This article reviews Sanger’s continued relevance, its integration with other technologies, recent innovations, and where it continues to deliver value in today’s modern lab workflows.
Strengths of Sanger Sequencing
Sanger still holds a place in the sequencing toolkit because it addresses needs that newer methods don’t always meet. “Despite the widespread adoption of NGS, Sanger sequencing remains the gold standard for DNA sequencing due to its unparalleled accuracy and reliability,” emphasized Adam Blatter, Product Manager at Promega. One of its key advantages, Blatter noted, is its capacity to generate long, accurate reads, ideal for applications requiring base-by-base validation. This makes it useful for confirming regions with homopolymer runs or resolving ambiguous bases that can be problematic for other sequencing methods.
“Sanger sequencing is particularly cost-effective and efficient for small-scale projects, such as sequencing individual genes or verifying specific mutations,” added Blatter. In these targeted applications, it provides accurate results with minimal setup, turnaround time, and data analysis.Additionally, Sanger sequencing is easy to use and delivers results quickly, making it useful in settings where NGS would be unnecessary or burdensome. This is especially valuable in settings where clear results are needed quickly and with reduced hands-on time.
Current Applications in the Lab
Today, Sanger sequencing continues to play an important role in a variety of applications across clinical and research settings. “Sanger sequencing is widely used in clinical diagnostics to confirm genetic variants identified by NGS, ensuring the accuracy of results before they inform patient care,” stated Blatter. Recent studies in immunodeficiency and newborn screening have demonstrated its reliability for confirming single-gene variants in targeted clinical applications1,2.
Blatter also emphasized that Sanger sequencing remains a routine part of molecular biology research, where it’s used to validate cloning constructs, sequence PCR amplicons, and confirm site-directed mutagenesis. But its utility extends well beyond research applications. “Additionally, it’s used in microbial identification, forensic analyses, and pharmacogenomics,” stated Blatter. “Laboratories often rely on Sanger sequencing for any application where high precision and reliability are critical.”
Integrating Sanger Sequencing into Modern Workflows
Sanger sequencing is a core component of many laboratory workflows. As Blatter explained, “Sanger sequencing complements NGS by serving as a validation tool to confirm variants and mutations detected in high-throughput sequencing projects.” In clinical genomics, Sanger sequencing is commonly used to validate pathogenic variants detected by NGS before any clinical action is taken. “This two-step approach combines the broad scope of NGS with the precision of Sanger sequencing,” he noted.
In addition to NGS, Blatter shared that Sanger sequencing is often combined with fragment analysis to gather the size and sequence data. This combination is particularly effective in microsatellite analysis and detecting insertions or deletions, where sequence integrity and fragment length are important.
During the development of mRNA therapeutics and cell therapies, Sanger sequencing is a key quality control (QC) tool. In mRNA applications, it is used to verify that synthetic mRNA sequences match their intended design to ensure product consistency. “For mRNA therapeutics, ensuring the accuracy of the mRNA sequence is paramount, as any errors can impact the efficacy or safety of the therapeutic product,” emphasized Blatter. Similarly, in engineered cell therapies, Sanger is essential for confirming that genetic edits have been correctly introduced. A 2024 study comparing tools for analyzing Sanger chromatograms from CRISPR-edited samples highlights its continued use in evaluating on-target modifications and estimating editing efficiency3. “By integrating Sanger sequencing into QC workflows, developers can ensure the integrity and safety of their therapeutic products before they proceed to clinical applications.”
Recent Innovations and Improvements
Sanger sequencing has come a long way since its early days using gel-based readouts, and the advances haven’t stopped. “Recent innovations focus on enhancing the accessibility and efficiency of Sanger sequencing,” stated Blatter. “Instruments like the Spectrum Compact have made significant strides by offering benchtop solutions that integrate both Sanger sequencing and fragment analysis in a single platform accessible for any research lab.” He also noted that this instrument simplifies Sanger workflows and minimizes lab space requirements, enabling more labs to perform high-quality genetic analysis without extensive infrastructure.
Similar to other technologies in the field, automation has been another key advancement. With fewer manual steps, automated workflows reduce manual input and potential inconsistencies. Furthermore, Blatter highlighted that newer chemistries and consumables have led to increases in accuracy, read lengths, and greater affordability. “Software enhancements have also streamlined data analysis, making it more user-friendly for a broader range of laboratory personnel,” stated Blatter.
Future Outlook
In the years ahead, Sanger sequencing is expected to remain a valuable method for a wide range of genetic applications. Blatter envisions it continuing to play a key role in precision medicine and clinical diagnostics, noting that “its unparalleled accuracy makes it indispensable for confirming critical genetic variants that can influence patient treatment plans.” As gene editing technologies like CRISPR become more routine in research and therapeutic development, he believes Sanger will continue to be the method of choice for validating on-target edits and detecting off-target effects at single-base resolution.
Blatter also shared that the growing demand for precise, individualized data in personalized medicine will continue to sustain Sanger’s role in targeted applications. Outside of its clinical utility, he emphasized its value in training the next generation of scientists. “Education and training in molecular biology will also continue to rely on Sanger sequencing as a foundational technology,” Blatter concluded. Its simplicity and dependable results make it especially useful for teaching core principles in molecular genetics.
References
- Chan, K. W., Wong, C. Y., Leung, D., Yang, X., Fok, S. F. S., Mak, P. H. S., Yao, L., Ma, W., Mao, H., Zhao, X., Liang, W., Singh, S., Barbouche, M. R., He, J. X., Jiang, L. P., Liew, W. K., Le, M. H. T., Muktiarti, D., Santos-Ocampo, F. J., Djidjik, R., … Lau, Y. L. (2022). Targeted Gene Sanger Sequencing Should Remain the First-Tier Genetic Test for Children Suspected to Have the Five Common X-Linked Inborn Errors of Immunity. Frontiers in immunology, 13, 883446. https://doi.org/10.3389/fimmu.2022.883446
- Hogner, S., Lundman, E., Strand, J., Ytre-Arne, M. E., Tangeraas, T., & Stray-Pedersen, A. (2023). Newborn Genetic Screening-Still a Role for Sanger Sequencing in the Era of NGS. International journal of neonatal screening, 9(4), 67. https://doi.org/10.3390/ijns9040067
- Aoki, K., Yamasaki, M., Umezono, R., Hamamoto, T., & Kamachi, Y. (2024). Systematic Comparison of Computational Tools for Sanger Sequencing-Based Genome Editing Analysis. Cells, 13(3), 261. https://doi.org/10.3390/cells13030261