- Sanger sequencing, developed by Frederick Sanger in the late 1970s, was the first widely adopted method for determining the nucleotide sequence of DNA.
- It is based on selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) during DNA replication. Initially, this method involved labor-intensive procedures with radioactive labeling and slab gel electrophoresis, which limited its speed, safety, and scalability.
- The need for higher throughput and efficiency led to the automation of Sanger sequencing, a major advancement that transformed molecular biology and genomics research.
- A critical innovation in automated Sanger sequencing was the use of fluorescently labeled dideoxynucleotides. In contrast to early methods where each of the four DNA bases required a separate radioactive reaction, fluorescent labeling allowed all four ddNTPs to be included in a single reaction, each tagged with a different fluorescent dye. During the sequencing reaction, the DNA polymerase incorporates both regular deoxynucleotides (dNTPs) and occasional ddNTPs, which terminate the elongation. Because each ddNTP is labeled with a distinct fluorescent color, the terminal base of each resulting DNA fragment can be identified by its fluorescence, enabling the sequence to be read directly and efficiently.
- Another major advancement was the replacement of slab gel electrophoresis with capillary electrophoresis (CE). Capillary electrophoresis uses narrow capillary tubes filled with a polymer matrix to separate DNA fragments by size. When an electric current is applied, shorter DNA fragments migrate faster through the capillary. As the fragments pass a detection window near the end of the capillary, a laser excites the fluorescent tags on the ddNTPs, and a detector captures the emitted light. The sequencing instrument converts these fluorescent signals into an electropherogram, a graph where each peak represents a base in the DNA sequence.
- The combination of fluorescent labeling and capillary electrophoresis allowed the entire sequencing process to be automated, greatly improving accuracy, speed, and throughput. Instruments such as the ABI Prism 3700 and similar platforms were capable of running 96 or more capillaries in parallel, generating hundreds of thousands of base pairs per day. This made automated Sanger sequencing the backbone of major genome projects in the 1990s and early 2000s, including the Human Genome Project.
- Although newer next-generation sequencing (NGS) technologies now dominate large-scale sequencing due to their massive parallelism and lower cost per base, automated Sanger sequencing remains widely used today for smaller-scale applications. These include sequencing individual genes, validating NGS results, identifying mutations, and performing clinical diagnostics and DNA barcoding. The combination of fluorescent ddNTPs and capillary electrophoresis continues to offer a highly accurate and reliable method for DNA sequencing, representing a pivotal evolution in the history of genomics.
