What is Sanger sequencing?

Sanger sequencing is a method that yields information about the identity and order of the four nucleotide bases in a segment of DNA. Also known also as the “chain-termination method”, it was developed in 1977 by Frederick Sanger and colleagues, and is still considered the gold standard of sequencing technology today since it provides a high degree of accuracy, long-read capabilities, and the flexibility to support a diverse range of applications in many research areas.

In the mid-1970s, Sanger wasn’t alone in the race to sequence DNA; almost in parallel, two American scientists, Maxam and Gilbert, developed a technique in which DNA is chemically treated to break the chain at specific bases. Following electrophoresis of the cleaved DNA, the relative lengths of the fragments—and thus the positions of specific nucleotides—can be determined and the sequence inferred [1]. This is considered the birth of first-generation sequencing. However, the advent of Sanger’s chain-termination method in 1977 would be the breakthrough that propelled sequencing into the future [1]; many years after its development, Sanger sequencing was used to sequence the entire human genome. (To learn more about the history of sequencing technologies, see the article titled “What is sequencing”.)

Sanger sequencing targets a specific region of template DNA using an oligonucleotide sequencing primer, which binds to the DNA adjacent to the region of interest. (There must be an area of known sequence close to the target DNA.)W In order to determine the sequence, Sanger sequencing makes use of chemical analogs of the four nucleotides in DNA. These analogs, called dideoxyribonucleotides (ddNTPs), are missing the 3´ hydroxyl group that is required for 5’ to 3’ extension of a DNA polynucleotide chain. By mixing ddNTPs that have been labeled with a different color for each base, unlabeled dNTPs, and template DNA in a polymerase-driven reaction, strands of each possible length are produced when the ddNTPs are randomly incorporated and terminate the chain. The extension products are then separated by electrophoresis, resolved to single-nucleotide differences in size. The chain-terminated fragments are detected by their fluorescent labels, with each color identifying one of the terminating ddNTPs. The sequence of the template DNA strand can thus be derived by analysis (Figure 1).

Automation of Sanger sequencing has been made possible with the development of a variety of DNA sequencing instruments and platforms; one of the latest innovations is the Applied Biosystems SeqStudio Genetic Analyzer, which uses a cartridge-based system rather than an individual reagent configuration. (For Research Use Only. Not for use in diagnostic procedures.)

The Sanger sequencing workflow involves amplifying the sequence by PCR and performing the sequencing reaction, capillary electrophoresis, and data analysis (Figure 2). (Note that there is a clean-up step following PCR and sequencing.)

During PCR and cycle sequencing, the DNA is first denatured (the double-stranded DNA template becomes single-stranded DNA). A subsequent annealing step allows for hybridization of the oligonucleotide primer close to the sequence of interest. In the extension step, the DNA polymerase extends the primer from its 3´ hydroxyl group to synthesize a new strand. An adenine base (A) is paired with every thymine (T) on the template and a cytosine (C) with every guanine (G) and vice versa. Occasionally, one of the four chain-terminating ddNTPs will be inserted by chance, stopping elongation of the DNA strand. The elongation reaction is repeated for 30–40 cycles.

Following sequencing clean-up, the newly synthesized DNA fragments are separated by electrophoresis. The fragments are run through a single long glass capillary filled with a gel polymer, where they migrate according to their length. By using an optimized combination of a very thin capillary, appropriate choice of gel or polymer, and electric field parameters, CE can separate DNA strands up to ~1,000 base pairs in length with single-nucleotide resolution.

As the fragments migrate through the capillary,a laser excites the fluorescent label on the ddNTP incorporated at the end of each terminated chain. Because each of the four ddNTPs is labeled with a different color, the signal emitted by each excited nucleotide corresponds to a specific base. Software generates a chromatograph showing the fluorescent peak of each labeled fragment.

Learn more about the steps of the Sanger sequencing workflow ›

Sanger sequencing has undergone many changes over the last 40 years, but it remains the most commonly used DNA sequencing technology worldwide. With 99.99% accuracy, it is the gold standard for most applications—both research and clinical. However, Sanger sequencing is best suited for medium- to low-throughput targeted sequencing projects; higher-throughput DNA sequencing technologies based on fundamentally different methods have emerged in the last decade. Called next-generation sequencing (NGS), these massively parallel technologies have revolutionized the study of genomics and molecular biology.

To find out which sequencing technology is right for your application, see the article How do I choose the right sequencing technology?.

To find out more about NGS, visit the NGS section of this Sequencing Learning Center.

1. Heather JM, Chain B (2016) The sequence of sequencers: The history of sequencing DNA. Genomics 107:1-8.