Sanger Sequencing

Sanger sequencing is a laboratory method that scientists use to determine the sequence of nucleotides within a piece of deoxyribonucleic acid, or DNA. Frederick Sanger, who studied biochemistry in the United Kingdom, and colleagues developed the technique in 1977. Sanger sequencing was one of the first methods to sequence long stretches of DNA accurately, allowing researchers to collect and analyze large amounts of genetic data. The technique uses modified nucleotides that terminate DNA synthesis at specific points, which results in fragments of varying lengths that researchers sort to determine the sequence of nucleotides. Sanger sequencing was one of the most widely used sequencing methods for thirty years after its creation and facilitated early genomic sequencing projects, such as the Human Genome Project, or HGP. As of 2025, Sanger sequencing remains a common technique despite the advent of newer sequencing technologies and plays a role in clinical settings and genetic counseling, a service that provides information on genetic conditions to people and their families.

Nucleic acids, namely DNA and ribonucleic acid, or RNA, are the molecules that store and transmit genetic information in living organisms. They consist of chains of nucleotides. Adenine, thymine, guanine, and cytosine are the four types of nucleotides in DNA. Sequencing refers to the process of determining the order of the nucleotides that make up the DNA molecule. It allows scientists to conduct genetic research, such as understanding the function of genes and identifying genetic mutations associated with diseases. 

Although by the 1950s scientists possessed an understanding of the general composition of DNA, the ability to read the sequence of nucleotides in either DNA or RNA was undeveloped. Researchers were only able to measure nucleotide concentrations, not order. Robert Holley, who studied transfer RNA, or tRNA, used analytical chemistry techniques and biochemical techniques to degrade specific types of RNA molecules based on their structural or sequence features. In 1965, Holley’s approach produced one of the first complete nucleic acid sequences of the tRNA that carries the amino acid alanine during protein formation in the yeast strain Saccharomyces cerevisiae.

Concurrently to Holley, Sanger and collaborators were developing a related technique based on the detection of radio-labeled partial-digestion fragments, or RNA molecules researchers partially cleave with an enzyme and tag with a radioactive marker for detection. The team published their results in 1967. Walter Fiers, a scientist who studied molecular biology in Belgium, used this technique to produce one of the first complete protein-coding gene sequences of the coat protein of the bacteriophage MS2 in 1972 and the complete RNA sequence of MS2 in 1976. Bacteriophages are viruses that infect bacteria.

In the 1970s, researchers began to adapt methods used in RNA sequencing to sequence DNA and tested their protocols on bacteriophages that use DNA as genetic material. Ray Wu and Dale Kaiser, who studied molecular biology at Stanford University in Stanford, California, noticed that DNA-based bacteriophage λ possessed overhanging cohesive ends. The ends of the DNA were single-stranded overhangs that could form sticky ends and were easier to manipulate. Wu and Kaiser used the enzyme DNA polymerase to attach nucleotides with radioactive labels to the sticky ends of the DNA sequence one at a time and used the labels to determine the sequence of the DNA. According to David Botstein, who was a geneticist and Kaiser’s colleague, that was one of the first instances of DNA sequencing. Researchers generalized the mechanics of Wu and Kaiser’s technique through the use of specific oligonucleotide primers, or short chains of nucleotides that provide starting points for the DNA polymerase to synthesize DNA. They used radioactive nucleotides to infer the order of nucleotides at any point in the sequence of the bacteriophage genomes, not just the end. Scientists could still only determine the order of bases of short stretches of DNA.

Researchers utilized gel electrophoresis, or a technique that separates particles in a gel medium based on their size, to improve the speed and accuracy of sequencing. In 1975, Sanger and colleague Alan Coulson developed the plus and minus system to sequence DNA. Like previous methods, the plus and minus technique used primers to designate a starting point for DNA synthesis and incorporation of radiolabeled nucleotides. Then, they performed two polymerization reactions, or reactions that create larger molecules called polymers from smaller molecules called monomers. The plus step treated the synthesized strand with one type of nucleotide, which resulted in all extensions of the DNA ending with that base. The minus step treated the synthesized stand with the remaining three types of nucleotides, which produced sequences up to the position before the next missing nucleotide.

The plus and minus system that Sanger and Coulson developed enabled the formation of strands with overlapping nucleotide sequences, which gel electrophoresis separated by size and enabled a direct readout of the sequence. Sanger and Coulson used the technique to determine two DNA sequences in the bacteriophage φX174. The main challenge with that method was its difficulty in determining the length of homopolymer runs, or sequences of the same nucleotide repeated. The technique produced bands for the start and end of those runs, but no bands appeared for positions within the run itself. Scientists had to estimate the length of the run by measuring the spacing between bands on the gel, which became less reliable as the runs got longer.

In 1977, Sanger and Coulson, alongside collaborator Steve Nicklen, used the chain-termination technique to develop Sanger sequencing and address the issues with the plus and minus system. The technique uses special bases called chain terminators or dideoxynucleotides or ddNTP, which are modified nucleotides that lack hydroxyl groups at the 3’ position. Those nucleotides are a critical component to extend the DNA strand further. Without that group, DNA polymerase cannot add more nucleotides once the technique incorporates ddNTP, which causes the DNA strand to terminate at that specific nucleotide.

As with the plus and minus system, Sanger sequencing begins with DNA synthesis using an oligonucleotide primer. As the DNA strand grows, the incorporation of a radiolabeled ddNTP halts its elongation, which produces DNA fragments of varying lengths. Each fragment ends at a nucleotide corresponding to the incorporated ddNTP. The process repeats using chain terminators of all four nucleotides in DNA: ddATP derived from adenine, ddTTP derived from thymine, ddCTP derived from cytosine, and ddGTP derived from guanine. Gel electrophoresis separates the resulting fragments and sorts them by size. Due to the radiolabeling, an X-ray film can visualize the ddNTPs. The position of each fragment indicates the sequence of the template strand. The length of the fragment corresponds to the position of the nucleotide in the DNA sequence. Scientists can read the bases sequentially from the bottom to the top of the gel.

Compared to previous techniques, Sanger sequencing enabled the sequencing of larger DNA fragments of 500 to 1,000 base pairs. In December 1977, Sanger, Coulson, and Nicklen announced their method for determining DNA nucleotide sequences in a paper titled “DNA Sequencing with Chain-Terminating Inhibitors” in the journal Proceedings of the National Academy of Sciences of the United States of America. They explain the biochemical mechanics of Sanger sequencing and demonstrate the validity of their discovery by sequencing the DNA of φX174. In their paper, Sanger, Coulson, and Nicklen also explain that their method is faster and more accurate than the plus and minus method.

Researchers working during the early 1980s used Sanger sequencing to produce new findings. In 1981, scientists used the Sanger technique to sequence human mitochondrial DNA. In 1982, Sanger and a team of researchers created one of the first determinations of a complete genome sequence of bacteriophage λ. Sanger and colleagues built upon the principles of previous sequencing methods, namely the idea of using DNA fragment length to indicate nucleotide position and the labeling of the final nucleotide in the fragment. They had developed a technique that was highly accurate and easy to use, which established Sanger sequencing as one of the most widely used sequencing technologies for thirty years after its creation.

In the following years, Sanger sequencing underwent refinement, including using fluorescent dyes to label ddNTPs instead of radioactive phosphorus or hydrogen isotopes. There was also a shift from using traditional gel electrophoresis, which uses a slab-like gel matrix to suspend particles, to capillary-based electrophoresis, which uses a thin capillary tube filled with a viscous buffer. Once the DNA is loaded, an electric current flows through the capillary, which causes the negatively charged DNA fragments to move toward the positive electrode located at the end of the capillary. Smaller DNA fragments move through the gel more quickly, while larger fragments move more slowly. A device called a capillary sequencing machine then shoots a laser through the capillary to induce fluorescence. A camera detects the pattern of color in the fluorescence and displays the data as a graph of different colored peaks, which corresponds to the base pairs in the sequence. Both traditional and capillary electrophoresis use an electric field to separate particles based on size. Capillary electrophoresis is faster, more efficient, and able to process smaller sample quantities. Those improvements contributed to the development of increasingly automated DNA sequencing machines. The biotechnology company Applied Biosystems was able to create one of the first commercial capillary sequencers in the mid-1980s.

Early DNA sequencing machines produced DNA reads of approximately 1,000 base pairs. Researchers developed techniques in the early 1980s to read large amounts of genetic material, such as shotgun sequencing, which involves the sequencing and subsequent joining of overlapping DNA fragments. The National Institutes of Health, or NIH, in Bethesda, Maryland, and Wellcome Trust Sanger Institute in Cambridge, England, headed the HGP to generate one of the first sequences of an individual human genome using Sanger sequencing. Sanger sequencing only sequences a single DNA fragment at a time, so HGP scientists conducted a shotgun phase. They divided the human chromosomes into small, overlapping DNA fragments. After sequencing and combining the fragments into a continuous series, the scientists initiated a finishing phase that involved filling in gaps and resolving ambiguous DNA sequences. Researchers used other laboratory techniques, such as polymerase chain reaction, or PCR, which produces many copies of specific sections of DNA, to better handle the large amounts of data. Still, the process was time-consuming and expensive. It lasted from 1990 to 2003 and cost three billion US dollars.

In 2005, researchers at the biotechnology company Solexa developed next-generation sequencing, or NGS, and introduced the technology for commercial use to address those inefficiencies. NGS operates as many miniscule Sanger sequencing reactions running in parallel. Compared to Sanger sequencing, NGS requires smaller sample inputs but retains a high accuracy level. It is also faster and more cost-effective, which makes it useful in genomic studies that process large amounts of data and precision medicine initiatives that create therapeutics tailored to particular genetic variations.

Despite the development of alternative sequencing techniques, Sanger sequencing remains one of the most accurate methods of DNA sequencing. Researchers widely use the technique to perform diagnostic sequencing of a single gene and identify familial genes that carry disease risk. Sanger sequencing plays a role in predictive genomic testing like the BRCA1 gene variant that affects breast cancer risk, carrier testing for parents when a child has an autosomal recessive condition like cystic fibrosis, and prenatal testing for known genetic conditions within a family. Sanger sequencing can also supplement NGS by confirming previously identified variants and filling in gaps in data. According to the National Health Service, or NHS, the public health care system of the United Kingdom, scientists consider Sanger sequencing to be the gold standard for accurately detecting single nucleotide variants and small insertions or deletions of nucleotides in the DNA sequence. Although Sanger sequencing is not practical for use in large-scale genomic projects, it is helpful in analyzing small numbers of gene targets for individualized clinical care.

Although scientists typically sequence whole genomes using other methods as of 2025, Sanger sequencing finds use in sequencing individual pieces of DNA for small-scale studies, clinical diagnostics, and validation of NGS results. Developed in 1977, Sanger sequencing was the primary method of sequencing until the introduction of NGS in the early 2000s. Sanger sequencing has allowed scientists to obtain a more comprehensive understanding of the genetic organization of the human genome, as indicated in its usage in the HGP and other early sequencing projects. In 1980, Sanger and American biochemist Walter Gilbert, who also developed a sequencing technique called the Gilbert-Maxam method, shared the 1980 Nobel Prize in Chemistry for their work in DNA sequencing. According to Ali Marian, a professor of internal medicine at McGovern Medical School in Houston, Texas, Sanger sequencing has revolutionized the field of molecular genetics and biology. Sanger sequencing has facilitated the understanding of the genetic basis of various diseases.

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