Sanger sequencing
Genomics · Molecular Biology · Biotechnology
Say it: SANG-er SEE-kwen-sing
Sanger sequencing is a method for reading the order of bases in DNA that uses chain-terminating nucleotides, developed by Frederick Sanger in the 1970s.
Also: chain-termination sequencing, dideoxy sequencing
Definition
What is Sanger sequencing?
Sanger sequencing, also called the chain-termination or dideoxy method, copies a single-stranded DNA template using DNA polymerase, a primer and the four normal nucleotides, plus a small amount of modified ones called dideoxynucleotides (ddNTPs). A ddNTP lacks the 3' hydroxyl group that the next nucleotide must bond to, so whenever the polymerase adds one, that copy stops growing. The result is a collection of fragments of every length, each ending in a known base.
In the modern version, each of the four ddNTPs carries a different fluorescent dye. The fragments are separated by size, now in thin capillary tubes rather than slab gels, and a laser reads the color at the end of each fragment in turn, giving the sequence directly. One reaction reads a single stretch of up to about 1,000 bases with very high accuracy.
Frederick Sanger and his team in Cambridge, UK, developed the method, and it earned him his second Nobel Prize in Chemistry in 1980. It was the workhorse of the Human Genome Project. Next-generation methods now handle big projects, but Sanger sequencing is still used to check single genes, confirm variants and verify cloned DNA.
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Terms that go with it
- Next-generation sequencingNext-generation sequencing (NGS) is a set of high-throughput methods that read the sequences of millions of short DNA fragments in parallel.
- DNA polymeraseDNA polymerase is an enzyme that builds new DNA by adding nucleotides one at a time to a growing strand, using an existing strand as a template.
- Gel electrophoresisGel electrophoresis is a lab technique that uses an electric field to pull DNA, RNA or proteins through a gel, separating them by size.
- NucleotideA nucleotide is a single unit of DNA or RNA, made of a nitrogen-containing base, a five-carbon sugar and one or more phosphate groups.
- Polymerase chain reactionThe polymerase chain reaction (PCR) is a lab technique that copies one chosen stretch of DNA over and over until there are billions of copies.
- GenomeA genome is the complete set of DNA in an organism or cell, including all of its genes and the stretches of DNA between them.
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Written for biofideX in plain words, after checking these references:
- OpenStax Biology 2e, 17.3 Whole-Genome Sequencing
- yourgenome (Wellcome Connecting Science): What is Sanger sequencing?
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