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Biology · Glossary

What is Sequencing by chain termination?

Definition 4.1 University Biology — Year 3 · Chapter 4 — Genomics and Sequencing

Sanger sequencing copies a single-stranded template from a primer with DNA polymerase in the presence of the four normal nucleotides and a small proportion of dideoxynucleotides, which lack the 3′' hydroxyl and so terminate the chain wherever they are incorporated. Each dideoxynucleotide carries a different fluorescent dye. The product is a mixture of fragments, one for every position of the template, each ending in a base of known identity; separated by size in a capillary of gel, they pass a detector in order of length, and the sequence of colours is the sequence of the template. A run reads 700 to 900 bp700\text{ to }900\,\mathrm{bp} with an error rate below 10−310^{-3}; it remains the method for verifying a construct or a single gene.

Left: Frederick Sanger, who devised the first practical methods for reading proteins and DNA and received a Nobel prize for each. Right: an autoradiograph of a sequencing gel from the pre-capillary era, one lane per base, the sequence read upward from the ladder of bands. Left: Frederick Sanger, who devised the first practical methods for reading proteins and DNA and received a Nobel prize for each. Right: an autoradiograph of a sequencing gel from the pre-capillary era, one lane per base, the sequence read upward from the ladder of bands.
Left: Frederick Sanger, who devised the first practical methods for reading proteins and DNA and received a Nobel prize for each. Right: an autoradiograph of a sequencing gel from the pre-capillary era, one lane per base, the sequence read upward from the ladder of bands.
Chain-termination sequencing. A dideoxy base ends the copy at every position where it is incorporated; the fragments, one per length, are separated by size and the colour of each terminal base is read in order.
Chain-termination sequencing. A dideoxy base ends the copy at every position where it is incorporated; the fragments, one per length, are separated by size and the colour of each terminal base is read in order.
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