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Sanger sequencing 2026-10-18

Sanger sequencing determines a DNA nucleotide sequence by extending a common sequencing primer and occasionally incorporating a dideoxynucleoside triphosphate. A normal deoxynucleoside triphosphate already has hydrogen in place of a

2^{'}

hydroxy group; it retains the

3^{'}

hydroxy group. A dideoxynucleoside triphosphate lacks that

3^{'}

hydroxy group too. It can join the growing strand because the preceding nucleotide supplies the reacting

3^{'}

hydroxy group. Once incorporated, its own

3^{'}

end cannot form the next phosphodiester bond. Thus the blocked step is the next addition, not attachment of the terminator itself.^[ref]

Figure 1.
A dNTP retains a $3^{'}$ hydroxy group; a ddNTP permits incorporation but blocks the next extension
. Original chemical-feature and chain-extension schematic. Both substrates have $2^{'}$ -H; only the dNTP has $3^{'}$ -OH. Boxes omit most chemical atoms and do not specify stereochemistry. The lower rows show incorporated units, which no longer carry the substrate's complete triphosphate group. The previous strand end attaches either substrate; the difference appears at the new terminal $3^{'}$ end. This is a conceptual drawing, not molecular imaging or experimental data. **Details**.

In the historical four-reaction format, each tube receives copies of the same template, the same sequencing primer, a DNA polymerase and all four ordinary deoxynucleoside triphosphates. The tubes differ in their terminating substrate: ddATP, ddCTP, ddGTP or ddTTP. In the A reaction, ordinary dATP can allow copying to continue past one A position, whereas incorporation of ddATP stops another copy there. Competition produces a family of endpoints, rather than every strand stopping at the first A. Adjusting substrate proportions makes later endpoints detectable. A lane identifies the terminating nucleobase in the new strand, not the complementary template letter.^[ref]

The readout requires gel electrophoresis capable of separating single-stranded DNA fragments differing by one nucleotide. Historical denaturing polyacrylamide gel electrophoresis suppressed strand folding and resolved these adjacent lengths; radioactive labels were detected by autoradiography. An ordinary agarose gel electrophoresis comparison with a 100-base pair DNA ladder does not supply that resolution. Four separate reaction lanes carry the identities in this format.^[ref]

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