= Sanger sequencing
{c}
{wiki}
= Chain-termination sequencing
{synonym}
= Dideoxy sequencing
{synonym}
<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.<Molecular Biology of the Cell, 4th edition by Bruce Alberts and others/Isolating, Cloning, and Sequencing DNA>[Alberts and others, Molecular Biology of the Cell, 4th edition (2002), Isolating, Cloning, and Sequencing DNA, Figure 8-36 and accompanying dideoxy-method explanation]{ref}
\Image[/sanger-dntp-ddntp-chain-extension.png]
{title=A <dNTP> retains a $3'$ <hydroxy group>; a <ddNTP> permits incorporation but blocks the next extension}
{description=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.}
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.<Genomes, 2nd edition by Terence A. Brown/Sequencing Genomes>[T. A. Brown, Genomes, 2nd edition (2002), section 6.1.1, Chain termination sequencing in outline]{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.<Genomes, 2nd edition by Terence A. Brown/Sequencing Genomes>[T. A. Brown, Genomes, 2nd edition (2002), section 6.1.1, Figure 6.1, Figure 6.2 and Technical Note 6.1: single-nucleotide resolution and autoradiographic readout]{ref}
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