dideoxynucleotide
/ dy-DEE-oxy-NEW-clee-oh-tide; ddNTP /
Sanger sequencing depends on copies of DNA that stop growing at controlled points. What makes them stop? A cleverly broken building block called a dideoxynucleotide. It looks almost exactly like a normal DNA letter — close enough that the polymerase will add it — but it is missing the one piece needed to attach the next letter, so the chain ends right there.
A normal DNA building block (a deoxynucleotide) has a sugar with a 3'-hydroxyl group (a 3'-OH) on it. That 3'-OH is the hook onto which the next nucleotide's phosphate is joined — it is what lets the chain keep growing. A dideoxynucleotide is the same building block but with the 3'-OH removed (hence 'di-deoxy': two oxygens missing rather than one). DNA polymerase can still add a dideoxynucleotide to a growing strand, because the rest of the molecule looks normal, but once it is in place there is no 3'-OH for the following nucleotide to attach to. The chain is terminated. By including a small, controlled amount of these terminators, Sanger reactions produce strands that stop at every possible position.
This single missing oxygen is the whole basis of chain-termination sequencing — a perfect example of how understanding a molecule's exact chemistry yields a powerful method. The same idea also underlies some antiviral drugs (chain-terminating nucleoside analogs that jam viral polymerases). The honest subtlety: termination is statistical, not at fixed sites — across millions of molecules, each position gets terminated in some fraction of them, which is exactly what builds the complete ladder of lengths.
Imagine adding to a copying reaction normal building blocks plus a sprinkle of fluorescent ddA, ddT, ddG, ddC. Most of the time a normal A is added and copying continues; occasionally a ddA goes in instead, and that strand stops with a red-tagged A on its end — marking exactly where an A sat.
Missing 3'-OH = no hook for the next base = chain stops here.
The only difference from a normal nucleotide is the missing 3'-hydroxyl — that single absent oxygen, not any added chemistry, is what stops the chain; the same chain-terminator principle is also used by some antiviral drugs.