Dideoxynucleotide (ddNTP) is analogous or similar to the deoxynucleotide triphosphate except that it does not have 3' -OR at its 3' -carbon position in the sugar. In practice, four reaction mixtures are set up, each containing all the four normal deoxynucleotides (dNTP) and one dideoxynucleotide.

In a given reaction, the ratio of dNTP and ddNTP is kept at approximately 100: 1, so that there is only one chance of termination at a given point in the nascent chain.

All reaction mixtures contain a template strand, a single primer (usually forward primer) taq polymerase and components of PCR. PCR reaction is carried out, but the only difference here is that the fragments that are generated are not of uniform length, but of varying length.

The DNA sequence can be deduced by reading the ladder produced by autoradiography of the polyacrylamide gel used to separate the fragments.
Currently by this method, DNA of length 100 bp can be read, but the sensitivity and length of DNA fragment that can be sequenced can be increased by using 35S- deoxynucleotide triphosphate. 35S-dNTP contains a sulphur atom at one of the oxygen position phosphate nucleotide molecule.The chain termination DNA sequencing method.