Sequencing more-limited regions of the genome can be done by a method called Di-deoxy sequencing or Sanger sequencing method. They use 2', 3'- di-deoxy triphosphates (ddNTPs) that vary from the deoxyribonucleotide substrates of DNA synthesis by missing a 3'-OH group. The di-deoxyribonucleotides are able to function as substrates for DNA polymerase and adjoin to the 3' end of the developing chain. Due to which, later additions cannot takes place and integration of a di-deoxynucleotide stops the growth of the DNA chain.
If a minute quantity of a certain di-deoxyribonucleotide is added in the production of DNA, it is infrequently included in the place of the related dNTP. This results in an immediate termination in the replication process. The length of the resultant fragment of DNA recognizes the location of the nucleotide that must have been incorporated.
An excess dTTP, dGTP, dCTP, and dATP are added with the small amount of ddATP. Then DNA polymerase is added to the reaction mixture. As the polymerase replicates the DNA, it irregularly introduces a ddATP residue in the place of a dATP residue. As a result, the developing chain of the DNA is terminated. Unsystematic incorporation of ddATP leads to the production of newly produced DNA fragments of different lengths, ending with A (ddA). The length of each fragment matches to the length from the 5' end of the primer to one of the Adenine residues in the sequence.