5.08: DNA Sequencing by the Dideoxy Method

Three stacked panels compare thymidine nucleotide analogs, each drawn as a five-membered deoxyribose sugar ring with an oxygen at top, carbons numbered 1' through 5', and a thymine base attached at the 1' carbon. Top panel, Deoxythymidine triphosphate (dTTP): a chain of three phosphate groups (P???P???P) is attached to the 5' carbon at the upper left. The 3' carbon bears an OH group; the 2' carbon bears an H. Middle panel, Dideoxythymidine triphosphate (ddTTP): identical to dTTP except the 3' carbon bears an H instead of OH; a red arrow points to this H, highlighting the missing hydroxyl that terminates DNA chain elongation. Bottom panel, Azidodideoxythymidine (AZT): the 5' position carries a hydroxyl (HO???) rather than a triphosphate chain, and the 3' carbon bears an azido group (N3) in place of OH. The 2' carbon again bears H, and thymine is attached at 1'. The key contrast is the 3' substituent: OH in the normal nucleotide, H in the dideoxy analog, and N3 in AZT.

The DNA to be sequenced is prepared as a single strand. This template DNA is supplied with

• a mixture of all four normal (deoxy) nucleotides in ample quantities
  • dATP
  • dGTP
  • dCTP
  • dTTP
• a mixture of all four dideoxynucleotides, each present in limiting quantities and each labeled with a "tag" that fluoresces a different color:
  • ddATP
  • ddGTP
  • ddCTP
  • ddTTP
• DNA polymerase I