Fig. 6. DNA sequence analysis by the chemical synthesis method of Sanger.25 All genetic information is encoded in the sequence of bases of DNA. The method developed by Sanger and colleagues is outlined here. A pure source of template DNA and a pure primer that anneals to the template are required. Synthetic reactions make copies of the template DNA. An amount of dideoxynucleotide is included in each of four separate reactions to terminate the newly synthesized DNA chains. In the figure, the terminators are represented by the Greek letters α, γ, ε, and τ, which represent dideoxyadenosine, dideoxycytosine, dideoxyguanosine, and dideoxythymine, respectively. In each reaction, the ratio of terminator to normal deoxynucleotide is tuned so that for each reaction adequate amounts of terminated chains from 1 to approximately 400 bases in size are synthesized. These chains also are radioactively labeled during this synthesis step. We resolve these chains by running a urea-polyacrylamide gel, which is capable of resolving DNA molecules differing in size by only one base. We load the gel with the four different reactions side by side. After electrophoresis for a few hours, we obtain an image of the radioactive bands by autoradiography. The pattern of bands looks like a ladder. The bands at the bottom are the shortest DNA molecules, and we “read” the DNA sequence by starting at the bottom of the gel and stepping up one band at a time, shifting from one of the four lanes to the next, moving to the immediately higher band. The direction of synthesis is always 5' to 3', and the order from bottom to top of the gel is consequently also 5' to 3'.