Figure 1. Sanger chain termination sequencing method. Comparison of the chemical structures of 2¢-deoxynucloside triphosphate and 2¢, 3¢-dideoxynucloside triphosphate. Note the substitution of the 3¢ hydroxyl group by a hydrogen in the dideoxy form, which prevents extension of the nascent DNA chain 5¢ to 3¢.

Figure 2. Schematic of the chain termination method as implemented with fluorescent detection of the four dideoxynucleotides. See text for further explanation. Because the sequence is read from newly synthesized fragments, it corresponds to the complementary strand of the initial template.

Figure 3. Hierarchical shotgun versus whole-genome shotgun, or top-down versus bottom-up, showing the different features of hierarchical mapping and shotgun sequencing and whole-genome shotgun strategies. In the hierarchical, top-down approach, the genome is progressively broken down into smaller segments. Initially, long-range framework maps were derived by genetic mapping of microsatellites and radiation hybrid mapping of any marker that could be amplified by polymerase chain reaction. Then the genome was organized in overlapping contigs of clones, notably BACs, which were positioned relative to the framework maps. The genome sequence is achieved by shotgun sequencing of single BACs sufficient to cover the entire map. In the whole-genome shotgun approach (bottom-up), the genome is randomly cloned directly into the sequencing vector. Sufficient clones are sequenced to provide five- to tenfold coverage of the entire genome and the genome sequence is assembled in a single step. The use of sequences from both ends of inserts of defined size provides paired reads that assist the assembly and link across gaps.