Frederick Sanger developed an alternative method, rather than using chemical cleavage reactions, Sanger opted for a method involving a third form of the ribose sugar (Sanger et al. 1977). Ribose has a hydroxyl group on both the 2′ and the 3′ carbons, whereas deoxyribose has only the one hydroxyl group on the 3′ carbon. There is a third form of ribose, dideoxyribose in which the hydroxyl group is missing from both the 2′ and the 3′ carbons (Fig. 2.2). Whenever a dideoxynucleotide incorporated into a polynucleotide, the chain irreversibly stops or terminates. The basic idea behind chain termination method developed in 1974 by Sanger was to generate all possible single-stranded DNA molecules complementary to a template that starts at a common 5′ base and extends up to 1 kilobase in the 3′ direction (Fig. 2.3). These single strands of DNA are labeled in such a way which allows the identity of the 3′-end base in each molecule. These molecules are separated according to size by electrophoresis and each band corresponding to a class of molecule differing in length by one nucleotide from the adjacent band (Fig. 2.4a, b).

The principle of automated DNA sequencing is same as Sanger’s method but the detection is different. In this automated method, the primer or the ddNTPs are labeled by incorporation of a fluorescent dye. Thus, rather than running the gel for a particular time and reading the results, the machine uses a laser to read the fluorescence of the dye as the bands pass a fixed point. Labeling of the ddNTPs is much more advantageous than the primer labeling because four ddNTPs each labeled with different dyes leads the sequencing reaction to run in a single tube and separated in a single lane, thus increasing the capacity of the machine. Automated DNA sequencers can sequence up to 384 DNA samples in a single batch and run up to 24 runs per day. DNA sequencers carry out capillary electrophoresis for size separation, detection and recording of dye fluorescence, and data output as fluorescent peak trace chromatograms. Since the capillary tubes have a high surface to volume ratio (25–100 mm diameter), it radiates heat readily, thus the samples do not over heat. Detection of the migrating molecules is accomplished by shining a light source through a portion of the tubing and detecting the light emitted from the other side (Fig. 2.5). In thermo cycling sequencing the reactions by thermo cycling, cleanup, and re-suspension in a buffer solution before loading onto the sequencer are performed separately. A number of commercial and non-commercial software packages can trim low-quality DNA traces automatically. These programs score the quality of each peak and remove low-quality base peaks (generally located at the ends of the sequence).

The genome sequencing usually deals with large-scale sequencing, e.g., whole chromosomes, very long DNA pieces, etc. For longer targets, such as chromosomes, common approaches consisting of cutting (with restriction enzymes) and shearing (with mechanical forces) the large DNA fragments into shorter DNA fragments are used. The fragmented DNA is cloned into a DNA vector and amplified in E. coli or other suitable organisms. Short DNA fragments purified from individual clones and sequenced individually called shotgun sequencing, followed by electronic assembly into one long contiguous sequence. The overlapping fragments are joined together to form a contig; two or more contigs assembled to make draft sequence. This stage contains gaps in the assembled sequence which can be filled by primer walking and nested deletion strategies. The next stage is the finishing process which involves filling in the gaps and correcting the more obvious errors and uncertainties. The finished sequence does not contain gaps and is accurate to a defined level. The final stage is annotation which identifies the protein coding sequence. The Human genome project was completed by implementing two approaches: clone-by-clone sequencing and whole genome shotgun sequencing.