DNA sequencing is used to determine the exact order of bases in DNA. The procedure uses DNA synthesis to produce copies of the target sequence much the same as in PCR. However, instead of synthesizing multiple copies of the complete sequence, some of the copies are forced to terminate before reaching the end. In fact, the conditions are set such that a percentage of the copies ends at each of the base positions in the sequence. This chain termination method is accomplished by adding a small proportion of dideoxynucleotides (ddNTPs) to the standard DNA synthesis reaction, which contains deoxynucleotides (dNTPs). Dideoxynucleotides are chemically modified at one end so they can be added to a new chain, but nothing can be added to them. Thus, the replication process stops whenever one of these bases is added. The resulting copies, varying by only one base in length, are separated by electrophoresis on a polyacrylimide gel. When processed to visualize the copies, a ladder-like pattern is seen, where each wrung of the ladder represents a DNA copy that differs from it's neighbors by one base .

The process of DNA Sequencing:
Double-stranded DNA is dissociated and a radioactive or fluorescent primer is annealed to the DNA (Figure 8-A); four separate reactions are performed to synthesize new DNA, each reaction contains all four deoxynucleotides and a small portion of one of the dideoxynucleotide bases (8-B); DNA is synthesized, terminating each time a ddNTP is incorporated (8-C); DNA from all four reactions is separated on a gel in side-by-side lanes to produce a sequence ladder (8-D); the sequence is read from the bottom up, and is the compliment (opposite) of the base identified in the gel.

As an alternative, automated sequencing machines use ddNTPs that are each tagged with a different color fluorescent dye. Instead of performing four different reactions, DNA synthesis occurs in one tube. The sequencing machine then uses a light sensor to read the gel, identifying the bases by their different colors.