Reversible Terminator

The blocking group at the nucleotide’s 3′ hydroxyl position prevents DNA polymerase from extending the newly synthesized strand immediately after incorporation. As a result, only one nucleotide is added during each sequencing cycle. This constraint converts DNA synthesis into a stepwise measurement process, allowing the identity of each successive base to be recorded in order.

The blocking group temporarily limits strand extension rather than permanently ending it. After the incorporated nucleotide has been identified, chemical cleavage removes the block and restores the strand’s ability to accept the next nucleotide. This reversible control is essential because it links one incorporation event to one imaging step while preserving the template for continued sequencing.

Each incorporated nucleotide carries a fluorescent label associated with its base identity. Imaging after incorporation detects the resulting fluorescent signal, revealing which nucleotide was added in that cycle. Repeating this observation across successive cycles produces an ordered series of signals, which can be used to determine the corresponding DNA sequence.

A sequencing cycle allows DNA polymerase to incorporate a nucleotide carrying both a fluorescent label and a 3′ blocking group. Imaging then identifies the incorporated base. Chemical cleavage removes the blocking group, preparing the growing strand for another incorporation event. Repetition of these linked steps builds the sequence from successive base calls.

Their one-base-per-cycle control creates a consistent, imageable sequence of incorporation events. Because each cycle yields a fluorescent observation and the block can be removed for continued synthesis, the process can generate ordered sequence information across many sequencing reactions. This supports high-throughput analysis rather than limiting sequencing to a single short measurement.

The resulting sequence information supports genome analysis, mutation detection, and other molecular biology investigations. In a biology research workflow, ordered fluorescent signals can help characterize genetic material and identify sequence differences. These applications depend on accurately connecting each observed signal with the nucleotide incorporated during its corresponding synthesis cycle.