Selection of an appropriate DNA polymerase is critical to the efficiency of the base addition step and must meet several criteria:
* Ability to efficiently incorporate FdNTP at consecutive positions
* Lack of 3–5 exonuclease and proofreading activity to prevent the removal newly incorporated FdNTP
* High fidelity to minimize mis-incorporations
* Good activity on templates which are mounted to surfaces (e.g. optical mapping surface)
In addition, different polymerase preference for different fluorochromes, linker length on fluorochrome-nucleotides, and buffer compositions are also important factors to be considered to optimize the base addition process and maximize number of consecutive FdNTP incorporations.