Therefore, we further improved the unnatural base pair system to bypass the need for the .amidotriphosphates, while maintaining the high selectivity (more than 99%) of the unnatural base pairing, in PCR.
The fidelity of unnatural base pairing in the polymerase reaction is determined by both the selectivity of the unnatural base substrates opposite the unnatural pairing partner bases and the misincorporation rate of the unnatural base substrates opposite the natural bases.
(1997) Theoretical and experimental study of isoguanine and isocytosine: Base pairing in an expanded genetic system.
In general, the creation of unnatural base pairs is initiated by designing the molecular structures or modifying the A-T and G-C pairs, based on a certain idea or concept underlying the mechanism of the selective complementarity of the natural base pairings in polymerase reactions.
(40,43,44) For the creation of unnatural base pairs, these factors should also be considered, besides the hydrogen-bonding pattern and shape-complementarity of the base pairings.
For replication studies, the steady-state kinetic parameters ([V.sub.max]/[K.sub.M] or [k.sub.cat]/[K.sub.M]) of cognate and non-cognate base pairings are determined by a conventional single-nucleotide insertion experiment, (47-49) which is useful to assess the enzymatic incorporation efficiency of a substrate, opposite a template base, into a primer strand (see Fig.
Primer extension and PCR amplification in the presence of all substrates of natural and unnatural bases are more practical assessments of unnatural base pairings in replication.