Expansion of the genetic alphabet by an unnatural base pair system provides a platform for the site-specific, enzymatic incorporation of extra, functional components into nucleic acids. Recently, several unnatural base pairs that exhibit high fidelity and efficiency in PCR have been developed. Functional groups of interest, such as fluorescent dyes, can be linked to the unnatural bases, and the modified base substrates are site-specifically incorporated into nucleic acids by polymerases. Furthermore, unique unnatural base pairs between fluorophore and quencher base analogs have been developed for imaging PCR amplification and as molecular beacons. Here, we describe the recent progress in the development of unnatural base pairs that function in PCR amplification and their applications as sensing and diagnostic tools.
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