A third base pair for the polymerase chain reaction: Inserting isoC and isoG

Abstract

Two additional bases (isoguanosine and isocytosine), generating a third base pair, have been implemented in PCR. Enzyme fidelity for the third base pair is demonstrated using molecular thermo-dynamic melting, chemical cleavage and molecular beacons. When amplifying as few as 15 targets containing multiple non-natural base pairs with 40 cycles of amplification, our results confirm sequence conservation. The additional sequence space provided by three base pairs allows for the construction of molecular tools that achieve higher complexity and better discrimination than those possible with natural DNA alone. © Oxford University Press 2004; all rights reserved.