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

Nucleic Acids Res. 2004 Mar 29;32(6):1937-41. doi: 10.1093/nar/gkh522. Print 2004.


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 thermodynamic 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.

Publication types

  • Evaluation Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Pairing
  • Cytosine / analogs & derivatives*
  • Cytosine / chemistry*
  • Cytosine / metabolism
  • DNA / biosynthesis
  • DNA / chemistry
  • DNA Primers
  • DNA-Directed DNA Polymerase / metabolism
  • Guanosine / chemistry*
  • Guanosine / metabolism
  • Molecular Probes / chemistry
  • Nucleic Acid Denaturation
  • Polymerase Chain Reaction / methods*


  • DNA Primers
  • Molecular Probes
  • isocytosine
  • Guanosine
  • isoguanosine
  • Cytosine
  • DNA
  • DNA-Directed DNA Polymerase