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Published online 29 March 2004

Nucleic Acids Research, 2004, Vol. 32, No. 6 1937-1941
© 2004 Oxford University Press

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

Scott C. Johnson, Christopher B. Sherrill, David J. Marshall, Michael J. Moser and James R. Prudent*

Research Department, EraGen Biosciences Inc., 918 Deming Way, Madison, WI 53717, USA

*To whom correspondence should be addressed. Tel: +1 608 662 9000; Fax: +1 608 662 9004; Email: jprudent{at}eragen.com
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors

Received January 22, 2004; Revised and Accepted March 10, 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.


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