Published online 28 September 2005
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The use of thymidine analogs to improve the replication of an extra DNA base pair: a synthetic biological system
1Department of Chemistry, University of Florida Gainesville, FL 32611-7200, USA 2Department of Anatomy and Cell Biology, University of Florida Gainesville, FL 32611-7200, USA
*To whom correspondence should be addressed at Department of Chemistry, University of Florida, PO Box 117200, Gainesville, FL 32611-7200, USA. Tel: +1 352 392 7773; Fax: +1 352 392 7918; Email: benner{at}chem.ufl.edu
Received August 3, 2005. Revised September 12, 2005. Accepted September 12, 2005.
Synthetic biology based on a six-letter genetic alphabet that includes the two non-standard nucleobases isoguanine (isoG) and isocytosine (isoC), as well as the standard A, T, G and C, is known to suffer as a consequence of a minor tautomeric form of isoguanine that pairs with thymine, and therefore leads to infidelity during repeated cycles of the PCR. Reported here is a solution to this problem. The solution replaces thymidine triphosphate by 2-thiothymidine triphosphate (2-thioTTP). Because of the bulk and hydrogen bonding properties of the thione unit in 2-thioT, 2-thioT does not mispair effectively with the minor tautomer of isoG. To test whether this might allow PCR amplification of a six-letter artificially expanded genetic information system, we examined the relative rates of misincorporation of 2-thioTTP and TTP opposite isoG using affinity electrophoresis. The concentrations of isoCTP and 2-thioTTP were optimal to best support PCR amplification using thermostable polymerases of a six-letter alphabet that includes the isoC–isoG pair. The fidelity-per-round of amplification was found to be 
98% in trial PCRs with this six-letter DNA alphabet. The analogous PCR employing TTP had a fidelity-per-round of only 93%. Thus, the A, 2-thioT, G, C, isoC, isoG alphabet is an artificial genetic system capable of Darwinian evolution.
Correspondence may also be addressed to A. Michael Sismour. Tel: +1 352 846 1856; Fax: +1 352 392 7918; Email: msismour{at}chem.ufl.edu
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