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Nucleic Acids Res. 2005 Sep 28;33(17):5640-6. Print 2005.

The use of thymidine analogs to improve the replication of an extra DNA base pair: a synthetic biological system.

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Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA.


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 approximately 98% in trial PCRs with this six-letter DNA alphabet. The analogous PCR employing TTP had a fidelity-per-round of only approximately 93%. Thus, the A, 2-thioT, G, C, isoC, isoG alphabet is an artificial genetic system capable of Darwinian evolution.

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