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Amplification, Mutation, and Sequencing of a Six-Letter Synthetic Genetic System

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Zunyi Yang†‡, Fei Chen†‡, J. Brian Alvarado†‡, and Steven A. Benner*†‡

Foundation for Applied Molecular Evolution (FfAME), 720 SW Second Avenue, Suite 201, Gainesville, Florida 32601, United States

The Westheimer Institute for Science and Technology (TWIST), 720 SW Second Avenue, Suite 208, Gainesville, Florida 32601, United States

J. Am. Chem. Soc., 2011, 133 (38), pp 15105–15112

DOI: 10.1021/ja204910n

Publication Date (Web): August 15, 2011

§ Author Contributions

These authors contributed equally to this work.

Section:

Biochemical Genetics

Abstract

The next goals in the development of a synthetic biology that uses artificial genetic systems will require chemistry–biology combinations that allow the amplification of DNA containing any number of sequential and nonsequential nonstandard nucleotides. This amplification must ensure that the nonstandard nucleotides are not unidirectionally lost during PCR amplification (unidirectional loss would cause the artificial system to revert to an all-natural genetic system). Further, technology is needed to sequence artificial genetic DNA molecules. The work reported here meets all three of these goals for a six-letter artificially expanded genetic information system (AEGIS) that comprises four standard nucleotides (G, A, C, and T) and two additional nonstandard nucleotides (Z and P). We report polymerases and PCR conditions that amplify a wide range of GACTZP DNA sequences having multiple consecutive unnatural synthetic genetic components with low (0.2% per theoretical cycle) levels of mutation. We demonstrate that residual mutation processes both introduce and remove unnatural nucleotides, allowing the artificial genetic system to evolve as such, rather than revert to a wholly natural system. We then show that mechanisms for these residual mutation processes can be exploited in a strategy to sequence “six-letter” GACTZP DNA. These are all not yet reported for any other synthetic genetic system.

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This article has been cited by 1 ACS Journal articles (1 most recent appear below).

Natural versus Artificial Creation of Base Pairs in DNA: Origin of Nucleobases from the Perspectives of Unnatural Base Pair Studies
Ichiro Hirao, Michiko Kimoto, and Rie Yamashige
Accounts of Chemical Research2012 Article ASAP
- Natural versus Artificial Creation of Base Pairs in DNA: Origin of Nucleobases from the Perspectives of Unnatural Base Pair Studies
  Ichiro Hirao, Michiko Kimoto, and Rie Yamashige
  Accounts of Chemical Research2012 Article ASAP
  Since life began on Earth, the four types of bases (A, G, C, and T(U)) that form two sets of base pairs have remained unchanged as the components of nucleic acids that replicate and transfer genetic information. Throughout evolution, except for the U to T ...
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