Sanger Gap Sequencing for Genetic Alphabet Expansion of DNA

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Sanger Gap Sequencing for Genetic Alphabet Expansion of DNA
Title:
Sanger Gap Sequencing for Genetic Alphabet Expansion of DNA
Other Titles:
ChemBioChem
Publication Date:
22 March 2020
Citation:
Kimoto, M., Soh, S.H.G. and Hirao, I. (2020), Sanger Gap Sequencing for Genetic Alphabet Expansion of DNA. ChemBioChem. doi:10.1002/cbic.202000057
Abstract:
Genetic alphabet expansion technology, by creating new replicable and functional DNA molecules with unnatural base pairs (UBPs), is the novel promising research area of xenobiology. Recently, this technology has rapidly advanced, resulting in the need for a sequencing method for DNA molecules containing UBPs. However, all of the conventional sequencing methods, such as Sanger methods, are for four-letter DNA molecules. Here, we present an improved Sanger sequencing method (Sanger gap sequencing method) for DNAs containing our UBP, Ds−Px, which appears as gaps in the sequencing peak patterns. By improving the sequencing reaction for the efficient Ds−Px pairing and using modified Px substrates, we developed a sequencing method with the increased processivity and clear gap patterns for multiple Ds−Px pairs in various sequence contexts. This method is useful for the UBP applications, such as high-affinity DNA aptamer generation and semi-synthetic organism creation involving UBPs. In addition, through this research, we found that the side chains of UBs greatly affect the efficiency of UB pairings in replication, suggesting the further development of UBPs.
License type:
PublisherCopyrights
Funding Info:
This work was supported by the Institute of Bioengineering and Nanotechnology, Biomedical Research Council, Agency for Science, Technology and Research (A*STAR), Singapore and A*STAR under its RIE2020 Advanced Manufacturing and Engineering Programmatic Grant scheme (A18A9b0060) and National Research Foundation of Singapore (NRF-CRP17-2017-07).
Description:
This is the peer reviewed version of the following article: Kimoto, M., Soh, S.H.G. and Hirao, I. (2020), Sanger Gap Sequencing for Genetic Alphabet Expansion of DNA. ChemBioChem. doi:10.1002/cbic.202000057, which has been published in final form at https://doi.org/10.1002/cbic.202000057. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.
ISSN:
1439-4227
1439-7633
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