Sanger Gap Sequencing for Genetic Alphabet Expansion of DNA

Sanger Gap Sequencing for Genetic Alphabet Expansion of DNA

ChemBioChem

doi:10.1002/cbic.202000057

http://dx.doi.org/10.1002/cbic.202000057

Michiko Kimoto, Si Hui Gabriella Soh, Ichiro Hirao

unnatural base pair, aptamers, DNA replication, Sanger gap sequencig, sequence determination

23 March 2020

Kimoto, M., Soh, S. H. G., & Hirao, I. (2020). Sanger Gap Sequencing for Genetic Alphabet Expansion of DNA. ChemBioChem, 21(16), 2287–2296. doi:10.1002/cbic.202000057

Genetic alphabet expansion technology, 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) for DNAs containing our UBP, Ds-Px, which appears as gaps in the sequencing peak patterns. By improving the sequencing reaction for efficient Ds-Px pairing and using modified Px substrates, we have developed a sequencing method with increased processivity and clear gap patterns for multiple Ds-Px pairs in various sequence contexts. This method is useful for UBP applications such as high-affinity DNA aptamer generation and semisynthetic 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, thus suggesting further development of UBPs.

Publisher Copyright

This research / project is supported by the A*STAR - RIE2020 Advanced Manufacturing and Engineering Programmatic Grant scheme
Grant Reference no. : A18A9b0060

This research is supported by core funding from: IBN
Grant Reference no. : -

This is the peer reviewed version of the following article: Kimoto, M., Soh, S. H. G., & Hirao, I. (2020). Sanger Gap Sequencing for Genetic Alphabet Expansion of DNA. ChemBioChem, 21(16), 2287–2296. doi:10.1002/cbic.202000057, which has been published in final form at http://dx.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.

https://oar.a-star.edu.sg/communities-collections/articles/17814

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