Simple and efficient synthesis of 5' pre-adenylated DNA using thermostable RNA ligase |
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Authors: | Zhelkovsky Alexander M McReynolds Larry A |
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Institution: | New England Biolabs, Division of RNA Biology, Ipswich, MA 01938-2723, USA. |
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Abstract: | We report a simple method of enzymatic synthesis of pre-adenylated DNA linkers/adapters for next-generation sequencing using thermostable RNA ligase from Methanobacterium thermoautotrophicum (MthRnl). Using RNA ligase for the reaction instead of the existing chemical or T4 DNA ligase-based methods allows quantitative conversion of 5'-phosphorylated single-stranded DNA (ssDNA) to the adenylated form. The MthRnl adenylation reaction is specific for ATP and either ssDNA or RNA. In the presence of Mg(+2), the reaction has a pH optimum of 6.0-6.5. Unlike reactions that use T4 DNA ligase, this protocol does not require synthesis of a template strand for adenylation. The high yield of the reaction simplifies isolation and purification of the adenylated product. Conducting the adenylation reaction at the elevated temperature (65°C) reduces structural constraints, while increased ATP concentrations allow quantitative adenylation of DNA with a 3'-unprotected end. |
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