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Nucleic Acids Res. 2011 Sep 1;39(17):e117. doi: 10.1093/nar/gkr544. Epub 2011 Jun 30.

Simple and efficient synthesis of 5' pre-adenylated DNA using thermostable RNA ligase.

Author information

  • 1New England Biolabs, Division of RNA Biology, Ipswich, MA 01938-2723, USA.

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.

PMID:
21724605
[PubMed - indexed for MEDLINE]
PMCID:
PMC3177227
Free PMC Article

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