title: Simple and efficient synthesis of 5' pre-adenylated DNA using thermostable RNA ligase.
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.
authors: New England Biolabs, Division of RNA Biology, Ipswich, MA 01938-2723, USA.
authors: Alexander M
authors: AM
authors: Zhelkovsky
authors: Larry A
authors: LA
authors: McReynolds
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D017014:Methanobacterium / Q000201:enzymology; D011130:RNA Ligase (ATP) / Q000378:metabolism*; D013696:Temperature
chemical_list: D004277:DNA, Single-Stranded; D000255:Adenosine Triphosphate; D011130:RNA Ligase (ATP)