Send to:

Choose Destination
See comment in PubMed Commons below
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.


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.

[PubMed - indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for HighWire Icon for PubMed Central
    Loading ...
    Write to the Help Desk