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

By Alexander M. Zhelkovsky and Larry A. McReynolds

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

Topics: Methods Online
Publisher: Oxford University Press
OAI identifier: oai:pubmedcentral.nih.gov:3177227
Provided by: PubMed Central

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Citations

  1. (2001). An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans.
  2. (2008). Archaeal RNA ligase is a homodimeric protein that catalyzes intramolecular ligation of single-stranded RNA and DNA.
  3. (2002). Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains.
  4. (1977). Bacteriophage T4 RNA ligase. Reaction intermediates and interaction of substrates.
  5. (2005). Cloning of small RNA molecules.
  6. (2009). Efficient chemical synthesis of AppDNA by adenylation of immobilized DNA-50-monophosphate.
  7. (2008). Efficient microRNA capture and bar-coding via enzymatic oligonucleotide adenylation.
  8. (2008). Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing.
  9. (2008). Improvement of DNA adenylation using T4 DNA ligase with a template strand and a strategically mismatched acceptor strand.
  10. (2005). Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties.
  11. (2002). Making AppDNA using T4 DNA ligase.
  12. (2003). Mfold web server for nucleic acid folding and hybridization prediction.
  13. (2007). OligoCalc: an online oligonucleotide properties calculator.
  14. (2004). Practical and general synthesis of 50-adenylated RNA
  15. (1972). Purification and properties of bacteriophage T4-induced RNA ligase.
  16. (2004). Structure and mechanism of RNA ligase.
  17. (2008). The structure of an archaeal homodimeric ligase which has RNA circularization activity.