Directed evolution of DNA polymerases for next-generation sequencing.

[Image: see text] We present the application of an activity-based phage display method to identify DNA polymerases tailored for next generation sequencing applications. Using this approach, we identify a mutant of Taq DNA polymerase that incorporates the fluorophore-labeled dA, dT, dC, and dG substrates ~50 to 400-fold more efficiently into scarred primers in solution and that also demonstrates significantly improved performance under actual sequencing conditions.

Improvement of DNA adenylation using T4 DNA ligase with a template strand and a strategically mismatched acceptor strand.

DNA with a 5'-adenylpyrophosphoryl cap (5'-adenylated DNA; AppDNA) is an activated form of DNA that is the biochemical intermediate of the reactions catalyzed by DNA ligase, RNA ligase, polynucleotide kinase, and other nucleic acid modifying enzymes. 5'-Adenylated DNA is also useful for in vitro selection experiments. Efficient preparation of 5'-adenylated DNA is therefore desirable for several biochemical applications.