Enzymatic assembly of overlapping DNA fragments
DG Gibson - Methods in enzymology, 2011 - Elsevier
Three methods for assembling multiple, overlapping DNA molecules are described. Each
method shares the same basic approach:(i) an exonuclease removes nucleotides from the
ends of double-stranded (ds) DNA molecules, exposing complementary single-stranded (ss)
DNA overhangs that are specifically annealed;(ii) the ssDNA gaps of the joined molecules
are filled in by DNA polymerase, and the nicks are covalently sealed by DNA ligase. The first
method employs the 3′-exonuclease activity of T4 DNA polymerase (T4 pol), Taq DNA …
method shares the same basic approach:(i) an exonuclease removes nucleotides from the
ends of double-stranded (ds) DNA molecules, exposing complementary single-stranded (ss)
DNA overhangs that are specifically annealed;(ii) the ssDNA gaps of the joined molecules
are filled in by DNA polymerase, and the nicks are covalently sealed by DNA ligase. The first
method employs the 3′-exonuclease activity of T4 DNA polymerase (T4 pol), Taq DNA …