| 摘要 |
A method is described for the efficient single step directional assembly of two or more DNA fragments into a construct, whereby fragments with complementary ends of at least 15 nucleotides are amplified by PCR or obtained by restriction cleavage, single-stranded overhangs are generated utilizing exonucleolitic activity of Exonuclease III or T4 DNA polymerase or a functional equivalent thereof; after exposure of the overhangs, the fragments are subjected to conditions facilitating annealing of the complementary overhangs, thus creating a single functional construct, and the construct is then directly transformed/transfected into host cells.
|
