| 摘要 |
<p>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 expose of the overhangs, the fragments are subjected to conditions facilitating annealing of the complementary overhangs, thus creating a single functional construct, then, the gaps which may have been created by the prior action of the exonuclease treatment, in the adjoining regions of the said construct are filled-in by T4 DNA polymerase or T7 DNA polymerase or by a functional equivalent thereof, and the construct is transformed and/or transfected into host cells with or without a prior ligation step.</p> |
