FIG. 4.

DNA polymerase catalyzed joining of linear molecules sharing different lengths of overlapping end homology. In vitro reactions contained PCR-amplified insert DNA (LucM0 to LucM333, labeled A), cut vector (pRP406/BstEII+PacI, labeled B), vaccinia virus single-strand DNA binding protein, and vaccinia virus DNA polymerase. After a 20-min incubation at 37°C the deproteinized reaction mixture was fractionated on a 0.8% agarose gel and the DNA was visualized by staining with ethidium bromide. Control reaction mixtures omitted the DNA polymerase (lane 2), the vector (lane 3), or the insert (lane 4). Size markers included circular (Form I and II) and linear (Form III) pRP406 species (lanes 17 and 18, respectively) and a 1-kbp DNA ladder. Shown inset are the proposed identities of various molecules.