Figure 2

Diagram of a Sticky RICE cloning reaction into pJL 43. Sticky RICE cloning used a mixture of DNA polymerase and ligase (and, optionally, polynucleotide kinase) with specially designed vector and insert (PCR product) DNAs to directionally ligate DNAs. Single stranded 3 nt, 5' overhangs were generated on pJL 43 by digestion the restriction endonuclease SapI (underlined). Vector was treated with phosphatase after digestion to remove phosphates from 5' ends of DNA. I. Purified PCR product [amplified with 5' phosphorylated primers that began with 5'GGCCWW and 5'GCWW (W = A or T)] was added to SapI cut pJL 43. II. A mixture of T4 DNA polymerase, the nucleotides dATP/dTTP and T4 DNA ligase were added to combined vector and PCR product. During this step the 5' overhangs of SapI cut pJL 43 were altered by the T4 DNA polymerase. A single G residue was removed from the 3' end of the left end of the SapI cut vector, to generate a 5' overhang of GGCC. A single A residue was added to the 3' end of the right end of the SapI cut vector, to generate a 5' overhang of GC. Similarly, the 3' to 5' exonuclease activity of T4 DNA polymerase in the presence of dATP and dTTP removed G or C residues from the 3' ends of the PCR product. Complementary 5' overhangs in vector and PCR product (insert) guided annealing of DNAs. III. Annealed DNAs were joined by T4 DNA ligase. Sequences of the PCR product are in bold type. Vector sequences in final joined product are in all caps. The recognition sequences for the restriction endonucleases StuI (AGGCCT) and Hind III (AAGCTT) were generated at the vector-insert junctions.