Figure 8.

Construction of the second-generation library of binary patterned sequences. α-Helices were elongated by insertion of combinatorially diverse sequences into the gene encoding sequence n86. In step 1, PCR primers were annealed to DNA encoding the C-terminal section of α-helix 1 and the N-terminal section of α-helix 2. New sequence was inserted (shaded regions) by PCR amplification of the entire plasmid. Following PCR, plasmids were recircularized by blunt end ligation. The combinatorially diverse product of step 1 was then used as the PCR template for step 2, and PCR primers were annealed to DNA encoding the end of α-helix 3 and the beginning of α-helix 4. Again, new sequence was inserted (shaded regions) by PCR amplification of the entire plasmid, and plasmids were recircularized by blunt-end ligation.