Figure 2.

DNA agarose gel (2%) showing process of high fidelity DNA shuffling of 1E2A and wild-type (wt) subtilisin E genes (5). Lanes: 1, AmpliSize DNA Size standards (Bio-Rad), from top to bottom: 2000, 1500, 1000, 700, 500, 400, 300, 200, 100, and 50 bp; 2, 1:1 mixture of 986-bp fragments from wt and 1E2A before DNase I digestion; 3, 1:1 DNA mixture of wt and 1E2A after 2 min of DNase I digestion in the presence of Mn²⁺; 4, fragment reassembly with Pfu polymerase; and 5, PCR amplification of the reassembly product with Taq/Pfu (1:1). Primers 5′-CCGAG CGTTG CATAT GTGGA AG-3′ (underlined sequence is NdeI restriction site) and 5′-CGACT CTAGA GGATC CGATT C-3′ (underlined sequence is BamHI restriction site) were used.