Figure 2. Construction and Diversification of Routing Gene Populations.

(A) Overlapping complementary oligonucleotides that span an entire gene (for example [Z–a]_1–8 and a_1–8′–Z_2–9′) were assembled into full gene products (“all a,” “all b,” etc.) by primerless PCR and subcloned. Equivalent amounts of the ten resulting plasmids (a_1–8, … , j_1–8) were mixed and used as template for eight separate PCR reactions with noncoding region primer pairs (Z_i/Z_{i +1}′) that flanked a single coding position. The eight degenerate PCR products (Z_n−x_n−Z_{n +1}) were assembled into a library of 10⁸ different genes by primerless PCR (right).

(B) To generate ssDNA, a T7 promoter (pT7) was appended to the 3′ end of the double strand DNA library. The minus strand of the library was transcribed using T7 RNA Polymerase (T7 RNAP), and reverse transcribed from a Z₁ primer using MMLV Reverse Transcriptase (MMLV RT) in a coupled reaction. The resulting DNA/RNA heteroduplex was treated with sodium hydroxide to hydrolyze the RNA, providing ssDNA.