Figure 1.
Selective rolling circle amplification (RCA) flowchart.
The heteroduplex, ccc-ds(U)DNA formed in Kunkel mutagenesis (A), is treated with UDG (B) and subsequently amplified with RCA (C) using random hexamers as primers. The resulting DNA concatemer is cut to plasmid-sized units (D) and re-circularized by self-ligation (E) for host transformation.
Figure 2.
In the CDR-H1 loop mutagenesis and VH-gene incorporation scFv gene was fused to gene 9-protein (g9p) of the filamentous phage. In the CDR-H3 loop mutagenesis scFv was fused in-frame to β-lactamase (TEM-1). EB104 was the mutagenesis primer in the CDR-H1 and EB120 in CDR-H3 mutagenesis, respectively. The primer pairs WO375-B1 and A1-pAK400rev were used in the analysis of transformants of the CDR-H3 mutagenesis. (ScFv) Single-chain variable fragment, (VL) immunoglobulin variable light domain gene, (VH) immunoglobulin variable heavy domain gene, (PelB) signal sequence for periplasmic excretion and (Lac P/O) Lac promoter.
Table 1.
Diversity based on sequencing in CDR-H1 loop mutagenesis and VH-gene incorporation experiments.
Figure 3.
The effect of UDG treatment on Kunkel and selective rolling circle amplification (RCA) mutagenesis.
CDR-H3 region was altered by Kunkel mutagenesis and further treated with +/− UDG and amplified with RCA. The mutant yield was studied by plating transformed samples. Successful CDR-H3 primer incorporation in scFv-β-lactamase gene resulted in ampicillin resistant clones (pie chart, black sector). The template clones were sensitive to ampicillin (pie chart, white sector). In theory, 22% of the mutated clones with 7× NNN codons are not ampicillin resistant due to primer-born STOP-codons (pie chart, grey sector).
Figure 4.
Digestion of 1 µg mutated phagemid DNA library pools with HindIII and SacII.
(A) Kunkel −UDG, (B) Kunkel + UDG, (C) Kunkel −UDG +RCA, (D) Kunkel +UDG +RCA with exoresistant random primers, (E) Kunkel +UDG +RCA with normal random primers, (−) SacII resistant control, (+) SacII sensitive control, (L) 1 kb Fermentas DNA Ladder.
Table 2.
Colony PCR screen of clones created with primer extension mutagenesis of CDR-H3 loop.
Figure 5.
Colony PCR analysis of the progeny of two primary clones (A.1 and B.1) originating from the Kunkel mutagenesis of CDR-H3 loop.
(I) PCR with wild-type-template- specific primer A1 and pAK400rev. 900 bp product is formed, if template is present. (II) SacII digestion analysis of PCR products (940 bp) primed with WO375 and B1 that hybridize outside the CDR-H3 loop. Both wild-type and mutated DNA is amplified, but only the wild-type DNA is cut to 814 bp and 126 bp fragments. (A.2a–d) Daughter colonies of A.1; (B.2a–d) Daughter colonies of B.1; (−) SacII resistant control, (+) SacII sensitive control, (L) 1 kb Fermentas DNA Ladder bands 1500–500 bp.