Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material

Diversity based on sequencing in CDR-H1 loop mutagenesis and VH-gene incorporation experiments.

<p>K: Kunkel, U: UDG and R: RCA.</p>a<p>AatII digestion was used to eliminate unmutated template clones. Before AatII digestion 7/10 were template clones according to restriction enzyme analysis.</p

Colony PCR screen of clones created with primer extension mutagenesis of CDR-H3 loop.

<p>Analysis I: colonies from cm-plates (all grow) amplified with WO375 & B1 (all amplified) and digested with SacII. Analysis II: colonies from cm & amp-plates (mutants grow) amplified with A1 & pAK400rev (wild type specific). K: Kunkel, U: UDG, R: RCA, cm: chloramphenicol and amp: ampicillin.</p

The effect of UDG treatment on Kunkel and selective rolling circle amplification (RCA) mutagenesis.

<p>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).</p

Selective rolling circle amplification (RCA) flowchart.

<p>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.</p

Target of mutagenesis.

<p>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.</p

Digestion of 1 µg mutated phagemid DNA library pools with HindIII and SacII.

<p>(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.</p