Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

Abstract

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.

Development of a specific affinity-matured exosite inhibitor to MT1-MMP that efficiently inhibits tumor cell invasionin vitroand metastasisin vivo

“…Since highly diverse CDR3 loops have been reported as the key determinant of specificity in antigen recognition [ 45 ], we carried out affinity selections using our most potent first generation scFv clones E3 and G1. New VH and VL libraries were constructed via randomizing blocks of five to six amino acid residues in the CDR3 loops [ 35 , 46 ]. Using affinity selections with steadily decreasing antigen levels in addition to off-rate selection with a large excess of non-biotinylated antigen, antibodies with changes in the CDR3 loops that decreased the dissociation rate and increased overall binding strength were favoured.…”

Uracil DNA Glycosylase Counteracts APOBEC3G-Induced Hypermutation of Hepatitis B Viral Genomes: Excision Repair of Covalently Closed Circular DNA

“…We followed their method with minor modifications. In brief, DHBV cccDNA purified by Hirt extraction (DpnI treated) was mixed with 8 DHBV-specific primers (Table S2); denatured at 95°C for 3 min; cooled sequentially at 50°C for 15 s, 37°C for 15 s, and room temperature; and reacted with the phi29 DNA polymerase and buffers (New England Biolabs) at 37°C for 16 h. Note that the DNA polymerase (phi-29 DNA pol) used in the RCA reaction can polymerize DNA even with a uracil-containing DNA template [70]. The RCA concatemerized product was converted to the monomeric full-length DHBV genome by digestion with EcoRI, where the DHBV sequence contains a single site, and was cloned into the replicon vector backbone at the EcoRI site; thus, the full-length DHBV genome in the original vector was replaced with the corresponding fragment from the purified cccDNA.…”

Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step

“…We have similar finding in this study and it suggests that additional selection pressure, such as heat treatment, can be applied to mutagenic pools to select clones that not only have higher affinities, but also remain thermostable. Third, the sizes of secondary libraries constructed in this study are limited by the low recombination rate with the Kunkel mutagenesis, and thus one might increase the frequency of recombinant clones in the secondary library by restriction enzyme digestion [ 63 ] or by rolling circle amplification [ 64 ].…”