Compartmentalized Self‐Replication for Evolution of a DNA Polymerase

Abstract

Compartmentalized self-replication (CSR) is an emulsion PCR-based method for the selection of DNA polymerases. E. coli host cells expressing a library of DNA polymerases are emulsified so that no more than a single cell is present in a single emulsion droplet. In a subsequent emulsion PCR step, the DNA polymerase protein, as well as the plasmid encoding it are released into the emulsion droplet and the genes that created the most active or abundant polymerase variants are exponentially amplified and can be passed to the next round of CSR. CSR is a powerful method for engineering of polymerases since it allows selection under a variety of conditions, including the use of non-standard substrates. In this unit, we provide a step-by-step procedure for the selection of polymerases, using as an example the selection of reverse transcriptase activity starting from a library of Thermococcus kodakaraensis (KOD) DNA polymerase variants. © 2018 by John Wiley & Sons, Inc.

Optimization of Metabolic Labeling with Alkyne-Containing Isoprenoid Probes

“…We have previously demonstrated that isoprenoid analogues (Fig. 1) C15AlkOP and C15AlkOPP served as good substrates for metabolic labeling in COS-7 cells whereas C15AlkOH was incorporated with much lower efficiency[22]. In that study, we showed how labeling efficiency can be optimized by varying the probe concentration.…”

Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase

“…Furthermore, several polymerase mutants were produced by adding site-directed mutagenesis into the exonuclease active site and C-terminal-conserved regions, which enhanced the incorporation of ribonucleotides and unnatural nucleotides (Blasco et al, 1992;Bonnin et al, 1999;Torres and Pinheiro, 2018). On the other hand, evolutionary methods are used for DNA polymerase engineering through the method of compartmentalized selfreplication (CSR) (Ghadessy et al, 2001;Ghadessy and Holliger, 2007;Abil and Ellington, 2018;Sakatani et al, 2019). For reaction conditions optimization, host DNA and environmental DNA may lead to false positive results in the amplification reaction, as phi29 DNA polymerase has high affinity for both single-strand and doublestranded DNA (Blanco et al, 1989).…”

Modified nucleic acids: replication, evolution, and next-generation therapeutics

“…A range of directed evolution strategies have been employed in the field. Compartmentalized self-replication (CSR) [ 139 – 141 ] challenges polymerases to PCR amplify their own gene inside an emulsion compartment. The exponential enrichment of highly active clones, as opposed to simple partitioning of active variants, makes CSR a powerful method.…”