A Compartmentalized Self-Replication Assay Is Developed to Identify Polymerases Capable of Amplifying Difficult Sequence Motifs

Polymerase chain reaction (PCR) – utilized in forensic science, clinical diagnosis, and recombinant DNA technologies – is a ubiquitous molecular biology technique that amplifies a targeted sequence DNA. PCR requires a heat-stable enzyme due to high reaction temperatures. Taq polymerase, naturally found in hot springs, is commonly used in PCR. However, Taq polymerase fails to amplify the G-quadruplexes – found in clinical targets and genome sequencing projects. Optimizing better Taq mutants capable of amplifying G-quadruplexes results in accurate genotyping and fully resolves genomes of interest. Compartmentalized self-replication (CSR) – a simple feedback loop of a polymerase that replicates its own encoding gene – segregates each mutant polymerase into its own droplet reaction within an emulsion, allowing polymerase genes to evolve toward more efficient replication. Droplet digital PCR (ddPCR), a science that exploits droplets – formed in a water-in-oil emulsion and served as individual test tubes, allows creation of multiple subunit reactions of CSR in order to screen for optimal mutants. Fluorescence is used to visualize the amplification of target DNA, showing that a new vector is developed as an assay for improving difficult sequencing polymerases. Besides, a challenging template, based on the G-quadruplex structure, is also identified. Nonetheless, there is a need to modify the promoter to express protein in the plasmids so that a library of Taq mutants can be built to screen and select better mutants using ddPCR.