![]() Step 7: The sequences of the enriched DNA templates are analyzed after several rounds of screening. Step 6: The template DNA on these selected microbeads are amplified to generate a new DNA pool with a higher ratio of the positive clone, and then subjected to another round of screening. A polymerase chain reaction (PCR) in water droplets with water-in-oil emulsion (emulsion PCR) facilitates parallel amplification of a single-molecule. The microbeads with the peroxidase mutant of interest and the corresponding DNA are separated according to the relative strengths of their fluorescent intensities. Step 5: After the fluorogenic assay, the microbeads are subjected to a selection process by using FACS (panel F). Emulsion PCR (EmPCR) is a commonly employed method for template amplification in multiple NGS-based sequencing platforms. Then, streptavidin-Cy5 is used to obtain a fluorescent signal from the biotin. During this assay, the peroxidase catalyzes the conversion of the biotin-labeled tyramide to the short-lived tyramide radical that forms a covalent bond with a nearby tyrosine or tryptophan on the HRP surface. Step 4: The enzyme-DNA library on the microbeads is recovered from the emulsion, followed by a tyramide-based fluorogenic assay (panel E POI: protein of interest). Alginate Microbeads are one of the most widely investigated cell encapsulation materials as they are biocompatible. In this way, the linkage of the genotype and phenotype on the same microbead is achieved. Emulsion PCR (ePCR) is an important technique that permits amplification of DNA molecules in physically separated picoliter-volume water-in-oil droplets, and thus avoids formation of unproductive chimeras and other artifacts between similar DNA sequences. From the template DNA on the microbead, the peroxidase is synthesized by CFPS in emulsion and immobilized on the same microbead via the scCro-DNA interaction inside the droplets (panel D). 'Solid-phase' emulsion PCR, using microbeads. Step 3: The recovered microbeads are diluted with the CFPS mixture to less than one microbead per droplet after emulsification. Emulsion PCR has been used for the directed evolution of DNA polymerases 4,5, single-molecule reverse-transcription PCR 6 and haplotyping 7. Step 2: The DNA library on the microbeads (panel C) is recovered via emulsion disruption. The template DNA is amplified on the microbead using emulsion PCR (panel B). Beads coated with primers and a DNA library are dispersed into droplets together with the second free primer. Step 1: A DNA pool (panel A) is diluted with the PCR mixture containing the primer/scaffold hairpin-immobilized microbeads to less than one template DNA per droplet after emulsification. Schematic of emulsion PCR (ePCR) on microbeads. Emulsion polymerase chain reaction (ePCR) enables parallel amplification of millions of different DNA molecules while avoiding bias and chimeric byproducts, essential criteria for applications including next generation sequencing, aptamer selection, and protein-DNA interaction studies.
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