Statistical Analysis of Non-Uniform Volume Distributions for DropLet-based Digital PCR Assays
| Autor: | Bryant S. Fujimoto, Thomas Schneider, Daniel T. Chiu, Jason E. Kreutz, Gloria S. Yen |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
Accuracy and precision
endocrine system Statistics as Topic 010402 general chemistry 01 natural sciences Biochemistry complex mixtures Polymerase Chain Reaction Catalysis Statistical power Article Colloid and Surface Chemistry Lab-On-A-Chip Devices Digital polymerase chain reaction Poisson Distribution Fusion Observational error Dynamic range Chemistry technology industry and agriculture General Chemistry eye diseases 0104 chemical sciences Volume (thermodynamics) Emulsion Emulsions Biological system |
| Popis: | We present a method to determine the concentration of nucleic acids in a sample by partitioning it into droplets with a nonuniform volume distribution. This digital PCR method requires no special equipment for partitioning, unlike other methods that require nearly identical volumes. Droplets are generated by vortexing a sample in an immiscible oil to create an emulsion. PCR is performed, and droplets in the emulsion are imaged. Droplets with one or more copies of a nucleic acid are identified, and the nucleic acid concentration of the sample is determined. Numerical simulations of droplet distributions were used to estimate measurement error and dynamic range and to examine the effects of the total volume of droplets imaged and the shape of the droplet size distribution on measurement accuracy. The ability of the method to resolve 1.5- and 3-fold differences in concentration was assessed by using simulations of statistical power. The method was validated experimentally; droplet shrinkage and fusion during amplification were also assessed experimentally and showed negligible effects on measured concentration. |
| Databáze: | OpenAIRE |
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