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Simple Bulk Readout of Digital Nucleic Acid Quantification Assays
Published on: September 24, 2015
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Microfluidics-based in situ padlock/rolling circle amplification system for counting single DNA molecules in a cell
Arisa Kuroda1, Yuri Ishigaki, Mats Nilsson
1Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University.
Summary
We created a simplified, automated microfluidic system for in situ padlock/rolling circle amplification (RCA) to count DNA fragments in cells. This automated method reduces reagent volume and costs while maintaining high product yield.
Area of Science:
- Biotechnology
- Molecular Biology
- Microfluidics
Background:
- In situ padlock/rolling circle amplification (RCA) is a technique for amplifying, visualizing, and quantifying DNA within cells.
- The conventional manual RCA method involves multiple steps, making it expensive and complex.
Purpose of the Study:
- To develop a novel, simplified, and automated microfluidic system for in situ RCA.
- To demonstrate the system's effectiveness in quantifying amplified mitochondrial DNA fragments in HeLa cells.
Main Methods:
- Optimization of reaction solution volumes and washing buffer composition for the microfluidic system.
- Application of the automated microfluidic RCA system to count mitochondrial DNA fragments in HeLa cells.
Main Results:
- The automated microfluidic RCA system achieved product yield comparable to the conventional manual method.
- Reagent volume was reduced to 10 μL, less than half of the conventional method's requirement.
- Successfully demonstrated the counting of amplified mitochondrial DNA fragments in HeLa cells.
Conclusions:
- The developed automated microfluidic system offers a simplified and cost-effective approach to in situ RCA.
- This novel method represents the first automated microfluidic system for in situ padlock/RCA.
- The system holds potential for improving the efficiency of pathological diagnoses.
