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Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation
Published on: December 7, 2013
Nanoliter reactors improve multiple displacement amplification of genomes from single cells
Yann Marcy1, Thomas Ishoey, Roger S Lasken
1Department of Bioengineering, Stanford University, Stanford, California, USA.
Plos Genetics
|September 26, 2007
Summary
Genomic sequencing from single bacterial cells is now possible using reduced-volume Multiple Displacement Amplification (MDA). This method improves DNA amplification quality and reduces bias, enabling high-throughput sequencing without cell culturing.
Area of Science:
- Microbiology
- Genomics
- Biotechnology
Background:
- Many environmental bacteria cannot be cultured in the lab, hindering genomic study.
- Genomic sequencing from single cells bypasses the need for culturing methods.
Purpose of the Study:
- To investigate the efficacy of reduced-volume Multiple Displacement Amplification (MDA) for single-cell genomic DNA amplification.
- To assess the impact of nanoliter reaction volumes on amplification quality and bias.
Main Methods:
- Individual Escherichia coli cells were isolated using a microfluidic device.
- Genomic DNA was amplified using MDA in 60-nl reactions.
- Amplification quality was assessed by qPCR and compared to standard 50-microl reactions.
Main Results:
- Reduced MDA reaction volumes (60-nl) lowered nonspecific DNA synthesis.
- Amplification bias was significantly reduced in nanoliter volumes, improving sequence representation.
- Single-cell amplicons from both microliter and nanoliter volumes yielded high-quality pyrosequencing data.
Conclusions:
- Microfluidic-based, reduced-volume MDA is a viable method for single-cell genome amplification.
- This approach offers a straightforward and efficient route to sequencing genomes directly from single bacterial cells.
