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A high-throughput method for detection of DNA in chloroplasts using flow cytometry.
Beth A Rowan1, Delene J Oldenburg, Arnold J Bendich
1Department of Biology, University of Washington, Seattle, WA 98195, USA. rowanb@u.washington.edu
Plant Methods
|March 27, 2007
Summary
A new flow cytometry method using SYTO 42 effectively quantifies chloroplast DNA (cpDNA) content changes during plant development. This high-throughput technique aids in studying cpDNA reduction in leaves.
Area of Science:
- Plant Molecular Biology
- Cell Biology
- Genetics
Background:
- Chloroplast DNA (cpDNA) content declines significantly during leaf development in some plant species.
- Investigating this cpDNA decline requires a high-throughput method for analyzing numerous tissue samples.
Purpose of the Study:
- To evaluate DNA-binding fluorophores for flow cytometric analysis of cpDNA content in Arabidopsis chloroplasts.
- To establish a high-throughput method for detecting cpDNA quantity changes during development.
Main Methods:
- Flow cytometry was used to analyze cpDNA content in Arabidopsis chloroplasts.
- Four DNA-binding fluorophores (DAPI, SYBR Green I, SYTO 42, SYTO 45) were tested.
- Results were validated using fluorescence microscopy and quantitative PCR (qPCR).
Main Results:
- SYTO 42 and SYBR Green I enabled flow cytometric detection of cpDNA content changes during development.
- SYTO 42 offered more sensitive detection, correlating well with qPCR data.
- qPCR analysis indicated a whole chloroplast genome copy number reduction, not selective degradation.
Conclusions:
- Flow cytometry with SYTO 42 is a suitable high-throughput method for assessing cpDNA content changes during plant development.
- This method can be used to sort chloroplasts based on their DNA content.
