title: Isolation, quantification, and analysis of chloroplast DNA.
abstract: Many areas of chloroplast research require methods that can assess the quality and quantity of chloroplast DNA (cpDNA). The study of chloroplast functions that depend on the proper maintenance and expression of the chloroplast genome, understanding cpDNA replication and repair, and the development of technologies for chloroplast transformation are just some of the disciplines that require the isolation of high-quality cpDNA. Arabidopsis thaliana offers several advantages for studying these processes because of the sizeable collection of mutants and natural varieties (accessions) available from stock centers and a broad community of researchers that has developed many other genetic resources. Several approaches for the isolation and quantification of cpDNA have been developed, but little consideration has been given to the strengths and weaknesses and the type of information obtained by each method, especially with respect to A. thaliana. Here, we provide protocols for obtaining high-quality cpDNA for PCR and other applications, and we evaluate several different isolation and analytical methods in order to build a robust framework for the study of cpDNA with this model organism.
authors.affiliation: Department of Biology, University of Washington, Seattle, WA, USA.
authors.forename: Beth A
authors.initials: BA
authors.lastname: Rowan
authors.forename: Arnold J
authors.initials: AJ
authors.lastname: Bendich
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D017360:Arabidopsis / Q000378:metabolism*; D002467:Cell Nucleus / Q000235:genetics; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000378:metabolism; D002736:Chloroplasts / Q000235:genetics*; D018742:DNA, Chloroplast / Q000032:analysis* / Q000302:isolation & purification*; D016521:Electrophoresis, Gel, Pulsed-Field; D004996:Ethidium / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008967:Molecular Biology / Q000379:methods*; D009693:Nucleic Acid Hybridization; D060888:Real-Time Polymerase Chain Reaction; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013194:Staining and Labeling
chemical_list: D002593:Cetrimonium Compounds; D018742:DNA, Chloroplast; D005456:Fluorescent Dyes; D007211:Indoles; C007293:DAPI; D000469:Alkaline Phosphatase; D004996:Ethidium; D000077286:Cetrimonium