title: Selection of chloroplasts by laser microbeam microdissection for single-chloroplast PCR.
abstract: Laser microbeam microdissection and laser pressure catapulting offer the possibility of separating cell compartments, thus allowing for contamination-free analysis. Using these methods, we were able to select single chloroplasts of Nicotiana tabacum. Starting from homogenized leaf material, chloroplasts were purified by differential centrifugation and applied directly onto a poly-ethylene-naphthalate membrane that was mounted on a microscope slide. Single chloroplasts were dissected under microscopic control and catapulted into a PCR tube. Subsequent PCR of a spacer region between the trnT and trnF genes verified the successful amplification of DNA from a single chloroplast. The advantage of this method compared to the use of capillaries or optical tweezers is that one is able to prepare high numbers of samples in a short time.
authors.affiliation: Department Biologie I, Bereich Biodiversitätsforschung: Systematische Botanik Ludwig-Maximilians-Universität Menzinger Str. 67, 80638 München, Germany. meimberg@botanik.biologie.uni-muenchen.de
authors.forename: H
authors.initials: H
authors.lastname: Meimberg
authors.forename: S
authors.initials: S
authors.lastname: Thalhammer
authors.forename: A
authors.initials: A
authors.lastname: Brachmann
authors.forename: B
authors.initials: B
authors.lastname: Müller
authors.forename: L A
authors.initials: LA
authors.lastname: Eichacker
authors.forename: W M
authors.initials: WM
authors.lastname: Heckl
authors.forename: G
authors.initials: G
authors.lastname: Heubl
mesh_terms: D001483:Base Sequence; D001709:Biotechnology; D002458:Cell Fractionation / Q000379:methods; D002736:Chloroplasts / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification; D007834:Lasers; D016133:Polymerase Chain Reaction / Q000379:methods*; D014026:Nicotiana / Q000235:genetics / Q000648:ultrastructure
chemical_list: D017931:DNA Primers; D018744:DNA, Plant