Selection of chloroplasts by laser microbeam microdissection for single-chloroplast PCR.
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.
Meimberg
H
H
Department Biologie I, Bereich Biodiversitätsforschung: Systematische Botanik Ludwig-Maximilians-Universität Menzinger Str. 67, 80638 München, Germany. meimberg@botanik.biologie.uni-muenchen.de
Thalhammer
S
S
Brachmann
A
A
Müller
B
B
Eichacker
L A
LA
Heckl
W M
WM
Heubl
G
G
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
D017931:DNA Primers; D018744:DNA, Plant