Selection of chloroplasts by laser microbeam microdissection for single-chloroplast PCR
Meimberg, H.; Thalhammer, S.; Brachmann, A.; Müller, B.; Eichacker, L.A.; Heckl, W.M.; Heubl, G.
Biotechniques 34(6): 1238-1243
2003
ISSN/ISBN: 0736-6205 PMID: 12813891 Document Number: 565582
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 and Taraxacum officinale. Starting from homogenized leaf material, chloroplasts were purified by differential centrifugation and applied directly onto a polyethylene-naphthalate membrane that was mounted on a microscope slide. Single chloroplasts were laser dissected under microscopic control and catapulted into a PCR tube using a single laser shot. 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 reliably in a short time.