Selection of Chloroplasts by Laser Microbeam Microdissection for Single-Chloroplast PCR

• Published in: BioTechniques Vol. 34; no. 6; pp. 1238 - 1243
• Main Authors: Meimberg, H, Thalhammer, S, Brachmann, A, Müller, B, Eichacker, L.A, Heckl, W.M, Heubl, G
• Format: Journal Article
• Language: English
• Published: Natick, MA Future Science Ltd 01.06.2003; Eaton; Taylor & Francis Group
• Subjects: Base Sequence; Biological and medical sciences; Biotechnology; Cell Fractionation - methods; Chloroplasts - genetics; Diverse techniques; DNA Primers - genetics; DNA, Plant - genetics; DNA, Plant - isolation & purification; Fundamental and applied biological sciences. Psychology; Lasers; Molecular and cellular biology; Nicotiana - genetics; Nicotiana - ultrastructure; Polymerase Chain Reaction - methods; Molecular biology; Technique
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/03346rr01

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 . 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 and 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.