biologia plantarum

Biologia plantarum 46:369-373, 2003 | DOI: 10.1023/A:1024322001786

Preparation of HMW DNA from Plant Nuclei and Chromosomes Isolated from Root Tips

H. Šimková¹, J. Číhalíková¹, J. Vrána¹, M.A Lysák¹, J. Doležel¹

¹ Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Olomouc, Czech Republic

Simple, fast and cost-effective method for preparation of DNA with high molecular weight (HMW DNA) from plant nuclei and mitotic chromosomes has been developed. The technique involves mechanical homogenization of formaldehyde-fixed root tips, purification of nuclei and/or chromosomes on sucrose gradient, embedding in low-melting-point agarose, and DNA isolation in agarose plugs. Alternatively, nuclei and chromosomes may be purified using flow cytometry. Majority of DNA obtained is megabase-sized and well digestible by restriction endonucleases. The method is highly efficient as microgram amounts of DNA can be obtained from only several milligrams of plant tissue. Handling negligible amounts of plant material reduces the consumption of chemicals. Furthermore, the use of root tips makes it possible to obtain high-quality DNA even from plant species with leaves that are rigid or rich in secondary metabolites such as polyphenols. It is expected that preparation of HMW DNA from root tip nuclei will facilitate long-range mapping and construction of large-insert DNA libraries also in these species. Successful isolation of HMW DNA from flow-sorted chromosomes opens a way for construction of chromosome-specific large-insert libraries in plants.

Keywords: DNA isolation; flow cytometry; large-insert DNA library; pulsed field gel electrophoresis; sucrose gradient

Subjects: banana, root apex, DNA; barley, root apex, DNA; broad bean, root apex, DNA; DNA preparation, library; flow cytometry; Hordeum vulgare; Musa acuminata; root apex, nuclei, chromosomes; Triticum aestivum; Vicia faba; wheat, root apex, DNA

Prepublished online: April 1, 2003; Published: November 1, 2003  Show citation