Preparation of HMW DNA from Plant Nuclei and Chromosomes Isolated from Root Tips |
| |
Authors: | H ?imková J ?íhalíková J Vrána MA Lysák J Dole?el |
| |
Institution: | (1) Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Sokolovská 6, CZ-77200 Olomouc, Czech Republic |
| |
Abstract: | 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.
This revised version was published online in July 2006 with corrections to the Cover Date. |
| |
Keywords: | DNA isolation flow cytometry large-insert DNA library pulsed field gel electrophoresis sucrose gradient |
本文献已被 SpringerLink 等数据库收录! |
|