Rapid preparation of megabase plant DNA from nuclei in agarose plugs and microbeads.

Conventional methods for preparation of megabase plant DNA require a protoplast preparation step (1—5). Preparation of protoplasts is tedious and time-consuming. In contrast isolation of nuclei from leaf tissue is simple. Many researchers have attempted to prepare megabase DNA from nuclei (e.g., 3-6) , but no success has been reported. As an alternative approach Guidet and Langridge (6, 7) described a method to prepare megabase DNA by directly embedding ground tissue in agarose plugs. This method, however, is not in general use for large DNA cloning, and cannot be used to prepare DNA in agarose microbeads. Here we describe a simple and rapid method for preparation of megabase DNA by isolating nuclei directly from whole plants or leaf tissue and embedding them in either agarose plugs or microbeads. The microbeads preparation is advantageous for pipetting and enzymatic manipulations (8—10). Plant nuclei were' isolated by grinding whole plants (Arabidopsis) or leaf tissue (rice) in liquid nitrogen, suspending the powder in nuclei isolation buffer and filtering through nylon meshes. We used a nuclei isolation buffer modified from previous reports (11 — 13) and pelleted the nuclei directly in this buffer without resort to percol gradients. For preparing DNA in agarose microbeads, we found that adding cold mineral oil to the agarose-mineral oil emulsion resulted in quick chilling, enhancing microbead size uniformity. The majority of Arabidopsis and rice DNA prepared by this method in agarose plugs or microbeads was larger than 2.5 Mbp, beyond the resolving power of our CHEF gel electrophoresis conditions (Figure 1). This demonstrates that most of the nuclei were maintained intact during the isolation. In contrast, when nuclei were isolated by homogenizing plant tissue in isolation buffer using a blender, a large number of nuclei were broken and no DNA of desired size could be obtained (data not shown). About 200-300 ng DNA could be obtained from 20 g Arabidopsis whole plants or 25 g rice leaves. In comparison to protoplast-based methods, this method is very simple and rapid, requiring only 1.5 h for all manipulations from intact plant tissue through the start of lysis. Since megabase DNA could be isolated reliably from both Arabidopsis and rice, this method should be readily used for other dicot and monocot species. The megabase DNA is amenable to digestion with both frequent and rare cutting restriction enzymes (Figure 2A). Digestion of Arabidopsis DNA with Sail and Smal produced a majority of fragments smaller than 200 kb, while digestion with Notl yielded much larger fragments. This is in agreement with a previous report (1). Hybridization of the digested DNA with DNA probes produced distinct band patterns (Figure 2B). In addition, digestion proceeded more rapidly for DNA prepared in microbeads.