Isolation, Cloning and Gel Blot Analysis of High Molecular Weight Wheat DNA

High molecular weight (HMW) DNA analysis is required for physical mapping, contiguous map construction and generating large insert libraries in cosmid, yeast artificial chromosome (YAC) or bacterial artificial chromosome (BAC) vectors (6,7). Procedures of HMW DNA isolation and gel-blot analysis using multi-copy probes have been described for wheat (5). Most of the current HMW DNA isolation procedures for wheat or other plants involve protoplast isolation, embedding them in low-melting agarose and lysis in agarose plugs (1,5). The protoplastbased procedures yield good quality DNA, but require expensive cell wall degrading enzymes and expertise in protoplast handling. Guidet et al. (4) described a direct method of HMW DNA isolation that involved embedding powdered leaf tissue in agarose, followed by cell lysis. Recently, a procedure for HMW DNA isolation from nuclei has been described for sorghum that is also suitable for wheat (7). We attempted unsuccessfully to use the previously described methods of HMW DNA isolation for gel-blot DNA hybridization analysis using single-copy probes. In this paper, we standardized or modified HMW DNA analysis methods to make them cost-effective, safer, reproducible and suitable for singlecopy probe analysis. To isolate HMW DNA, young leaves were ground to a fine powder in liquid nitrogen using a mortar and a pestle. The powder was transferred to a tube, kept at 60°C and quickly mixed with 20 mM Tris-HCl, pH 8.0, 30 mM NaCl, 300 mM EDTA, pH 9.0, and 1.5% low-melting agarose (CHEF grade from Bio-Rad, Hercules, CA, USA) at a concentration of 0.5 g leaf tissue/mL. An equal volume of hot mineral oil (50°C) was mixed with the slurry, and the mixture was immediately pipetted into 100–200 mL of ice-cold stirring TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The agarose beads were then allowed to settle, and the TE was replaced with 10 volumes (of beads) of lysis buffer (10 mM TrisHCl, pH 8.0, 500 mM EDTA, pH 9.0, 1% Sarkosyl and 0.1 mg/mL proteinase K) for 36–48 h with one change of the buffer. The beads were washed with three to five changes of TE at 50°C for 2–4 h. The DNA in beads was stored in TE buffer at 4°C for more than one year without any degradation. The HMW DNA contained in the agarose beads was purified as follows: a 1% high-melting-point agarose gel (Bio-Rad) was prepared in 0.5× TBE (45 mM Tris-borate, 1 mM EDTA, pH 8.0). A two-inch-wide piece of gel, including the wells, was cut out and replaced by 1% CHEF-grade low-melting-point agarose (Bio-Rad) solution prepared in 0.5× TBE. A 0.5-cm slot was made across the low-melting agarose gel by using a comb that had Scotch tape on its teeth. The agarose beads containing DNA were loaded in the slot that was then sealed with 1% high-melting-point agarose. The gel was run using a HULA® gel apparatus (Hoefer Pharmacia Biotech, San Francisco, CA, USA) at the following conditions: 0.5× TBE running buffer, for 40 h at 180 V, 60–120-s ramped pulse, 120° angle and 11°C. The gel was stained in ethidium bromide solution, and the HMW DNA band (ca. 0.5 cm wide) was cut out and dialyzed in two to three changes of TE at 50°C for 2–4 h. The gel piece containing the purified DNA was cut into approximately 0.5× 1.0-cm sized pieces (plugs). The purified DNA was stored at 4°C in TE for more than two years without any degradation. For restriction enzyme digestion, each plug containing purified HMW DNA was cut into four to six pieces and incubated in 300 μL of restriction enzyme buffer (plug volume was approximately 80 μL with 30 μL 10× buffer and 190 μL sterile distilled water) on ice for 1–3 h. The buffer was then replaced with 75 μL of enzyme solution (7.5 μL 10× buffer, 30–40 U of enzyme and sterile distilled water to the volume) and incubated at recommended temperature for 8–12 h. For partial digestion, the buffer solution was replaced with 60 μL of enzyme solution