Protection of megabase-sized chromosomal DNA from breakage by DNase activity in plant nuclei.

A prerequisite for constructing a physical map that spans an entire plant chromosome is isolation of intact chromosome-sized DNA. Two methods are commonly used to isolate megabase (Mb)-sized DNA from plant materials, i.e., from protoplasts (1) or from nuclei (3,5). Isolated protoplasts or nuclei are embedded in low-melting-point agarose to form either plugs or microbeads. DNA molecules are isolated and manipulated in situ to protect them from physical shearing. Rice and Arabidopsis DNA molecules isolated by these methods have been used for the construction of yeast artificial chromosome (YAC) or bacterial artificial chromosome (BAC) libraries (3,5). However, most DNA molecules that migrated into gels were less than 2.5 Mb in size. Furthermore, these previous studies did not show whether or not the chromosomal DNA that remained in the sample well was intact in the presence of a buffer that contained magnesium ions but without added restriction enzymes. We have used both the protoplast method (1) and the nuclei method (3,5) in attempts to isolate intact chromosomal DNA molecules from Arabidopsis, rice, corn and tomato plants, but we found large amounts of broken DNAs, presumably due to the presence of endogenous DNase activity. To find conditions by which this DNase activity can be inhibited while preserving the activity of several common restriction enzymes used in the manipulation of chromosome-sized DNA, we tested several potential DNase inhibitors and different combinations of two inhibitors. We found that the combination of 160 mM L-lysine HCl plus 4 mM EGTA fulfills both requirements. Figure 1 shows the result of a Southern blot hybridization analysis of Arabidopsis chromosomal DNA embedded in agarose plugs after pulsed-field gel electrophoresis (PFGE) (4) using a CHEF Mapper System (Bio-Rad, Hercules, CA, USA). Arabidopsis nuclei were isolated, embedded and treated according to a commonly used method (5). Briefly, our nuclei isolation method was based on the published procedures (3,5) but modified as follows: 20 g of leaves were ground into fine powder in liquid nitrogen, which was transferred into ice-cold wash buffer [homogenization buffer (HB; 10 mM Tris-HCl, 80 mM KCl, 10 mM EDTA, 1 mM spermidine, pH 9.4, 0.5 M sucrose) plus 0.5% Triton X-100 and 0.15% β-mercaptoethanol], mixed well and filtered through cheesecloth and Miracloth (Calbiochem-Novabiochem, La Jolla, CA, USA) and centrifuged at 1800× g for 25 min. After washing in wash buffer, the pellet was resuspended in 400 μL of HB and mixed with 450 μL of 1% low-melting-point agarose in HB at 42°C. The mixture was made into 10 plugs (each containing 85 μL). The plugs were incubated in lysis buffer (0.5 M EDTA, pH 9.0, 1% sodium lauryl sarcosine, 0.1 mg/mL proteinase K) at 55°C for 24 h, followed by treatment with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) plus 0.1 mM phenylmethylsulfonyl fluoride (PMSF) for 1 h, three times. The plugs were finally kept in 0.5 M EDTA, pH 8.0, at 4°C. PFGE was carried out using 0.6% SeaKem Gold Agarose Gel (FMC BioProducts, Rockland, ME, USA) in 0.5× TBE (0.045 M Tris-boric acid, 0.001 M EDTA, pH 8.0) at 14°C. The pre-run PFGE conditions were 2.2 V/cm with switch time 1.5 to 22.5 min over 10 h. The resolution PFGE conditions were 1.5 V/cm with switch time 15 to 90 min over 48 h. The probe was 32P-labeled Arabidopsis ribosomal DNA (rDNA). The PFGE pre-run described was done to remove the chloroplast and mitochondrial DNA and any broken nuclear DNA. After the pre-run PFGE, the agarose plugs were removed from the wells, equilibrated in appropriate buffers and incubated at 37°C overnight under one of several different conditions, which include: Mg++-containing REACT 4 buffer (Life Technologies, Gaithersburg, MD, USA) plus KpnI (Figure 1, lane 3), REACT 4 buffer only (lane 4) and TE only (lane 5). Following this incubation, each agarose plug was again subjected to PFGE but for a longer time for resolution of DNA molecules. The gel was stained with ethidium bromide, exposed to UV light, and the DNA fragments were transferred onto a Nytran Membrane (Schleicher & Schuell, Keene, NH, USA) and hybridized with 32P-labeled Arabidopsis rDNA (rDNA, including intergenic