ConstructionofBACLibraries：MegabaseDNAIsolation

Megabase DNA Isolation

Megabase-size DNA isolation from plants

To construct large insert DNA libraries in BAC and YAC vectors, methods must be developed to isolate very high molecular weight DNA - megabase-size DNA - from plants. To isolate such DNA, protoplasts or nuclei must first be embedded in agarose plugs or microbeads. The agarose acts as a solid porous matrix which allows for the diffusion of various reagents for DNA purification and subsequent manipulations while preventing the DNA from being sheared (Schwartz and Cantor, 1984). Microbeads are preferred over plugs because the use of beads increases the surface area surrounding the tissue sample by approximately 1000 folds, thereby allowing for more efficient and rapid diffusion of chemicals and enzymes into and out of the agarose beads (Cook, 1984, Overhauser and Radic, 1987, Wing et al, 1993). Once embedded, the protoplasts, or nuclei are lysed and proteins degraded in the presence of 0.5 M EDTA, 1% sarcosyl, and 0.1-1.0 mg/ml of proteinase-K at 50°C (Schwartz and Cantor, 1984). After cell lysis and protein degradation, the remaining DNA is suitable for enzymatic modifications.

Most protocols for the isolation of megabase-size DNA from plants utilize the protoplast method (Cheung and Gale, 1990, Ganal, et al, 1989, Honeycutt, et al, 1992, Sobral, et al, 1990, van Daelen, et al, 1989, Wing, et al, 1993, Woo et al,1995). Although the protoplast method yields megabase-size DNA of high quality, the process is costly and labor intensive. For example, to prepare protoplasts from tomato, young leaves are manually feathered with a razor blade before being incubated for 4-5 hours with cell wall degrading enzymes (Ganal and Tanksley, 1989). With sorghum, Woo et al (1995) found the best way to generate high yields of protoplasts for megabase-size DNA isolation is to rub carborundum on both sides of the leaves with a paintbrush, 50 strokes/side, before a 4-5 hour incubation with cellulysin. Thus the amount of time before embedding in agarose can be between 7-9 hours, depending on the amount of leaf material being processed. Furthermore, since each plant species requires a different set of conditions to generate protoplasts the method will only work if a high yielding protoplast method has been developed for a given plant species.

Some investigators have tried with varying degrees of success to prepare megabase-size DNA from nuclei (Hatano, et al, 1992). Zhang et al (1995) have recently developed a nuclei method that works well for several divergent plant taxa. Fresh or frozen tissue is homogenized with a blender or mortar and pestle, respectively. Nuclei are then isolated and embedded as above. The quality of the DNA is as good as DNA prepared from protoplasts, is often more concentrated, and was shown to contain lower amounts of chloroplast DNA. The primary advantage of method is that it is economical and not as labor intensive as the protoplast method. The amount of time required to isolate nuclei and embed in agarose routinely takes less than two hours.

Preparation of Megabase-size DNA from Plant Nuclei (from Zhang et al. 1995)

A. Plant materials

• Plant leaves or whole plants of divergent species, including grasses, legumes, vegetables, and trees can be used as materials for preparation of megabase-sized DNA by this method. The tissue can be either frozen in liquid nitrogen and stored in a -70°C freezer or kept fresh on ice before use.

B. Reagents

• 10 x homogenization buffer (HB) stock: 0.1 M Trizma base, 0.8 M KCl, 0.1 M EDTA, 10 mM spermidine, 10 mM spermine, final pH 9.4-9.5 adjusted with NaOH. The stock is stored at 4°C.

• 1 x HB: A suitable amount of sucrose is mixed with a suitable volume of 10x HB stock. The final concentration of sucrose is 0.5 M and HB stock is 1x. The resultant 1 x HB is stored at 4°C. Before use, b-mercaptoethanol is added to 0.15%.

• 20% Triton X-l00 in 1 x HB: Triton X-100 is mixed with 1 x HB without b-mercaptoethanol to 20%. The solution is stored at 4°C.

• Wash Buffer (1 x HB plus 0.5% Triton X-100): Prepared by mixing 1 x HB without b-mercaptoethanol with Triton X-l00 and stored at 4°C. Before use, b-mercaptoethanol is added to 0.15%.

• Lysis buffer: 0.5 M EDTA; pH 9.0-9.3, 1% sodium lauryl sarcosine, and 0.1 mg/ml proteinase K. The proteinase K powder is added just before use.

C. Preparation of intact nuclei

• For homogenization of the plant tissue, two methods can be used.

1A. Homogenization of Fresh Tissues

• a. Wash about 15 g of fresh tissue with tap water, and if necessary, cut into suitable pieces for homogenization with a kitchen blender (Osterizer 10 Speed Blender).

• b. Homogenize the tissue in 200 ml ice cold 1 x HB plus b-mercaptoethanol in the kitchen blender at speed 4 or "puree" for 10 to 30 seconds.

• c. Filter the homogenate into an ice cold 250 ml centrifuge bottle through two layers of cheesecloth and one layer of miracloth. Retrieve the remaining nuclei by squeezing the homogenate with gloved hands.

• d. Add 5 ml 1 x HB plus 20% Triton X-l00 (the final concentration of Triton X-l00 is 0.5%) to each bottle (200 ml 1 x HB buffer), gently mix the contents, and incubate on ice for 20 minutes.

1B. Homogenization of Frozen Tissues

• a. Grind about 15 g of the frozen tissue into a powder in liquid nitrogen with a mortar and pestle and immediately transfer into an ice cold 500 ml beaker containing 200 ml ice-cold 1 x HB plus b-mercaptoethanol and 0.5% Triton X-l00.

• b. Gently swirl the contents with a magnetic stir bar for 10 minutes on ice and filter into an ice-cold 250 ml centrifuge bottle as above.

2. Pellet the homogenate prepared by either of the above two methods by centrifugation with a fixed-angle rotor at 1,800 g at 4°C for 20 minutes.

3. Discard the supernatant fluid and add approximately 1 ml of ice cold wash buffer.

4. Gently resuspend the pellet with assistance of a small paint brush soaked in ice cold wash buffer. After the pellet is resuspended, add an additional 30 ml of ice cold wash buffer.

5. To remove the particulate matter remaining in the suspension, filter the resuspended nuclei into a 50 ml centrifuge tube through two layers of miracloth by gravity.

6. Centrifuge the contents at 57 g (500 rpm), 4°C for 2 minutes to remove intact cells and tissue residues.

7. Transfer the supernatant fluid into a fresh centrifuge tube and pellet the nuclei by centrifugation at 1,800 g, 4°C for 15 minutes in a swinging bucket centrifuge.

8. Wash the pellet 1 to 2 additional times by resuspension in wash buffer followed by centrifugation at 1,800 g, 4°C for 15 minutes.

9. After the final wash, resuspend the pelleted nuclei in a small amount (about 1 ml) of 1 x HB without b -mercaptoethanol with a paint brush. Count the nuclei, if possible, under a phase contrast microscope and adjust the nuclei concentration to approximately 4 x 10⁷ nuclei/ml with addition of the 1 x HB without b-mercaptoethanol, and store on ice.

D. Embedding the nuclei in agarose plugs and microbeads

• 1. Prepare a 1% LMP agarose in 1 x HB without b-mercaptoethanol and keep in a 45°C water bath.

2A. Embedding the nuclei in agarose microbeads

• a. Warm 20 ml of light mineral oil in a 50 ml Falcon tube to 45°C in a water bath (about 15 minutes)

• b. Pour 150 ml of ice cold 1 x HB without b-mercaptoethanol into a 500 ml beaker and place the beaker in an ice water bath on top of a magnetic stir plate. Vigorously swirl the solution using a magnetic stir bar.

• c. Place a 500 ml flask into a 45°C water bath and prewarm for at least 5 minutes.

• d. Prewarm the nuclei to 45°C in a water bath (about 5 minutes).

• e. Mix the prewarmed nuclei suspension with an equal volume of 1% LMP agarose in 1 x HB without b-mercaptoethanol kept in a 45°C water bath, pour into the prewarmed 500 ml flask, and add 20 ml of prewarmed light mineral oil at 45°C.

• f. Shake the contents of the flask vigorously for 2-3 seconds and immediately pour into the 500 ml beaker containing the swirling 150 ml of ice cold 1 x HB without b-mercaptoethanol. Continue to swirl the contents for 5-10 minutes on ice. This allows for the agarose microbeads to be more uniform in size.

• g. Harvest the agarose microbeads by centrifugation at 1,200 g, 4°C for 15-20 minutes in a swinging bucket centrifuge.

• h. Discard the supernatant fluid and resuspend the microbeads in 5-10 volume of lysis buffer.

• 2B. Embedding the nuclei in agarose plugs

• a. Prewarm the nuclei to 45°C in a water bath (about 5 minutes).

• b. Mix the nuclei with an equal volume of 1% low-melting point (LMP) agarose using a cut off pipet tip.

• c. Aliquot the mixture into ice cold plug molds on ice with the same pipette tip, 100 ul per plug. When the agarose is completely solidified, transfer the plugs into 5-10 volumes of lysis buffer.

3. Incubate the agarose microbeads or plugs in the lysis buffer for 24-48 hours at 50°C with gentle shaking.

4. Wash the plugs or the beads once in 0.5 M EDTA, pH 9.0-9.3 for one hour at 50°C, once in 0.05 M EDTA, pH 8.0 for one hour on ice, and store in 0.05 M EDTA, pH8.0, at 4°C.

E. Digestion of DNA Embedded in Agarose Microbeads

• 1. Before use, wash the agarose microbeads or the plugs containing HMW DNAs three times in 10 - 20 volumes of ice cold TE (10 mM Tris-HCl, pH8.0, 1 mM EDTA pH 8.0) plus 0.1 mM phenylmethyl sulfonyl fluoride (PMSF) and three times in 10 - 20 volumes of ice cold TE on ice, one hour each wash. The washed beads can be stored at 4°C for several months without significant degradation.

2. For digestion of agarose beads, mix the followings in a sterile microcentrifuge tube:

ddH2O	25 ul
Beads	50 ul*
10 x enzyme buffer	10 ul
40 mM spermidine	10 ul

Mix the beads well before pipetting and pipet the beads with a cut-off tip. The amount of beads to be digested is dependent on the concentration of DNA embedded in the agarose beads.

3. Incubate on ice for one hour, add 5 ul (approximately 10 units/ug DNA) of restriction enzyme for complete digestion and add 5 ul of each enzyme dilution series for partial digestion.

4. Incubate on ice for additional 30 minutes to allow the enzyme access to the DNA in the agarose beads and then transfer the reaction mixture to the recommended temperature for enzyme activity.

5. For complete digestion, incubate the reaction for at least 3 hours at the appropriate temperature. For partial digestion, incubate the beads for one hour.

6. Stop the reaction by adding 1/10 volume of 0.5 M EDTA, pH 8.0 and then place on ice or store at 4°C.

F. PFGE analysis and Southern blotting

• 1. Load the digested HMW DNA in agarose microbeads into an 1% agarose gel in 0.5 x TBE with a cut-off tip and seal the beads in position with the same molten agarose as the gel.

2. Perform pulsed-field gel electrophoresis as described below.

3. Stain the gel with ethidium bromide for 20 minutes, destain for one hour, photograph, and nick the DNA in the gel with 60 mJoules of UV light (254 nm) using the GS Gene Linker (BioRad, USA).

4. Blot the DNA in the gel onto Hybond-N+ membrane (Amersham, USA) with 1.5 M NaCl, 0.5 M NaOH for 40 hours.

5. Wash the southern blot in 2 x SSC for 10 minutes with gentle shaking. The blot can be directly used for hybridization or wet wrapped with plastic and stored at 4°C.