Isolation of High Molecular Weight DNA from Soybeans Using GELase™ Gel-Digesting Preparation, EPICENTRE® Biotechnologies

EPICENTRE Forum 2 (1)

|Isolation of High Molecular Weight DNA from Soybeans Using GELase™ Gel-Digesting Preparation|

Luojing Chen and Alan Atherly, Department of Zoology & Genetics, Iowa State University, Ames, IA 50011

One of the critical steps in the construction of genomic libraries using yeast artificial chromosomes (YACs) or cosmids is isolation of high molecular weight DNA for cloning. As described in the following procedure, we have successfully used GELase Gel-Digesting Preparation to obtain approximately 2,200 kb-sized soybean DNA fragments in high yields for constructing the genomic library of this DNA using a cosmid vector (SupCos I).

To isolate high molecular weight DNA from soybeans, we first prepared protoplast DNA agarose plugs according to the method of Honeycutt et al.^1-3 Harvested young expanding leaves were sterilized with 10% bleach and then rinsed with distilled water. The sterilized leaves were cut into approx. 3 mm slices with a scalpel and immersed in protoplast isolation buffer (PIB): 0.04 M mannitol, 6 mM CaCl₂, 3 mM MES (2-[N-morpholino] ethanesulfonic acid), 0.7 mM NaH₂PO₄, pH 5.6, supplemented with 1% cellulysin (Calbiochem, La Jolla, CA), and 0.2% pectolyase Y23 (Karlan Chemicals, Santa Rosa, CA).¹ 10 ml of PIB were used per gm of fresh leaves. The mixture was incubated at 28°C in the dark for 4-5 hr with gentle shaking (50 rpm).

The resulting protoplasts were pelleted by low speed centrifugation at 200 x g for 5 min, washed twice with 0.5 M mannitol, and filtered through 70 µm nylon mesh. The filtered protoplasts were pelleted again as before and resuspended in PIB to a concentration of approximately 4 x 10⁷ protoplasts per ml. The resuspended protoplasts at 37°C were mixed with an equal vol of 2% low-melting-point (LMP) agarose (prepared in PIB and cooled to 40°C) to form plugs. The plugs were then incubated at 50°C in ESP buffer (0.5 M EDTA, 1% N-lauroylsarkosyl, 1 mg/ml Proteinase K, pH 9.0) for 24 hr without shaking, and then stored at 4°C.

The DNA-agarose plugs were then loaded onto a 1% LMP agarose gel in 0.5X TBE buffer for pulsed field gel electrophoresis (40 sec pulse at 200 V for 18 hr).³ Following electrophoresis, the gel was stained with ethidium bromide and visualized under long wavelength UV light (Lane 1 in Figure 1). The high molecular weight (approx. 2,200 kb) DNA bands were excised with a razor blade into a tube, and the slices were immersed for 30 min in 2-3 vol of 1X GELase Buffer (50X GELase Buffer is supplied with GELase Gel-Digesting Preparation). Following this buffer exchange step, excess buffer was removed from the slices with a pipet. The slices were transferred to a clean tube and incubated at 70°C until the agarose was completely melted. The melted agarose was cooled to 43°C in a water bath, and GELase was added to a concentration of 0.125 U per gm gel. The mixture was incubated at 43°C for 20 hr without shaking to digest the agarose. Following agarose digestion, the reaction mixture containing the DNA could be used directly in restriction enzyme digestion and cloning without further manipulation.

Figure 1. Pulsed-field gel electrophoresis of high molecular weight soybean DNA. DNA was prepared and separated by gel electrophoresis as described. Lane 1, total DNA. Lane 2, DNA recovered using the GELase procedure from the approx. 2,200 kb band indicated in Lane 1.

Using this GELase procedure, recovery of 2.2 megabase DNA from the agarose gel was estimated to be at least 80%. As shown in comparing Lane 2 with Lane 1 in Figure 1, the recovered DNA remained at the same high molecular weight as before the treatment, indicating that DNA is recovered without degradation using GELase Gel-Digesting Preparation.

G. Chu et al. (1986) Separation of Large DNA Molecules by Contour-clamped Homogeneous Electric Fields, Science 234: 1582.
R.J. Honeycutt et al. (1992) Analysis of Large DNA from Soybean (Glycine Max L. Merr) by Pulsed-field Gel Electrophoresis, Plant J. 1: 133.
L. Chen et al. (1994) Mechanical Shear of High Molecular Weight DNA in Agarose Plugs, BioTechniques 16: 228.