Use of GELase™ Gel-Digesting Preparation for Improved EM of High Molecular Weight DNA Extracted from an Agarose Gel, EPICENTRE® Biotechnologies

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|Use of GELase™ Gel-Digesting Preparation for Improved EM of High Molecular Weight DNA Extracted from an Agarose Gel|

Philip Serwer, Dept. Biochemistry, The Univ. of Texas Health Science Center, San Antonio, TX 78284-7760

Electron microscopy (EM) of high molecular weight DNA fractionated by pulsed-field agarose gel electrophoresis (PFGE) is used to determine DNA size and conformation (linear, circular, or branched). EM is also used to detect and characterize proteins or other non-DNA macromolecules bound to DNA.¹ However, several factors can distort the sample during EM of PFGE-purified DNA: (a)Contaminating agarose fibers may mimic DNA.² (b)Hydrodynamic shear-induced degradation may occur during extraction of large DNA fragments (greater than approximately 40 kb) from the gel.^3,4 For example, electroelution of 80-120 kb concatemeric DNA⁵ from agarose gels following PFGE^2,6,7 can cause both degradation and loss of DNA.⁸ To determine whether extraction of DNA from agarose gels using a GELase-based procedure avoids these problems, we used 40 kb bacteriophage T7 DNA with an attached T7 capsid as a model system. The capsid bound to DNA was a marker to help confirm the identity of molecules observed by EM.

To obtain T7 capsid-DNA complexes, DNA was expelled from bacteriophage T7 in 0.1 M NaCl, 10 mM Tris-HCl, pH 7.4, 0.2 mM EDTA by raising the temperature to 51°C.¹ Approximately two-thirds of the released DNA molecules were free of capsid; the remaining DNA had capsid bound at a variable location. After expulsion of DNA, capsid-DNA complexes were separated from capsid-free DNA by PFGE (field inversion mode) for 24 hr using 3 V/cm at 15°C with a 48 sec. forward pulse and 16 sec. reverse pulse on 1.5% SeaPlaque® agarose (FMC BioProducts) cast in 0.01 M sodium phosphate, pH 7.4, and 1 mM EDTA. To obtain as much DNA as possible, samples were loaded on the agarose gel in amounts close to the upper limit permitted without band distortion. For a 1.5% gel, we used 2 µg total DNA per cm² of surface area at the gel entry point.¹

Following PFGE, DNA bands were located by ethidium bromide staining. By use of a flattened plastic soda straw, agarose plugs were removed at the position of either the capsid-DNA complex or capsid-free DNA. Each plug (approximately 100 µl) was melted by incubation at 65°C for 10 min in a 1 ml plastic centrifuge tube. The melted plug was equilibrated at 40°C and digested at 40°C with 1 µl GELase Gel-Digesting Preparation (1 U/µl). Neither capsid-DNA complex nor free DNA recovered by GELase digestion underwent an observable change in band mobility during a second separation by PFGE. Thus the GELase digestion did not cause detectable DNA degradation.

To avoid shear-induced degradation of the DNA, the GELase enzyme was not removed following digestion. To observe GELase-purified capsid-DNA complexes, cytochromec was bound to the DNA component; the complex was then negatively stained. GELase-purified DNA (2 µl) was added to 1 µl formamide and 0.5 µl of 500 µg/ml cytochromec. This mixture was negatively stained using 1% uranyl acetate.¹ Although an unusual procedure for DNA preparation, negative staining offers the clearest visualization by EM of the capsids attached to the DNA.

As seen in Figure 1, EM of these negatively stained, GELase-purified T7 DNA samples revealed fibers, almost all (94%) of which had an attached capsid. We concluded that these fibers were DNA. After scanning 600 grid squares and observing over 2,000 DNA molecules, we saw only 2 fibers that could have been agarose. These few, presumably agarose, fibers might be removed by more extensive GELase digestion, either by using more enzyme, or perhaps by a more extensive melting of the agarose plug.

Fig. 1. EM of bacteriophage T7 DNA purified using GELase Gel-Digesting Preparation. T7 capsid-DNA complexes were prepared as described. Most of a single capsid-DNA complex is shown; the capsid is indicated by an arrow. The insert contains an enlarged image of the region near the capsid. The length of both bars is 0.12 µm.

In subsequent studies, we have similarly purified the 170 kb linear DNA of bacteriophage T4 with only 10%-15% breakage. Although we have not yet attempted to recover DNA fragments longer than 170 kb using GELase digestion, we have successfully pipetted DNA as long as 1,000 kb without detectable degradation.⁹ Thus, by use of GELase Gel-Digesting Preparation recovery should be possible for undegraded DNA at least this large, following PFGE.

References

P. Serwer, S.J. Hayes, E.T. Moreno and C.H. Park (1992) A Small (58-nm) Attached Sphere Perturbs the Sieving of 40-80 Kilobase DNA in 0.2-2.5% Agarose Gels: Analysis of Bacteriophage T7 Capsid-DNA Complexes by Use of Pulsed Field Electrophoresis, Biochemistry 31: 8397.
P. Serwer (1990) Sieving by Agarose Gels and Its Use During Pulsed-Field Electrophoresis, Biotechnology and Genet. Eng. Rev. 8: 319.
R.E. Adam and B.H. Zimm (1977) Shear Degradation of DNA, Nuc. Acids Res. 4: 1513.
B. Zimm and H.R. Reese (1990) The Degradation of T7 DNA in Converging Flow, Nuc. Acids Res. 18: 4469.
M. Son, S.J. Hayes and P. Serwer (1988) Concatemerization and Packaging of Bacteriophage T7 DNA in vitro: Determination of the Concatemers' Length and Appearance Kinetics by Use of Rotating Gel Electrophoresis, Virology 162: 38.
C.R. Cantor, C.L. Smith and M.K. Mathew (1988) Pulsed Field Gel Electrophoresis of Very Large DNA Molecules, Ann. Rev. Biophys. & Biophys. Chem. 17: 287.
E. Lai, B.W. Birren, S.M. Clark, M.I. Simon and L. Hood (1989) Pulsed Field Gel Electrophoresis, BioTechniques 7: 34.
M. Son and P. Serwer, unpublished observations.
D. Louie and P. Serwer (1991) Effects of Temperature on Excluded Volume-Promoted Cyclization and Concatemeriza-tion of Cohesive-Ended DNA Longer than 0.04 Mb, Nuc. Acids Res. 19: 3047.