Digestion of Agarose-Embedded DNA
Pulsed field gel electrophoresis (PFG) techniques have made possible the resolution of DNA molecules up to several million base pairs in length (1,2). Manipulation of naked DNA of this size in liquid creates double-stranded breaks due to mechanical shear forces. To avoid this fragmentation, DNA can be embedded in an agarose matrix. Intact cells are immobilized in agarose, and then treated to disrupt their cell walls and remove cellular protein. Subsequently, the DNA-containing agarose plug is manipulated in much the same way as DNA in solution.
Restriction endonuclease digestion of agarose-embedded DNA has proved to be a valuable tool in the physical mapping of chromosomes (3,4). Most endonucleases can function in the presence of agarose (5). Since diffusion within agarose is more limited than in liquid reactions, higher concentrations of restriction endonucleases and longer incubation times are generally required for complete DNA digestion. The extended times, together with the larger size of the target DNA, increase the necessity for highly purified restriction endonucleases lacking contaminating nuclease activities.
Limited enzyme diffusion into the agarose matrix may account for diminished enzyme performance on agarose-embedded DNA. To reduce this effect, plugs should be made with low-melting-point agarose at concentrations of 0.5-1.0%. A 16 hour preincubation of the reaction (at 4°C) is useful in cases where enzyme stability is compromised by incubation at 37°C for extended periods of time. Also adding additional restriction enzyme 30 minutes before placing the digest at the recommended digestion temperature can sometimes be beneficial. Gentle mixing of the reaction has also been reported to aid in the diffusion of enzyme into the plug
Digestion Protocol for Agarose-Embedded DNA
1) Cut 10-20 µl (1x3x5 mm) from a plug of agarose-embedded DNA (1-2 µg) with a scalpel or razor blade and immerse in 1.0 ml of TE [10 mM Tris-HCl, 0.5 mM EDTA] in a microcentrifuge tube. Chill on ice for 15-30 min, occasionally inverting the tube to ensure equilibration. (This wash lowers the EDTA concentration in the plug).
2) Carefully remove the TE with a 1.0 ml pipet tip and tap the tube to place the plug at the bottom. Add 100 µl of 1X restriction endonuclease buffer and chill on ice for 15-30 minutes (this wash equilibrates the agarose-embedded DNA in endonuclease digestion conditions).
3) Replace the 1X restriction endonuclease buffer with fresh buffer and add the appropriate restriction endonuclease. Incubate at the recommended reaction temperature.
Note: Higher enzyme concentrations and longer incubation times must be used to achieve complete DNA cleavage in agarose as compared to solution. As a general rule, use 20 units for a 4 hour incubation or 5 units for a 16 hour incubation. Most restriction endonucleases will not cut to completion in less than 2 hours, regardless of the number of units present (see information about specific enzymes).
4) Following the restriction endonuclease digestion, chill the tube on ice and aspirate the enzyme and buffer. Equilibrate the plug with 1.0 ml of PFG running buffer on ice for 15-30 minutes, inverting the tube occasionally.
5) Carefully aspirate all but 100 µl of the PFG running buffer from the tube and load the plug onto the running gel with a clean spatula.
6) Perform PFG according to the manufacturer's instructions for your equipment. Conditions for optimizing DNA separation by PFG have been published (6).
- Lai, E. et al. (1989) BioTechniques 7, 34-42.
- Birren, B.W. et al. (1989) Anal. Biochem. 177, 282-285.
- Carle, G.F. and Olson, M.V. (1984) Nucl. Acids Res. 12, 5647-5664.
- Guzman, P. and Ecker, J.R., (1988) Nucl. Acids Res. 16, 11091-11105.|
- Davis, T.B. and Robinson, D.P., New England Biolabs, unpublished observations.
- Birren, B.W. et al. (1988) Nucl. Acids Res. 16, 7563-7582