Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels

Abstract

The DNA of bacteriophage T7 is cut into seven unique fragments by the restriction endonuclease DpnII (or the equivalent MboI), 19 fragments by HpaI, and eight additional fragments by the combination of the two enzymes. The relative location of each fragment in the T7 DNA has been determined by a combination of techniques. If it is assumed that the length of any DNA molecule equals the sum of the lengths of the fragments produced from it by cleavage, and that electrophoretic mobility through agarose gels is a smooth function of the length of the DNA, then the known relationships between fragments provide enough conditions to define accurately the relative molecular weight of each fragment in the set. Absolute molecular weights are based on that of full-length T7 DNA. The fragments provide a convenient set of length standards covering the entire range from about 100 to 40,000 base-pairs (the length of T7 DNA). A horizontal slab gel system for electrophoresis on agarose gels is described. In this system, gels of very low concentrations do not distort during electrophoresis and accurate relative mobilities of large DNAs are obtained. Excellent resolution can be obtained for DNAs of molecular weights up to at least 26·5×106, a difference of less than 10% being readily resolved even for molecules of this size. Agarose and polyacrylamide gels can be prepared in alkaline solvents that denature native DNA and completely unfold the single strands. The fragments of T7 DNA have the same relative mobilities whether subjected to electrophoresis as single strands in alkaline gels or as double-stranded DNA in neutral gels, and resolution is comparable in the two states. Thus, electrophoresis in alkaline gels can provide accurate molecular weights for linear, single-stranded DNAs, and should be useful in analyzing DNA for single-strand breaks, depurinations or topological differences such as ring forms. In both neutral and alkaline gels, the relative mobilities of DNAs shorter than about 1000 base-pairs (or bases) are essentially insensitive to changes in voltage gradient, at least over the range of voltage gradients commonly employed. However, relative mobilities become increasingly sensitive to voltage gradient the larger the DNA, with DNAs longer than about 20,000 base-pairs (or bases) being severely affected. This effect is probably due to the ease with which large DNA molecules can be deformed from their equilibrium conformations, thus permitting them to penetrate channels in the gel that would exclude them in their unperturbed conformations. As a practical matter, this means that low voltage gradients must be used for separations of large DNAs by gel electrophoresis.