Determination of microbial genome sizes by two-dimensional denaturing gradient gel electrophoresis

In two-dimensional denaturing gradient gel electrophoresis, DNA is digested with a restriction endonuclease and the resulting DNA fragments are separated as a function of size by conventional agarose gel electrophoresis. Following this first dimension electrophoresis, the fragment distribution is placed at the top of a denaturing gradient slab gel and electrophoresis is carried out parallel to the gradient direction. This second dimension separation is a complex function of the base sequence of each fragment. Analysis of the DNA fragment distribution as a function of fragment size allows the DNA size to be calculated. This method has been applied to calculate three microbial genome sizes: Mycoplasma capricolum, 724 kb; Acholeplasma laidlawii, 1646 kb; and Hemophilus influenzae, 1833 kb.