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Nucleic Acids Research, 1989, Vol. 17, No. 8 2889-2895
© 1989

MOLECULAR BIOLOGY

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

Saibal K. Poddar and Jack Maniloff*

Department of Microbiology and Immunology, University of Rochester Medical Center Box 672, Rochester, NY 14642, USA

*To whom correspondence should be addressed

Received November 4, 1988. Revised March 14, 1989. Accepted March 14, 1989.

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.


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