Quantitative gel electrophoresis of DNA: resolution of overlapping bands of restriction endonuclease digests.

ABSTRACT

Accurate quantitation of the distribution of DNA digested to completion by a restriction endonuclease and separated in an electrophoretic gel is greatly facilitated by electronic imaging systems employing charge-coupled device detectors. Such quantitation permits the extraction of more accurate values of the lengths of both the parent molecular species and the fragments produced by a restriction endonuclease. We describe a method that determines the number of components in bands containing two or more daughter fragments that are of similar length and hence not resolved by gel electrophoresis. We also report a constrained deconvolution procedure based on the equal number of daughter molecules in a terminal digestion by a restriction endonuclease that resolves the distribution of each of the overlapping species in a composite band on a gel and hence increases the accuracy of the lengths assigned to each.