A new method for mapping nucleic acid sequence homology by electron microscopy

Abstract

We describe here a new method for the electron microscopic mapping of sequence homology in nucleic acids. Specific segments of the T7 chromosome have been isolated following digestion with the restriction endonuclease from Hemophilus aegyptious (Haey). Denatured segments are annealed to the l-strand of T7 DNA; treatment of the hybrid with glyoxal allows only guanosine residues in the single-chain region to the reacted, producing an adduct which will no longer hydrogen bond with its complement on the r-strand. When the segment is displaced and the glyoxalated l-strand allowed to renature with the r-strand, "H" shaped structures are produced in which the duplex region corresponds to the position of sequence homology with the segment. The conditions employed for glyoxalation do not detectably disrupt duplex regions as small as 400 base pairs. This procedure should be generally useful for observing sequence homology in more complex DNA molecules containing duplex regions which can be specifically enriched for and their arrangement determined by electron microscopy.