Determination of the patterns of hexanucleotide distribution along DNA by electron microscopy

Abstract

Hexanucleotides containing modified bases (5-methylcytosine and 2-aminoadenine instead of cytosine and adenine) with increased capacities to bind complementary DNA sequences were used to map the distribution of their complementary sequences in a DNA target using electron microscopy. The method used hexamers to initiate DNA polymerse directed DNA synthesis at complementary sequences along a template. DNA synthesis was limited to about 200 residues by using a low concentration of deoxynucleotide precursors. During DNA synthesis a biotin ligand was incorporated to facilitate the subsequent binding of an electron-dense label (streptavidin-labeled colloidal gold particles) into newly synthesized DNA chains. The method can be implemented with commercially available products. The results demonstrate that the approach can be used to compare primary structural features of DNA fragments. The principles of the method can be adapted to a variety of single molecule detection methods such as electron, scanning tunneling, or atomic force microscopies.