Mapping Z-DNA tracts in plasmid DNA using electron microscopy and gold-labeled antibodies.
Using alternating poly(dG-dC).poly(dG-dC) and electron microscopy (EM), a method has been developed for detecting regions of Z conformation in DNA preparations. The procedure was developed with poly(dG-dC).poly(dG-dC) which had been converted to the Z conformation with MnCl2 and mild heat treatment. Conditions were found for reaction of this DNA with polyclonal anti-Z antibodies from rabbit, and further reaction of this mixture with gold-labelled anti-rabbit antibodies from mouse. Spreading of these samples onto air-water interfaces and examination by EM revealed gold particles aligned along strands of poly(dG-dC).poly(dG-dC). The method was refined and simplified using monoclonal antibodies and tested with the 2.2-kb plasmid, pDHg16, carrying a single tract of alternating d(G-C)23. Treatment with MnCl2 and mild heat was not necessary, as the superhelicity of this molecule ensured that the d(G-C) tract was in the Z conformation. Conditions were found for successful conjugation of mouse monoclonal anti-Z antibodies with colloidal gold (G10), 10.7-nm average diameter. The conjugate was then reacted with superhelical pDHg16, stabilized in polyethylene glycol and cross-linked with glutaraldehyde. Examination by EM showed gold particles at one site on the negatively superhelical circular DNA molecule. When these molecules were linearized with PstI, gold particles were found to occur at an average position 35% +/- 3% from one end. This location agrees well with the known position of the center of the alternating d(G-C) tract with respect to the PstI restriction site (36.8%).
Inman
R B
RB
Institute for Molecular Virology, University of Wisconsin, Madison 53706.
Jackson
J F
JF
D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D004247:DNA / Q000648:ultrastructure*; D004269:DNA, Bacterial / Q000276:immunology / Q000648:ultrastructure*; D006046:Gold; D010957:Plasmids / Q000235:genetics*; D011089:Polydeoxyribonucleotides / Q000235:genetics
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D004269:DNA, Bacterial; D011089:Polydeoxyribonucleotides; C019530:poly(dC-dG); D006046:Gold; D004247:DNA