DIHETERODUPLEX FORMATION USING GOLD-LABELED SINGLE-STRANDED PCR FRAGMENTS AND ITS APPLICATION IN ELECTRON-MICROSCOPY

Heteroduplex analysis is commonly used to map homoIogous sequences in DNA:DNA or DNA:RNA hybrids in spread preparations by electron microsco py. However, the standard procedures are not suitable to detect the or ientation of a fragment with a defined sequence in a hybrid molecule. Here, we describe an alter native protocol for the visualization of DN A:DNA ''diheteroduplex'' structures based on digoxigenin/anti-digoxige nin gold labeling that allows determination of the position and orient ation of a fragment. Single-stranded polymerase chain reaction (PCR) g enerated fragments labeled at their 3' ends are hybridized to double-s tranded plasmid DNA. Electron microscopy of spread preparations visual izes the gold label and, in combination with morphometric measurements , it is possible to determine the position and orientation of the frag ment with the diheteroduplex molecule.