Diheteroduplex formation using gold labeled single-stranded PCR fragments and its application in electron microscopy

Heteroduplex analysis is commonly used to map homologous sequences in DNA:DNA or DNA:RNA hybrids in spread preparations by electron microscopy. However, the standard procedures are not suitable to detect the orientation of a fragment with a defined sequence in a hybrid molecule. Here, we describe an alternative protocol for the visualization of DNA:DNA "diheteroduplex" structures based on digoxigenin/anti-digoxigenin gold labeling that allows determination of the position and orientation of a fragment. Single-stranded polymerase chain reaction (PCR) generated fragments labeled at their 3' ends are hybridized to double-stranded plasmid DNA. Electron microscopy of spread preparations visualizes the gold label and, in combination with morphometric measurements, it is possible to determine the position and orientation of the fragment with the diheteroduplex molecule.