DOP-PCR amplification of probe DNA for whole-mount FISH in Drosophila.

It is sometimes useful to amplify DNA before labeling it. Degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) allows amplification of any complex DNA, regardless of sequence. DOP-PCR is performed using a single primer that has a fixed sequence at its 5' end and several degenerate bases near its 3' end that allow it to anneal at low stringency to many sites in complex DNA. Following several PCR cycles with an annealing temperature of only 30°C, the annealing temperature is raised so that only products with the primer sequence at each end are amplified in the reaction. This protocol describes the use of DOP-PCR to amplify probe DNA for whole-mount fluorescent in situ hybridization (FISH) in Drosophila. Primary DOP-PCR amplification may be used for microdissected material, small volumes of melted gel slice, or small quantities of other DNA template. A procedure for secondary DOP-PCR amplification is also provided.