Individual DNA bands obtained by RAPD analysis of canine genomic DNA often contain multiple DNA sequences

The random amplified polymorphic DNA (RAPD) technique has been widely applied for genetic studies of plants, insects, and fungi, and recently has been used for studies in animals including dogs. To convert the RAPD marker into a classical PCR marker, the RAPD-PCR products are size-separated in an agarose gel and a specific DNA band is selected for potential association with a trait of interest. The DNA fragments present in the desired band are then cloned and sequenced using primers specific to the cloning vector, and the sequence is used to design a pair of classical PCR primers. Often a "positive clone" is identified based solely on a match of the size of the insert in the clone with the uncharacterized DNA band originally selected. We observed that single DNA bands obtained from RAPD-PCR using canine genomic DNA often contain DNA fragments of similar size but of different sequences. Based on this observation, we report here a modification of the protocol for RAPD analysis which will ensure that a promising RAPD marker selected based on initial screening is not lost for lack of a comprehensive investigation in the later experimental analysis.