Reproducible amplification of RAPD markers from vertebrate DNA

Randomly amplified polymorphic DNA (RAPD) is a modification of PCR that uses short, randomly generated primers to amplify genomic DNA. Generally, many bands of mixed intensity (i.e., strong, faint, fuzzy or sharp) are generated with each primer. Mixed-intensity bands are inherent with the RAPD technique because (i) the target DNAs are undefined, (ii) one or more copies of the target DNA may exist per genome and (iii) the percentage of hybridization of primer to target may vary. The problem of mixed-intensity bands exacerbates the well-known sensitivity of PCRs to reagent and template concentrations, pH and other reaction parameters. These complications have discouraged many investigators from using RAPD. Our goal was to optimize the RAPD amplification conditions for vertebrate DNA. We present the optimized protocol along with an experimental strategy for obtaining reproducible, interpretable RAPD banding patterns in vertebrates.