Plant Genome III Conference

PRE-AMPLIFICATION RESTRICTION ENZYME DIGESTION OF COMPLEX EUKARYOTIC GENOMES INCREASES THE UTILITY OF MICROSATELLITE-BASED SPAR

Microsatellites, or Simple Sequence Repeat (SSR)-based primers amplify multiple DNA markers from diverse eukaryotic genomes in Single Primer Amplification Reactions (SPARs). However, many primers produce so many bands that the patterns become unpredictable and thus much potential information content is lost. The number of primers amplifying uninterpretable patterns increases with the increasing complexity of eukaryotic genomes and requires an expensive and time consuming search for useful primers. The present study shows that the problem can be resolved by Pic-Amplification Restriction Enzyme Digestion (PARED) of genomic DNA. Of the six-, five- and four-base pair recognizing enzymes tested the four-base pair cutters produced the best results. Methods were developed to eliminate the DNA precipitation step following restriction enzyme digestion. PARED not only simplified the DNA fingerprints but produced different DNA markers by the use of various restriction enzymes. This method reduces the need for a large number of primers in many genetic studies. The study has increased the utility of SSR-SPAR across a wider spectrum of eukaryotic species. The technology is not limited to SSR-SPAR but can be applied to any SPAR which uses primers of multicopy origin.