Detection of microsatellite polymorphisms without cloning

Microsatellites or simple sequence repeats provide an attractive source of genetic polymorphisms for both mammals and plants. Microsatellites can be detected by PCR using minute amounts of starting material and they provide codominant markers with a high degree of allelic polymorphism. However, these markers are obtained through a difficult and labor-intensive procedure. In contrast, RAPD markers are easy to develop and less expensive to assay but they are usually genetically dominant and the degree of polymorphism is low. To compensate for the weaknesses of these two approaches, we have combined microsatellites and RAPDs into a new method to detect and map co-dominant polymorphisms without cloning and sequencing. Our method is based on the random distribution of nucleotide sequences immediately flanking the simple sequence repeats. A primer consisting of a 5' anchor and 3' repeats is end-labeled with gamma super(33)P-dATP and used to amplify genomic DNA in the presence or absence of decamers of arbitrary sequences (RAPD primers). The resulting products are resolved in denaturing polyacrylamide gels and, since only the repeat primer is labeled, only the amplification products derived from the anchored primer is detected. To facilitate the annealing of two primers with different Tm and to reduce the amplification mediated by the random 10mer alone, we employed a modified termally asymmetric PCR profile (3). The program was designed to switch between high and low annealing temperatures during the PCR reaction. Since the Tm's of the anchored primers are usually 10-15 degree C higher than those of the RAPD primers, in the PCR cycles with higher annealing temperature only the anchored primer should anneal efficiently, whereas at low annealing temperature cycles both anchored microsatellite and RAPD primers should anneal. Thus, DNA sequences from microsatellite loci are preferentially amplified. We named these polymorphisms RAMPs (random amplified microsatellite polymorphisms) to differentiate them from specific microsatellites with unique primers.