Rapid and precise genotyping of porcine microsatellites

Microsatellites are useful markers for genetic mapping and linkage analysis because they are highly polymorphic, abundant in genomes and relatively ea sily scored with polymerase chain reaction (PCR). A rapid genotyping system for microsatellites was developed, which included multiplex PCRs, multiple use of Hydrolink(TM) gels, automated fluorescent detection of fragments on an A.L.F. DNA sequencer, automatic assignment of alleles to each locus and verification of genotypes with a self-developed computer program "Fragtest ". Eight multiplex PCRs have been developed to genotype 29 microsatellites for genetic and quantitative trait loci (QTL) mapping on pig chromosomes 6, 7, 12 and 13. Three to six microsatellites could be amplified in one multi plex PCR. Each multiplex reaction required only different concentrations of each pair of primers and a low concentration of dNTP (100 mu m). A dNTP co ncentration of 100 mu m proved to be optimal for the coamplification of mic rosatellites under the concentration of 1.5 mM MgCl2. Using four internal s ize standards added in each sample, the 5% Hydrolink gel could subsequently be used up to five times (total running time of 500 min) on the A.L.F. aut omated sequencer without significant loss of resolution and precision of fr agment length analysis. Automatic assignment of alleles on each locus using "Fragtest" significantly increased the efficiency and precision of the gen otyping. This system is thus a rapid, cheap, and highly discriminating geno typing system.