Fluorescence-based semi-automated gene scan with microsatellite markers by multiplex PCR techniques

OBJECTIVE: To develop a high output and low cost multiplex PCR approach to facilitating large scale gene scan and gene typing with microsatellite markers. METHODS: 5-15 pairs of primers of microsatellite loci were added in one single tube with 5ul reaction volume, containing 10 mmol/L Tris-HCl (pH8.3), 50 mmol/L KCl,0. 1mg/ml gelatin, 3.0 mmol/L MgCl2, 200micromol/L dNTPs (each), 0.25U Taq DNA polymerase, Taq Start antibody and DNA templates. PCR thermocycles were carried out on Perkin Elmer Gene Amp PCR System 9600 with touch-down algorithm (the annealing temperature was decreased by 0.5 C in each cycle) to meet the different demands of different primers. RESULTS: Up to 10-15 loci were labeled by different fluorescents or by the same fluorescent, but their PCR amplification fragments did not overlap in size. Almost all target microsatellite loci were successfully co-amplified in a 5 microliter reaction volume,electrophoresized and analyzed in a single gel lane with Perkin Elmer ABI 373A DNA sequencer, and 9 microsatellite loci were well genotyped. CONCLUSION: This high output and low cost genotyping protocol is applicable to gene mapping, human evolution and forensic analysis.