Quintuplex PCR-amplification of microsatellites

We tested the potential usefulness of quintuplex PCR-amplification of STRs (D16S537, D8S320, FGA, D11S554 and THO1) for paternity testing, first by constructing 20 false family trios, each with a non-biological father, to estimate the actual exclusion rate, and next by a retrospective study of 20 paternity cases which had been previously tested by RFLP analysis and typing of conventional genetic markers. The five STR loci have a combined average exclusion power of 99.8%. The forensic efficiency values for four of the five STRs were based on typing of a Japanese population of 300 individuals. The observed rate of exclusion was in close agreement with the expected rate for the Japanese population. In 12 of the 20 cases, paternity was not excludable by any of the loci. The probability of paternity calculated for the alleged father was > 99% in the inclusion cases. In the remaining 8 cases, paternity was excluded by at least 2 of the STR loci. The results of STR typing agreed with the data from RFLP analysis and conventional genetic typing. Taking advantage of five STRs sharing AA in the tetranucleotide repeats (AAAG or AATG), we detected PCR products simultaneously with a digoxigenin-labeled (AAAG)6 oligonucleotide probe after sequencing-gel electrophoresis. The quintuplex PCR-amplification of STRs is a rapid and efficient method for paternity testing and individual identification.