EVALUATION OF AN AUTOMATED DNA PROFILING SYSTEM EMPLOYING MULTIPLEX AMPLIFICATION OF 4 TETRAMERIC STR LOCI

We have examined the performance and reproducibility of an automated D NA profiling system which is based on the multiplex amplification of 4 tetrameric STR loci - HUMVWFA31/A, HUMTH01, HUMF13A1 and HUMFES/FPS. The system was able to type 100 pg of purified, undegraded, genomic DN A. At lower concentrations of DNA (below 100 pg), allelec drop-out occ urred due to stochastic differences in allele copy number. Minor varia tion of individual PCR reagent concentrations or cycling temperatures did not result in a significant effect on the efficiency of amplificat ion of any of the 4 loci in the quadruplex system. More substantial va riation of reagent concentrations or cycling temperatures outside the optimum range of the system resulted in a reduction or complete loss o f signal for one or more loci. This was also observed at high ionic st rength or extreme pH. However, under all reagent concentrations and co nditions studied, no artefact bands that could potentially result in t he mistyping of a sample were apparent within the read region (130-240 bases) of the gel. Evaluation of both native and denaturing polyacryl amide gels revealed that, although native gels displayed faster run ti mes, the sizing precision of such gels for certain STR loci was lower than that of denaturing gels. Also, artefact bands may be present with in the read region of native gels. In conclusion the quadruplex amplif ication system decribed, coupled with automated fluorescence-based det ection on denaturing polyacrylamide gels, appeared to be a robust and reliable system for individual identification.