DNA TYPING WITH FLUORESCENTLY TAGGED SHORT TANDEM REPEATS - A SENSITIVE AND ACCURATE APPROACH TO HUMAN IDENTIFICATION

Human identification through DNA analysis has faced tremendous changes in the past seven years. The advent of the polymerase chain reaction (PCR) technology coupled with the discovery of amplifiable minisatelli tes and microsatellites known as amplified fragment length polymorphis ms and short tandem repeats (STRs), respectively, allow allelic profil es to be obtained with minute amounts of target DNA even in a degraded state. Very recently, a new dimension in DNA typing analysis was open ed with the development of instruments for automated real-time analysi s of fluorescent amplification products. In order to derive an automat ed approach to DNA typing, STR systems were evaluated for sensitivity and accuracy using the Gene Scanner and compared to other DNA typing m ethods currently in use. Eight different STR systems (encompassing tri -, tetra- and pentanucleotide repeats) were investigated, and conditio ns for their amplification with fluorescence-tagged primers, resolutio n on polyacrylamide gels and analysis on a fluorescent DNA fragment an alyzer were optimized. Using these conditions, discrete allelic profil es were obtained following amplification of DNA extracted from various cell lines, liquid blood, dry bloodstains and hair samples. Amplifica tion from serial dilutions of template DNA indicated that the minimal amount of target DNA required to detect a fluorescent signal on the Ge ne Scanner for any of the eight STR systems examined is approximately 100 picograms. The level of precision obtained for real-time allele si ze determination was observed to be +/- 0.2 to 0.5 base pair (intragel ) and +/- 0.5 to 1.5 base pairs (intergel). Consequently, PCR-based DN A typing with fluorescent STR primers and automated analysis provides the enhanced level of precision, accuracy and sensitivity required for forensic casework analysis. Moreover, this approach offers significan t advantages for the routine processing of large numbers of DNA sample s, greatly facilitates and expedites the generation of allelic profile databases and enables investigators to perform the simultaneous surve y of several different loci from single individuals and/or forensic sa mples.