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 minisatellites and microsatellites known as amplified fragment length polymorphisms and short tandem repeats (STRs), respectively, allow allelic profiles to be obtained with minute amounts of target DNA even in a degraded state. Very recently, a new dimension in DNA typing analysis was opened with the development of instruments for automated real-time analysis of fluorescent amplification products. In order to derive an automated approach to DNA typing, STR systems were evaluated for sensitivity and accuracy using the Gene Scanner and compared to other DNA typing methods currently in use. Eight different STR systems (encompassing tri-, tetra- and pentanucleotide repeats) were investigated, and conditions for their amplification with fluorescence-tagged primers, resolution on polyacrylamide gels and analysis on a fluorescent DNA fragment analyzer were optimized. Using these conditions, discrete allelic profiles were obtained following amplification of DNA extracted from various cell lines, liquid blood, dry bloodstains and hair samples. Amplification from serial dilutions of template DNA indicated that the minimal amount of target DNA required to detect a fluorescent signal on the Gene Scanner for any of the eight STR systems examined is approximately 100 picograms. The level of precision obtained for real-time allele size 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 DNA 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 significant advantages for the routine processing of large numbers of DNA samples, greatly facilitates and expedites the generation of allelic profile databases and enables investigators to perform the simultaneous survey of several different loci from single individuals and/or forensic samples.
Frégeau
C J
CJ
Royal Canadian Mounted Police, Central Forensic Laboratory, Biology Research and Development Support Unit.
Fourney
R M
RM
D000483:Alleles; D001483:Base Sequence; D002170:Canada; D002460:Cell Line; D004247:DNA / Q000032:analysis* / Q000097:blood; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D005554:Forensic Medicine; D005990:Glycerol; D006197:Hair / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012091:Repetitive Sequences, Nucleic Acid; D016622:Silver Staining
D005456:Fluorescent Dyes; D004247:DNA; D005990:Glycerol