R. M. Fourney, C.J. Fregeau, K.L. Bowen, G.G. Shutler, J.C. Elliott, L. Klevan, J.
Hartley, A. Hu and G.R. Carmody
Biology Research and Development, Central Forensic Laboratory, Royal Canadian Mounted
Police, Ottawa, Ontario, Canada K1G 3M8
ABSTRACT
The anticipated change from conventional RFLP analysis and PCR dot blot format to detection of STR alleles based on size determination using various detection methods (i.e. silver staining or fluorescent labeling) requires careful determination of the limitations of instrument precision and evaluation of potential biological allele sequence variation. The measurements of instrument precision and reproducibility of representative alleles from seven STR systems (FGA, D21S11, THO1, vWA, FES/FPS, F13A1, Amelogenin and several prototype STR systems) were determined using different internal molecular weight standards (i.e. ABD GS 2500, ABD GS 500 and BRL prototype 540 base ladder). Allele sizes were calculated by the local Southern method and reproducibility assessed from replicate sample runs. Precision was plotted across the seven loci for each representative allele between individual lanes with the same gel (intragel) and compared to independent sample runs between gels (intergel). Intragel precision was determined to be 0.5 bases regardless of the STR locus and intergel precision 0.75 bases (taking into account a worst case scenario). Differences in precision of allele size estimation were noted using different internal lane standards. This presumably is due to characteristics of the marker (i.e. a dual label on both strands versus single strand tagging; the potential for split peaks after denaturing electrophoresis; strategic spacing of rungs in the molecular weight ladder standards). Acceptable standards of measurement precision for declaration of a match are discussed in context with internal lane standards.
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