Validation of Highly Polymorphic Fluorescent Multiplex Short Tandem Repeat Systems Using Two Generations of DNA Sequencers

Abstract

HumD21S11 and HumFGA (multiplex 1B) have been evaluatedWe reported earlier (24,25) the development of a fluorescencefor use in forensic identification using the Applied Biosystems based multiplex DNA profiling system comprising two highly Model 373A and Prism 377 DNA Sequencers, respectively. Experiments were aimed at defining the limit of target DNA polymorphic STR loci, HumD21S11 (26) and HumFGA (27), and required for reliable profiling, the level of degradation that would the amelogenin gender determination system (28,29). In the present still permit amplification of the short tandem repeat (STR) loci report, we document the extensive validation studies performed examined, and the robustness of each locus in the multiplexes after and casework experience gained using this triplex system on the samples were exposed to environmental insults. In addition, the Applied Biosystems Model 373A DNA Sequencer. Validation specificity of the multiplexes was demonstrated using nonhuman DNAs. Forensically relevant samples such as cigarette butts, chewexperiments were also conducted using a modified triplex comprising gum, fingernails and envelope flaps were processed using both ing HumD3S1358 (30), HumD21S11 and HumFGA on the secondan organic extraction procedure and a QIAamp protocol. DNAs and generation ABI Prism 377 DNA Sequencer. Initial experiments resultant multiplex STR profiles were compared. The validation of were designed to determine the optimal amount of target DNA for the triplex STR systems was extended to include over 140 nonproreliable amplification results on both analytical instruments. As a bative casework specimens and was followed with a close monitoring of initial casework (over 300 exhibits). Our results document complement to previous reports documenting the suitability (quanthe robustness of these multiplex STR profiling systems which, tity and quality) of DNA extracted from specimens under extreme when combined with other multiplex systems, could provide a environmental conditions for RFLP profiling (31-34) or power of discrimination of approximately 0.9999. 16,[35][36][37][38][39][40], this paper further examined adverse conditions relevant to fluorescence-based amplifica-

Forensic evaluation of the QIAshredder/QIAamp DNA extraction procedure

“…Several authors have reported that the QIAamp DNA Mini Kit performed as well as or better than other extraction protocols [3,[7][8][9]. Results from the present study indicated that the use of the QIAshredder column in conjunction with the QIAamp system may further improve the amount of DNA recovered from blood and saliva on swabs (Table 1), as well as the quality of the subsequent SGM Plus profiles (data not showed).…”

“…First, more DNA was extracted from the chewing gum using the Chelex protocol (Sample D of the food remains; Table 1). Fregeau et al [8] reported that a PC protocol overcame the QIAamp procedure to recover DNA from this type of sample. This could result from interferences between the silica matrix and one of the chewing-gum components.…”

“…Others, such as Chelex [4,5], enable to recover substantial amounts of DNA, but may be less efficient to remove PCR inhibitors [6,7]. Several authors [3,[7][8][9] have reported that a relatively new extraction procedure, based on Silica membrane (QIAamp DNA Mini Kit), may overcome Chelex and PC protocols for some categories of samples. Sinclair and McKechnie [10] further showed that the treatment of stamps and envelop flaps with a homogenizing column (QIAshredder) can further improve the QIAamp extraction.…”

Genetics and Genomics of Core Short Tandem Repeat Loci Used in Human Identity Testing

“…While early work with STRs involved detection on silver‐stained polyacrylamide gels (17), the community has embraced fluorescence detection methods involving first gel electrophoresis (10,12,13) and then capillary electrophoresis with such instruments as the ABI 310 and ABI 3100 Genetic Analyzers (18). Over the years, the ABI 373 and 377 gel‐based DNA sequencers have also played a significant role in forensic DNA typing (19). …”

Trends in DNA forensic analysis

“…A detailed description of developmental validation requirements are given in [5]. Indeed, an extensive validation process always accompanied implementation of new methodologies, such as multiplexed loci systems (based on Promega and ABI kits) and new running procedures [29][30][31][32][33][34][35][36][37]. The discriminating power of new STR systems was examined using multiple population studies [38][39][40][41].…”