The specifications of forensic genotyping assays

The choice of genetic markers and assay technologies needs to be carefully considered if these technologies are to be employed in forensic genotype data banking. A decade ago, the forensic science community recognized in autosomal STRs a general genotyping technology with the potential for responding to data banking and casework needs (Kimpton et al., 1993; Urquhart et al., 1994; Lygo et al., 1994). Since then, forensic laboratory facilities have developed considerable experience and invested heavily in an installed base of this technology. The platform has proven robust and cost-effective, performs well in a variety of operational contexts and, importantly, allows for casework mixed-source samples to be analysed. Although many other genetic markers, e.g. mtDNA (Hagelberg et al., 1991; Sullivan et al., 1992), SNPs (Gill, 2001; Gill et al., 2004) and Y-STRs (Hall and Ballantyne, 2003; Schoske et al., 2004), have been developed and are put to use in specific operational contexts, the current autosomal STR system is likely to remain the reference platform for forensic DNA analysis platforms for the foreseeable future (Jobling and Gill, 2004). This will not impede development work on wet chemistry components of this assay as throughput improvements are expected to emerge from the implementation of thermal cycling and electrophoresis capabilities embarked on micro-fabricated devices.

Single tandem repeat genotyping is accomplished through multiplex PCR reactions designed to amplify up to 16 genetic markers or 32 different allelic targets in a single reaction. Its range of alleles and the presence of 1 bp variants confer high discrimination potential to the system and can be resolved with electrophoresis instrumentation designed for DNA sequencing applications. Several multiplexes configured for forensic application are commercially available and produce electropherograms balanced for intra- and inter-locus signal strength over a variety of forensic sample types. Precision and accuracy in allele calling are achieved on electrophoresis platforms with floating bins through the use of commercially available allelic ladders. A variable and generally small percentage of samples will either experience some random processing anomaly or present a rare but normal feature, both of which will be detected on electro-pherograms. These anomalies (e.g. 'spike', pull-up, saturated and split peaks, elevated baseline, heterozygote ratio imbalance, elevated stutter, profile slope), at times, may interfere with accurate allele calling, or cause the affected sample to fall outside of quality control specifications. Rare features may prove to be variant alleles, tri-allelic loci or unfavourable heterozygous ratio at a given locus as a consequence of a sequence polymorphism under a primer annealing site. The interpretation uncertainty associated with both random processing anomalies and rare genetic features is normally resolved by re-working the sample through the analytical platform: the latter condition will be replicated, the former will be resolved.

From an automation design perspective, the STR technology is stable - a desirable attribute as it limits the scope and costs of software maintenance/ upgrades. The labour-intensive procedure involved in STR typing offers substantial opportunities for quality, reliability and productivity improvements through the introduction of automated solutions.

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