Automated fluorescence analysis of polymerase chain reaction (PCR)-amplified short tandem repeat (STR) systems by capillary electrophoresis (CE) is becoming an established tool both in forensic casework and in the implementation of both state and national convicted offender DNA databases. A new capillary electrophoresis instrument, the ABI Prism 310 Genetic Analyzer, along with the Performance Optimized Polymer 4 (POP-4) provides an automated and precise method for simultaneously analyzing ten fluorescently labeled STR loci from a single PCR amplification kit, which provides a power of discrimination of approximately one in five billion from a single PCR amplification. Data are presented on sizing precision, sizing accuracy, and resolution for the STR loci in the AmpFlSTR Profiler kit. Sizing accuracy is highly dependent on the electrophoresis system, and therefore the reporting of alleles based on the nucleotide size obtained from an electrophoresis system is not recommended for forensic work. The precision of the 310 capillary electrophoresis system, coupled with software developed for automated genotyping of alleles based on the use of an allelic ladder, allows for accurate genotyping of STR loci. Sizing precision of < or = 0.16 nucleotide standard deviation was obtained with this system, thus allowing for accurate genotyping of length variants that differ in length by a single nucleotide.

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Laboratory procedures used in short tandem repeat (STR) analysis were subjected to various scenarios that assessed reliability and identified potential limitations. These validation studies were designed as recommended by the Technical Working Group on DNA Analysis Methods (TWGDAM) and the DNA Advisory Board (DAB) (17,18). Various DNA samples were amplified by the polymerase chain reaction (PCR) using AmpFᐉSTR™ PCR Amplification Kits (i.e., AmpFᐉSTR Green I, Profiler™, Profiler Plus™, and COfiler™ kits), detected with ABI Prism instrumentation, and analyzed using GeneScan and Genotyper software. Data acquired in these studies reinforced an existing body of knowledge and expertise regarding application and interpretation of STR typing in the forensic science community. Consistent STR genotypes were detected in various body tissues and fluids. Inter-laboratory comparisons produced concordant genotype results. Quantitative interpretational aids for DNA mixtures were characterized. Ability of the typing systems to type potentially compromised samples reliably was evaluated. Nonprobative case evidentiary DNA was successfully amplified, genotyped, and interpreted. Potential limitations or cautionary factors in the interpretation of minimal fluorescence intensity were demonstrated. Differential amplification between loci was observed when PCR was inhibited; preferential amplification typically was not. Single AmpFᐉSTR locus amplification did not offer consistent benefit over AmpFᐉSTR multiplexing, even in cases of DNA degradation or PCR inhibition. During rigorous evaluation, AmpFᐉSTR PCR Amplification Kits reproducibly yielded sensitive and locusspecific results, as required in routine forensic analyses.

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Despite compelling epidemiological evidence that folic acid supplements reduce the frequency of neural tube defects (NTDs) in newborns, common variant association studies with folate metabolism genes have failed to explain the majority of NTD risk. The contribution of rare alleles as well as genetic interactions within the folate pathway have not been extensively studied in the context of NTDs. Thus, we sequenced the exons in 31 folate-related genes in a 480-member NTD case-control population to identify the full spectrum of allelic variation and determine whether rare alleles or obvious genetic interactions within this pathway affect NTD risk. We constructed a pathway model, predetermined independent of the data, which grouped genes into coherent sets reflecting the distinct metabolic compartments in the folate/one-carbon pathway (purine synthesis, pyrimidine synthesis, and homocysteine recycling to methionine). By integrating multiple variants based on these groupings, we uncovered two provocative, complex genetic risk signatures. Interestingly, these signatures differed by race/ethnicity: a Hispanic risk profile pointed to alterations in purine biosynthesis, whereas that in non-Hispanic whites implicated homocysteine metabolism. In contrast, parallel analyses that focused on individual alleles, or individual genes, as the units by which to assign risk revealed no compelling associations. These results suggest that the ability to layer pathway relationships onto clinical variant data can be uniquely informative for identifying genetic risk as well as for generating mechanistic hypotheses. Furthermore, the identification of ethnic-specific risk signatures for spina bifida resonated with epidemiological data suggesting that the underlying pathogenesis may differ between Hispanic and non-Hispanic groups.