David D. Duncan

Automated analysis of sequence polymorphism in STR alleles by PCR and direct electrospray ionization mass spectrometry

Short tandem repeats (STRs) are the primary genetic markers used for the analysis of biological samples in forensic and human identity testing. The discrimination power of a combination of STRs is sufficient in many human identity testing comparisons unless the evidence is substantially compromised and/or there are insufficient relatives or a potential mutation may have arisen in kinship analyses. An automated STR assay system that is based on electrospray ionization mass spectrometry (ESI-MS) has been developed that can increase the discrimination power of some of the CODIS core STR loci and thus provide more information in typical and challenged samples and cases. Data from the ESI-MS STR system is fully backwards compatible with existing STR typing results generated by capillary electrophoresis. In contrast, however, the ESI-MS analytical system also reveals nucleotide polymorphisms residing within the STR alleles. The presence of these polymorphisms expands the number of alleles at a locus. Population studies were performed on the 13 core CODIS STR loci from African Americans, Caucasians and Hispanics capturing both the length of the allele, as well as nucleotide variations contained within repeat motifs or flanking regions. Such additional polymorphisms were identified in 11 of the 13 loci examined whereby several nominal length alleles were subdivided. A substantial increase in heterozygosity was observed, with close to or greater than 5% of samples analyzed being heterozygous with equal-length alleles in at least one of five of the core CODIS loci. This additional polymorphism increases discrimination power significantly, whereby the seven most polymorphic STR loci have a discrimination power equivalent to the 10 most discriminating of the CODIS core loci. An analysis of substructure among the three population groups revealed a higher θ than would be observed compared with using alleles designated by nominal length, i.e., repeats solely. Two loci, D3S1358 and vWA produced θ estimates of 0.0477 and 0.0234, respectively, when the expanded allele complement (i.e., nominal allele and SNPs) was considered compared to 0.0145 and 0.01266, respectively when only nominal repeat number was considered. These differences may indicate underlying population specific allele distributions exist within these populations. A system of nomenclature has been developed that facilitates the databasing, searching and analyses of these combined data forms.

Rapid and High-Throughput pan-Orthopoxvirus Detection and Identification using PCR and Mass Spectrometry

The genus Orthopoxvirus contains several species of related viruses, including the causative agent of smallpox (Variola virus). In addition to smallpox, several other members of the genus are capable of causing human infection, including monkeypox, cowpox, and other zoonotic rodent-borne poxviruses. Therefore, a single assay that can accurately identify all orthopoxviruses could provide a valuable tool for rapid broad orthopovirus identification. We have developed a pan-Orthopoxvirus assay for identification of all members of the genus based on four PCR reactions targeting Orthopoxvirus DNA and RNA helicase and polymerase genes. The amplicons are detected using electrospray ionization-mass spectrometry (PCR/ESI-MS) on the Ibis T5000 system. We demonstrate that the assay can detect and identify a diverse collection of orthopoxviruses, provide sub-species information and characterize viruses from the blood of rabbitpox infected rabbits. The assay is sensitive at the stochastic limit of PCR and detected virus in blood containing approximately six plaque-forming units per milliliter from a rabbitpox virus-infected rabbit.

Microbial Forensic Analysis of Trace and Unculturable Specimens

Publisher Summary This chapter demonstrates the use of whole genome amplification of DNA polymerase based on multiple displacement amplification for microbial forensic PCR/ESI-MS analysis of environmental air. It also demonstrates the utility of WGA of trace DNA specimens coupled with either Roche 454 sequencing technology or Pacific Biosciences single molecule real-time sequencing technology. Challenges remain, however. Whole genome amplification by multiple displacement amplification will amplify all DNA in a specimen, which can create challenges for detection and characterization of an agent of interest in the presence of a large amount of background. Soil samples and clinical specimens present such challenges. For example, 1 μl of human blood contains 1000 copies of the human genome so identifying a pathogen in this background would necessitate >1000 human genome projects of sequencing to find the pathogen DNA. Fortunately, there are many methods for the separation of organisms by size that can be used to separate viruses from bacteria from eukaryotic cells prior to DNA extraction and WGA. Next Gen sequencing technologies, combined with whole genome amplification, offer the forensic investigator new capabilities for the rapid microbial forensic characterization of trace and noncultivable specimens that can identify key signatures that can lead to attribution, as well as identifying bioengineered and emerging threats.