Internal Validation of RapidHIT® ID ACE Sample Cartridge and Assessment of the EXT Sample Cartridge*†.

A new rapid DNA solution, the RapidHIT® ID, can accommodate two different sample cartridges, ACE, for the analysis of a single swab and EXT, for the analysis of DNA extracts. An efficient internal validation designed for low-throughput rapid DNA is described. An evaluation of the EXT sample cartridge is also described. Each cartridge generated profiles with sufficient data quality to meet CODIS eligibility in fewer than 120 min. The results exhibited 100% correlation when compared to conventional DNA typing methods. Precision, reproducibility, stochastic, mixture, and contamination experiments produced expected results. Sensitivity of the ACE sample cartridge was acceptable for buccal swab analysis. The sensitivity of the EXT sample cartridge is discussed. The ACE validation and the EXT evaluation utilized a minimalist, cost-saving, efficient design to generate a validated RapidHIT® ID instrument capable of producing genetic profiles from both extracted forensic DNA samples and buccal swab samples within 120 min.

Quantifying in vivo somatic mutations using transgenic mouse model systems.

This chapter describes the use of the bacteriophage cII positive selection somatic mutational assay with the Muta™Mouse transgenic model system. The assay is similar to others involving a transgenic target, including the cII and lacI assays in the Big Blue(®) Mouse, lacZ in the MutaMouse, and the gpt delta assay. Briefly, high-molecular-weight DNA is purified from the tissue of interest and used as substrate during in vitro packaging reactions, where the λ transgenes are excised from the genome and assembled into viable phage. Phage containing the mutational targets is then adsorbed into an appropriate bacterial host, and mutations sustained in vivo are detected and quantified by either standard recombinant screening or selection assays. Mutant frequencies are reported as the ratio of mutant phage to total phage units analyzed. The λ-based transgenic mouse assays are used to study and characterize in vivo mutagenesis as well as for mutagenicity assessment of chemicals and other agents. These models permit the enumeration of mutations sustained in virtually any tissue of the mouse and are both sensitive and robust. Application of the assays is simple, not requiring resources beyond those commonly found in most academic laboratories.

Strain discrimination among B. anthracis and related organisms by characterization of bclA polymorphisms using PCR coupled with agarose gel or microchannel fluidics electrophoresis.

The bclA gene codes for the protein backbone of the exosporium glycoprotein BclA of B. anthracis. BclA has a central collagen-like region formed by polymorphic GXX repeats and conserved amino- and carboxy-termini. It is noted here that the bclA gene is also present in the genome of Bacillus cereus and Bacillus thuringiensis. There is considerable size heterogeneity among the BclA proteins, both for species and strains, due to different numbers of GPT repeats and [GPT]5GDTGTT repeats (BclA repeats). PCR products that included the entire variable region were analyzed by conventional agarose gel electrophoresis and by micro-channel fluidics (MCF) LabChip to assess differences in molecular weight (MW). Both methods provided discrimination at the strain level for B. cereus group organisms. Results obtained by MCF electrophoresis were superior to conventional agarose gel analysis demonstrating improved reproducibility and much faster analysis time. The expression of a carbohydrate-rich exosporium (corresponding to BclA) in other members of the B. cereus group, in addition to B. anthracis, was also demonstrated ultra-structurally. Analysis of sequence variability within the bclA gene CLR revealed even greater potential for strain and species identification.

Quantifying in vivo somatic mutations using transgenic mouse model systems.

This chapter describes the use of the bacteriophage cII positive selection assay with the MutaMouse transgenic model system. The assay is similar to others involving a transgenic target, including the cII and lacI assays in the Big Blue(R) Mouse, lacZ in the MutaMouse, and the gpt delta assay. Briefly, high-molecular-weight DNA is purified from the tissue of interest and used as substrate during in vitro packaging reactions, in which the lambda transgenes are excised from the genome and assembled into viable phage. Phage containing the mutational targets are then adsorbed into an appropriate bacterial host, and mutations sustained in vivo are evidenced by either standard recombinant screening or selection assays. Mutant frequencies are reported as the ratio of mutant phage to total phage units analyzed. The lambda-based transgenic mouse assays are used to study and characterize in vivo mutagenesis, as well as for mutagenicity assessment. The models permit the enumeration of mutations sustained in virtually any tissue of the mouse and are sensitive and robust. Application of the assays is simple, not requiring resources beyond those commonly found in most academic laboratories.