A modular injection system, multilevel sampler, and manifold for tracer tests.

Ground water injection and sampling systems were developed for bacterial transport experiments in both homogenous and heterogeneous unconsolidated, surficial aquifers. Two types of injection systems, a large single tank and a dynamic mixing tank, were designed to deliver more than 800 L of amended ground water to the aquifer over 12 hours, without altering the ground water temperature, pH, Eh, or dissolved gas composition. Two types of multilevel samplers (MLSs) were designed and installed. Permanent MLSs performed well for the homogenous surficial aquifer, but their installation procedure promoted vertical mixing, which could obfuscate experimental data obtained from vertically stratified, heterogeneous aquifers. A novel, removable MLS was designed to fit in 2- and 4-inch wells. Expandable O-rings between each sampling port hydraulically isolated each port for sample collection when a nut was tightened at the land surface. A low-cost vacuum manifold system designed to work with both MLS designs used 50 mL centrifuge tubes to efficiently sample 12 MLS ports with one peristaltic pump head. The integrated system was developed and used during four field campaigns over a period of three years. During each campaign, more than 3000 ground water samples were collected in less than one week. This system should prove particularly useful for ground water tracer, injection, and push-pull experiments that require high-frequency and/or high-density sampling.

Natural and iatrogenic variation in hepatitis B virus.

The existence of HBV as quasispecies is thought to be favoured by the infidelity of HBV RT, which would account for the emergence of the many natural mutants with point substitutions. RT infidelity may also underlie the hypermutation phenomenon. Indeed, the oft-reported point mutation in the preC gene that leads to failure of HBeAg synthesis may be driven by a hypermutation-related mechanism. The presence of mutants with deletions and insertions involving single nucleotides and oligonucleotides at specific positions in the genome, and of mutants with deletions of even longer stretches particularly in the C gene, suggests that other mutagenic mechanisms operate. Candidates include slippage during mispairing between template and progeny DNA strand, the action of cellular topoisomerase I, and gene splicing using alternative donor and acceptor sites. Natural substitutions, deletions or insertions involving the Cp/ENII locus in the X gene can significantly alter the extent of viral replicative activity. Similar mutations occurring at other locations of Cp/ENII, and at B-cell epitope sites of the S gene are associated with failure to detect serological markers of HBV infection. HBV variation can also arise from recombination between coinfecting strains. S gene mutations that become evident following HBIG administration and HBV vaccination are all point substitutions, as are mutations in functional RT domains of the P gene after treatment with viral RT-inhibitory drugs. Widespread and long-term use of prophylactic and therapeutic agents may potentially generate serologically occult HBV variants that might become difficult to eradicate.

Further validation of a multiplex STR system for use in routine forensic identity testing.

A polymerase chain reaction- (PCR) based short tandem repeat (STR) system has recently been developed for use in routine forensic identity testing. The methodology involves the simultaneous amplification of alleles at four loci on different chromosomes, followed by the fluorescent detection of products using an automated DNA sequencer. The adoption of this technology into operational casework offers several advantages over systems currently in use, particularly the ability to obtain results from very old or small samples, reduced operator time when compared with conventional DNA (single locus probe) analysis and the potential for automation. Validation studies were incorporated into the development work on this system. The scope of these studies has been extended by further investigation carried out in this laboratory to test the reliability of the system under normal operational procedures. It was demonstrated that the precision of size determination was sufficient for the discrimination of alleles and size windows for allelic designation were established. A collaborative exercise carried out in conjunction with two independent laboratories demonstrated the robustness of allelic designation. Having tested both the DNA quantification and amplification techniques against DNA samples from a wide range of animal and microbial species, it was confirmed that results are only obtained from higher primate DNA. The PCR methodology was tested with both simulated and real casework samples (over 250 in total). Reportable results were obtained from most items yielding extracted DNA. Approximately 20% of the casework items from which no grouping (ABO, PGM) nor SLP results were obtained, gave reportable STR results. A method for the routine purification of DNA extracts which failed to amplify was established and validated for use in forensic casework. The STR multiplex system developed by Kimpton et al. proved robust and reliable when tested under the operational procedures in place in this laboratory.