Recent developments in DNA evidence

DNA evidence has made a significant contribution to criminal investigations in Australia and around the world since it was widely adopted in the 1990s (Gans & Urbas 2002). The direct matching of DNA profiles, such as comparing one obtained from a crime scene with one obtained from a suspect or database, remains a widely used technique in criminal investigations. A range of new DNA profiling techniques continues to be developed and applied in criminal investigations around the world (Smith & Urbas 2012). This paper is the third in a series by the Australian Institute of Criminology (AIC) on DNA evidence. The first, published in 1990 when the technology was in its relative infancy, outlined the scientific background for DNA evidence, considered early issues such as scientific reliability and privacy and described its application in early criminal cases (Easteal & Easteal 1990). The second, published in 2002, expanded on the scientific background and discussed a significant number of Australian cases in a 12-year period, illustrating issues that had arisen in investigations, at trial and in the use of DNA in the review of convictions and acquittals (Gans & Urbas 2002). There have been some significant developments in the science and technology behind DNA evidence in the 13 years since 2002 that have important implications for law enforcement and the legal system. These are discussed through a review of relevant legal cases and the latest empirical evidence. This paper is structured in three sections. The first examines the scientific techniques and how they have been applied in police investigations, drawing on a number of recent cases to illustrate them. The second considers empirical research evaluating DNA evidence and databases and the impact DNA has on investigative and court outcomes. The final section discusses significant cases that establish legal precedent relating to DNA evidence in criminal trials where significant issues have arisen or new techniques have been applied that have not yet been widely discussed in the literature. The paper concludes by reflecting on implications for policy and practice.

DNA markers linked to the R(2) rust resistance gene in sunflower (Helianthus annuus L.) facilitate anticipatory breeding for this disease variant.

Pre-emptive breeding for host disease resistance is an effective strategy for combating and managing devastating incursions of plant pathogens. Comprehensive, long-term studies have revealed that virulence to the R (2) sunflower (Helianthus annuus L.) rust resistance gene in the line MC29 does not exist in the Australian rust (Puccinia helianthi) population. We report in this study the identification of molecular markers linked to this gene. The three simple sequence repeat (SSR) markers ORS795, ORS882, and ORS938 were linked in coupling to the gene, while the SSR marker ORS333 was linked in repulsion. Reliable selection for homozygous-resistant individuals was efficient when the three markers, ORS795, ORS882, and ORS333, were used in combination. Phenotyping for this resistance gene is not possible in Australia without introducing a quarantinable race of the pathogen. Therefore, the availability of reliable and heritable DNA-based markers will enable the efficient deployment of this gene, permitting a more effective strategy for generating sustainable commercial cultivars containing this rust resistance gene.

Reversal of handedness in DNA: A stable link between RU and LZ helices

Two types of left-handed zig-zag (LZ) helices were obtained following stereochemical guideline. They are referred to as LZ1 and LZ2 helices. LZ1 helices have conformations similar to those found in the single crystals of d(C-G)3 and d(C-G)25,6. Z-character is more prominent in LZ2 than in LZ1 helix. The conformations of a stable link between RU and LZ helical fragments are given. The link involves inverted stacking arrangement of the bases: a characteristic feature of all RL models proposed by us

A comparison of DNA pools constructed following whole genome amplification for two-stage SNP genotyping designs

Genotyping in DNA pools reduces the cost and the time required to complete large genotyping projects. The aim of the present study was to evaluate pooling as part of a strategy for fine mapping in regions of significant linkage. Thirty-nine single nucleotide polymorphisms (SNPs) were analyzed in two genomic DNA pools of 384 individuals each and results compared with data after typing all individuals used in the pools. There were no significant differences using data from either 2 or 8 heterozygous individuals to correct frequency estimates for unequal allelic amplification. After correction, the mean difference between estimates from the genomic pool and individual allele frequencies was .033. A major limitation of the use of DNA pools is the time and effort required to carefully adjust the concentration of each individual DNA sample before mixing aliquots. Pools were also constructed by combining DNA after Multiple Displacement Amplification (MDA). The MDA pools gave similar results to pools constructed after careful DNA quantitation (mean difference from individual genotyping .040) and MDA provides a rapid method to generate pools suitable for some applications. Pools provide a rapid and cost-effective screen to eliminate SNPs that are not polymorphic in a test population and can detect minor allele frequencies as low as 1% in the pooled samples. With current levels of accuracy, pooling is best suited to an initial screen in the SNP validation process that can provide high-throughput comparisons between cases and controls to prioritize SNPs for subsequent individual genotyping.

Separation of 5-methylcytosine-rich DNA using immobilized antibody

Anti-(5-methylcytosine) antibodies were immobilized on glutaraldehyde-activated Indion 48-R, a polystyrene resin with amino groups. Immobilized antibody was very stable and could be used several times without any apparent change in the initial binding capacity of the antibody-matrix. Fractions of total DNA from various animal and plant sources were retained on this column and could be eluted quantitatively with 1.0 m NaCl. The bound fraction was further characterized for its 5-methylcytosine content by restriction enzyme digestion patterns.

Comparative analysis of Tritrichomonas foetus (Riedmuller, 1928) cat genotype, T. foetus (Riedmuller, 1928) cattle genotype and Tritrichomonas suis (Davaine, 1875) at 10 DNA loci

The parasitic protists in the genus Tritrichomonas cause significant disease in domestic cattle and cats. To assess the genetic diversity of feline and bovine isolates of Tritrichomonas foetus (Riedmuller, 1928) Wenrich and Emmerson, 1933, we used 10 different genetic regions, namely the protein coding genes of cysteine proteases 1,2 and 4-9 (CP1, 2, 4-9) involved in the pathogenesis of the disease caused by the parasite. The cytosolic malate dehydrogenase 1 (MDH1) and internal transcribed spacer region 2 of the rDNA unit (ITS2) were included as additional markers. The gene sequences were compared with those of Tritrichomonas suis (Davaine. 1875) Morgan and Hawkins, 1948 and Tritrichomonas mobilensis Culberson et al., 1986. The study revealed 100% identity for all 10 genes among all feline isolates (=T. foetus cat genotype), 100% identity among all bovine isolates (=T. foetus cattle genotype) and a genetic distinctness of 1% between the cat and cattle genotypes of T. foetus. The cattle genotype of T. foetus was 100% identical to T. suis at nine loci (CP1, 2,4-8, ITS2, MDH1). At CP9, three out of four T. suis isolates were identical to the T. foetus cattle genotype, while the T. suis isolate SUI-H3B sequence contained a single unique nucleotide substitution. Tritrichomonas mobilensis was 0.4% and 0.7% distinct from the cat and cattle genotypes of T. foetus, respectively. The genetic differences resulted in amino acid changes in the CP genes, most pronouncedly in CP2, potentially providing a platform for elucidation of genotype-specific host-pathogen interactions of T. foetus. On the basis of this data we judge T. suis and T. foetus to be subjective synonyms. For the first time, on objective nomenclatural grounds, the authority of T. suis is given to Davaine, 1875, rather than the commonly cited Gruby and Delafond, 1843. To maintain prevailing usage of T. foetus, we are suppressing the senior synomym T. suis Davaine, 1875 according to Article 23.9, because it has never been used as a valid name after 1899 and T. foetus is widely discussed as the cause of bovine trichomonosis. Thus bovine, feline and porcine isolates should all be given the name T. foetus. This promotes the stability of T. foetus for the veterinary and economically significant venereal parasite causing bovine trichomonosis. (C) 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Polymorphism and conformational flexibility of DNA: right and left handed duplexes

Left handed duplexes are shown to be in agreement with the X-ray intensity data of A-, B- and D-forms of DNA. The structures are stereochemically satisfactory because they were obtained following a stereochemical guideline derived from theory and single crystal structure data of nucleic acid components. The same stereochemical guideline also led to right handed duplexes for B- and D-forms of DNA which have stereochemically preferred conformation and hence are superior to those given by Arnott and coworkers.

Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife

Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking log of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value = 1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated species in situ. This real-time PCR assay offers an alternative highly specific and sensitive diagnostic method for use in Trichinella wildlife surveillance and could be adapted to wildlife hosts of any region. (C) 2012 Elsevier B.V. All rights reserved.

Possible involvement of a calcium-stimulated ATP-hydrolyzing activity associated with mycobacteriophage I3 in the DNA injection process.

Ca2+ ions are necessary for the successful propagation of mycobacteriophage I3. An assay for the phage DNA release in the presence of an isolated cell wall preparation from the host was established, and in this system Ca2+ ions also stimulated the release of DNA. The inhibition of phage DNA injection caused by Tween 80 (polyoxyethylene sorbitan monooleate), a nonionic detergent routinely used in mycobacterial cultures, was reversed by Ca2+. The presence of a phage-associated ATP-hydrolyzing activity was demonstrated. This enzyme was stimulated by Ca2+ ions and inhibited by Tween 80. From this and the behavior of the two agents at the level of DNA injection, as well as the fact that phage I3 has a contractile tail structure, we conclude that the phage-associated ATPase is involved in the DNA injection process.

A need for re-examination of the fibre diffraction data of A-DNA

A-DNA pattern, obtained using a flat plat camera, was indexed by Fuller Image on the basis of a c-face centred monoclinic cell with A = 22.24 Å, B = 40.62 Å, C = 28.15 Å and β = 97.0°. A precession photograph of A-DNA which gives an undistorted picture of the lattice, showed that the unit cell parameters as given by Fuller Image were not quite correct. The precession photograph showed a strong meridional reflection (R = 0.00 Å−1) on the 11th layer line. But the occurrence of the meridional reflection on the 11th layer line could not be explained on the basis of the cell parameters given by Fuller Image ; using those cell parameters the reflection which comes closest to the meridian on 11th layer line is at R = 0.025 Å−1. However, a simple interchange of a and b values accounted for the meridional reflection on 11th layer line. The corrected cell parameter refined against 28 strong spots are A = 40.75 Å, B = 22.07 Å, C = 28.16 Å and β = 97.5°. In the new unit cell of A-DNA, the packing arrangement of the two molecules is different from that in the old one. Nonetheless, our earlier contention is again reaffirmed that both right and left-handed A-DNA are stereochemically allowed and consistent with the observed fibre pattern.

Determining changes in the distribution and abundance of a Rhyzopertha dominica phosphine resistance allele in farm grain storages using a DNA marker

BACKGROUND: The lesser grain borer, Rhyzopertha dominica (F.), is a highly destructive pest of stored grain that is strongly resistant to the fumigant phosphine (PH3). Phosphine resistance is due to genetic variants at the rph2 locus that alter the function of the dihydrolipoamide dehydrogenase (DLD) gene. This discovery now enables direct detection of resistance variants at the rph2 locus in field populations. RESULTS: A genotype assay was developed for direct detection of changes in distribution and frequency of a phosphine resistance allele in field populations of R. dominica. Beetles were collected from ten farms in south-east Queensland in 2006 and resampled in 2011. Resistance allele frequency increased in the period from 2006 to 2011 on organic farms with no history of phosphine use, implying that migration of phosphine-resistant R. dominica had occurred from nearby storages. CONCLUSION: Increasing resistance allele frequencies on organic farms suggest local movement of beetles and dispersal of insects from areas where phosphine has been used. This research also highlighted for the first time the utility of a genetic DNA marker in accurate and rapid determination of the distribution of phosphine-resistant insects in the grain value chain. Extending this research over larger landscapes would help in identifying resistance problems and enable timely pest management decisions. © 2013 Society of Chemical Industry © 2013 Society of Chemical Industry 69 6 June 2013 10.1002/ps.3514 Rapid Report Rapid Report © 2013 Society of Chemical Industry.

Requirement for calcium ions in mycobacteriophage I3 DNA injection and propagation

Ca2+ ions are absolutely necessary for the propagation of mycobacteriophage I3 in synthetic medium. These ions are required for successful infection of the host and during the entire span of the intracellular development of the phage. A direct assay of the phage DNA injection using 32[P] labelled phage, showns that Ca2+ ions are necessary for the injection process. The injection itself is a slow process and takes 15 min to complete at 37°C. The bacteria infected in presence of Ca2+ tend to abort if the ions are subsequently withdrawn from the growth medium. The effect of calcium withdrawal is maximally felt during the early part of the latent period; however, later supplementation of Ca2+ ions salvage phage production and the mature phage progeny appear after a delayed interval, proportional to the time of addition of Ca2+.

Electrotransformation of Haemophilus parasuis with in vitro modified DNA based on a novel shuttle vector

The objective of the present study was to establish a valid transformation method of Haemophilus parasuis, the causative agent of Glasser's disease in pigs, using a novel H. parasuis-Escherichia coli shuttle vector. A 4.2 kb endogenous plasmid pYC93 was extracted from an H. parasuis field isolate and completely sequenced. Analysis of pYC93 revealed a region approximately 800 bp showing high homology with the defined replication origin oriV of pLS88, a native plasmid identified in Haemophilus ducreyi. Based on the origin region of pYC93, E. coli cloning vector pBluescript SK(+) and the Tn903 derived kanamycin cassette, a shuttle vector pSHK4 was constructed by overlapping PCR strategy. When electroporation of the 15 H. parasuis serovar reference strains and one clinical isolate SH0165 with pSHK4 was performed, only one of these strains yielded transformants with an efficiency of 8.5 x 10(2) CFUhlg of DNA. Transformation efficiency was notably increased (1.3 x 10(5) CFU/mu g of DNA) with vector DNA reisolated from the homologous transformants. This demonstrated that restriction-modification systems were involved in the barrier to transformation of H. parasuis. By utilizing an in vitro DNA modification method with cell-free extracts of the host H. parasuis strains, 15 out of 16 strains were transformable. The novel shuttle vector pSHK4 and the established electrotransformation method constitute useful tools for the genetic manipulation of H. parasuis to gain a better understanding of the pathogen. (C) 2011 Elsevier B.V. All rights reserved.

DNA-based detection and characterisation of strictly anaerobic beer-spoilage bacteria

Megasphaera cerevisiae, Pectinatus cerevisiiphilus, Pectinatus frisingensis, Selenomonas lacticifex, Zymophilus paucivorans and Zymophilus raffinosivorans are strictly anaerobic Gram-stain-negative bacteria that are able to spoil beer by producing off-flavours and turbidity. They have only been isolated from the beer production chain. The species are phylogenetically affiliated to the Sporomusa sub-branch in the class "Clostridia". Routine cultivation methods for detection of strictly anaerobic bacteria in breweries are time-consuming and do not allow species identification. The main aim of this study was to utilise DNA-based techniques in order to improve detection and identification of the Sporomusa sub-branch beer-spoilage bacteria and to increase understanding of their biodiversity, evolution and natural sources. Practical PCR-based assays were developed for monitoring of M. cerevisiae, Pectinatus species and the group of Sporomusa sub-branch beer spoilers throughout the beer production process. The developed assays reliably differentiated the target bacteria from other brewery-related microbes. The contaminant detection in process samples (10 1,000 cfu/ml) could be accomplished in 2 8 h. Low levels of viable cells in finished beer (≤10 cfu/100 ml) were usually detected after 1 3 d culture enrichment. Time saving compared to cultivation methods was up to 6 d. Based on a polyphasic approach, this study revealed the existence of three new anaerobic spoilage species in the beer production chain, i.e. Megasphaera paucivorans, Megasphaera sueciensis and Pectinatus haikarae. The description of these species enabled establishment of phenotypic and DNA-based methods for their detection and identification. The 16S rRNA gene based phylogenetic analysis of the Sporomusa sub-branch showed that the genus Selenomonas originates from several ancestors and will require reclassification. Moreover, Z. paucivorans and Z. raffinosivorans were found to be in fact members of the genus Propionispira. This relationship implies that they were carried to breweries along with plant material. The brewery-related Megasphaera species formed a distinct sub-group that did not include any sequences from other sources, suggesting that M. cerevisiae, M. paucivorans and M. sueciensis may be uniquely adapted to the brewery ecosystem. M. cerevisiae was also shown to exhibit remarkable resistance against many brewery-related stress conditions. This may partly explain why it is a brewery contaminant. This study showed that DNA-based techniques provide useful tools for obtaining more rapid and specific information about the presence and identity of the strictly anaerobic spoilage bacteria in the beer production chain than is possible using cultivation methods. This should ensure financial benefits to the industry and better product quality to customers. In addition, DNA-based analyses provided new insight into the biodiversity as well as natural sources and relations of the Sporomusa sub-branch bacteria. The data can be exploited for taxonomic classification of these bacteria and for surveillance and control of contaminations.

Mitochondrial DNA diversity of Cleruchoides noackae (Hymenoptera: Mymaridae): a potential biological control agent for Thaumastocoris peregrinus (Hemiptera: Thaumastocoridae)

Carpintero and Dellap, (Hemiptera: Thaumastocoridae) is a native Australian sap-feeding insect that has become invasive and seriously damaging to commercially grown in the Southern Hemisphere. Lin and Huber (Hymenoptera: Mymaridae) was recently discovered as an egg parasitoid of the Thaumastocoridae in Australia. Mitochondrial DNA (mtDNA; cytochrome oxidase subunit I, COI) sequence diversity amongst 104 individuals from these native populations revealed 24 sequence haplotypes. The COI haplotypes of individuals collected from the Sydney and Southeast Queensland clustered in distinct groups, indicating limited spread of the insect between the regions. Individuals collected from Perth in Western Australia were represented by four COI haplotypes. Although this population is geographically more isolated from other populations, two COI haplotypes were identical to haplotypes found in the Sydney region. The results suggest that has recently been introduced into Perth, possibly from the Sydney area. The high mtDNA diversity and limited spread that is suggested for is in contrast to the lack of geographic associated mtDNA diversity and extensive spread of . If implemented as a biological control agent, this factor will need to be considered in collecting and releasing .