Bayesian mixture modelling for glass refractive index measurement.

Glass is a common type of physical evidence in forensic science. Broken glass recovered from a suspect may have similar physical characteristics to glass collected at a crime scene and therefore can be used as evidence. Statistical treatment of this evidence involves computing a measure of the weight of evidence. This may be done in a Bayesian framework that incorporates information from the circumstances of the crime. One of the most crucial quantities in this calculation is the assessment of the relative rarity of the characteristics of the glass, essentially the probability distribution used to model the physical characteristics of recovered glass. Typical characteristics used in casework are the elemental composition of glass and the refractive index measurement. There is a considerable body of scientific literature devoted to the modelling of this information. For example a kernel density estimation has been used to model the background population of glass based on the refractive index measurement and a multivariate Gaussian finite mixture model has been used to model the elemental composition of glass. In this paper, we present an alternative approach, the Dirichlet Process Mixture Model, to model the glass refractive index measurement in a Bayesian methodology. A key advantage is that using this method allows us to model the probability density distribution of refractive index measurements in a more flexible way.

RNA/DNA co-analysis from human skin and contact traces--results of a sixth collaborative EDNAP exercise.

The European DNA profiling group (EDNAP) organized a sixth collaborative exercise on RNA/DNA co-analysis for body fluid/tissue identification and STR profiling. The task was to identify skin samples/contact traces using specific RNA biomarkers and test three housekeeping genes for their suitability as reference genes. Eight stains, a skin RNA dilution series and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 22 participating laboratories using RNA extraction or RNA/DNA co-extraction methods. Two sets of previously described skin-specific markers were used: skin1 pentaplex (LCE1C, LCE1D, LCE2D, IL1F7 and CCL27) and skin2 triplex (LOR, KRT9 and CDSN) in conjunction with a housekeeping gene, HKG, triplex (B2M, UBC and UCE). The laboratories used different chemistries and instrumentation. All laboratories were able to successfully isolate and detect mRNA in contact traces (e.g., human skin, palm-, hand- and fingerprints, clothing, car interiors, computer accessories and electronic devices). The simultaneous extraction of RNA and DNA provides an opportunity for positive identification of the tissue source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling. The skin markers LCE1C and LOR and the housekeeping gene marker B2M were detected in the majority of contact traces. Detection of the other markers was inconsistent, possibly due to the low amounts and/or poor quality of the genetic material present in shed skin cells. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid/tissue identification method that can easily be combined with current STR typing technology.

Exploring the recovery and detection of messenger RNA and DNA from enhanced fingermarks in blood.

Often in the examination of bloodstained fingermarks discussion occurs around whether to prioritise the fingerprint evidence or focus on the biological evidence. Collecting a sample for genetic profiling may result in the loss of ridge detail that could have been used for fingerprint comparison. Fingermark enhancement and recovery methods along with sample collection methods could also compromise downstream genetic analysis. Previous forensic casework has highlighted circumstances where, after enhancement had been performed, it would have been extremely valuable to both identify the body fluid and generate a DNA profile from the same sample. We enhanced depletion series of fingermarks made in blood, using single treatments consisting of aqueous amido black, methanol-based amido black, acid yellow and leucocrystal violet, and exposure to long wave UV light. We then extracted the DNA and RNA for profiling, to assess the recovery and detection of genetic material from the enhanced fingermarks. We have shown that genetic profiling of bloodstained fingermarks can be successful after chemical enhancement; however it may still be necessary to prioritise evidence types in certain circumstances. From our results it appears that even with visible bloodstained fingermarks, leucocrystal violet can reduce the effectiveness of subsequent messenger RNA profiling. Aqueous amido black and acid yellow also have adverse effects on messenger RNA profiling of depleted fingermarks with low levels of cellular material. These results help with forensic decision-making by expanding knowledge of the extent of the detrimental effects of blood-enhancement reagents on both DNA profiling and body fluid identification using messenger RNA profiling.

RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains: results of a fourth and fifth collaborative EDNAP exercise.

The European DNA Profiling Group (EDNAP) organized a fourth and fifth collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling. The task was to identify dried menstrual blood and vaginal secretion stains using specific RNA biomarkers, and additionally test 3 housekeeping genes for their suitability as reference genes. Six menstrual blood and six vaginal secretion stains, two dilution series (1/4-1/64 pieces of a menstrual blood/vaginal swab) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 24 participating laboratories, using RNA extraction or RNA/DNA co-extraction methods. Two novel menstrual blood mRNA multiplexes were used: MMP triplex (MMP7, MMP10, MMP11) and MB triplex (MSX1, LEFTY2, SFRP4) in conjunction with a housekeeping gene triplex (B2M, UBC, UCE). Two novel mRNA multiplexes and a HBD1 singleplex were used for the identification of vaginal secretion: Vag triplex (MYOZ1, CYP2B7P1 and MUC4) and a Lactobacillus-specific Lacto triplex (Ljen, Lcris, Lgas). The laboratories used different chemistries and instrumentation and all were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA allowed for positive identification of the tissue/fluid source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling, also from old and compromised casework samples. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid identification method that can easily be combined with current STR typing technology.

Differentiation of drug and non-drug Cannabis using a single nucleotide polymorphism (SNP) assay.

Cannabis sativa is both an illegal drug and a legitimate crop. The differentiation of illegal drug Cannabis from non-drug forms of Cannabis is relevant in the context of the growth of fibre and seed oil varieties of Cannabis for commercial purposes. This differentiation is currently determined based on the levels of tetrahydrocannabinol (THC) in adult plants. DNA based methods have the potential to assay Cannabis material unsuitable for analysis using conventional means including seeds, pollen and severely degraded material. The purpose of this research was to develop a single nucleotide polymorphism (SNP) assay for the differentiation of "drug" and "non-drug"Cannabis plants. An assay was developed based on four polymorphisms within a 399 bp fragment of the tetrahydrocannabinolic acid (THCA) synthase gene, utilising the snapshot multiplex kit. This SNP assay was tested on 94 Cannabis plants, which included 10 blind samples, and was able to differentiate between "drug" and "non-drug"Cannabis in all cases, while also differentiating between Cannabis and other species. Non-drug plants were found to be homozygous at the four sites assayed while drug Cannabis plants were either homozygous or heterozygous.

An easily automated, closed-tube forensic DNA extraction procedure using a thermostable proteinase.

Most standard procedures for extracting DNA from forensic substrates involve manipulations that expose the sample to potential contamination and which reduce yields. Furthermore, most methods require centrifugation and/or solvent extraction steps that render them difficult to automate. We describe a simple closed-tube DNA extraction procedure using a proteinase from the thermophilic Bacillus species EA1 that produces good DNA yields from a wide range of forensic substrates. The reaction is controlled by a temperature shift regime programmed into a thermal cycler and so eliminates the need for solvent extraction or column purification. The new method is ideally suited to forensic samples where exposure to extraneous contaminating DNA must be avoided. In addition, The simplicity of the procedure makes it suitable for automation.

Allele frequencies for the four major sub-populations of New Zealand at three STR loci--HUMTH01, HUMTPOX and CSF1PO.

Allele frequencies for the three STR loci included in the GenePrint CTT multiplex system (HUMTH01, HUMTPOX, HUMCSF1PO) have been determined for the four major sub-populations of New Zealand.

Allele frequencies for the four major sub-populations of New Zealand at the 10 AMPFlSTR SGM Plus loci.

Allele frequencies for the 10 STR loci included in the AMPFlSTR SGM Plus multiplex system have been determined for the four major sub-populations of New Zealand.

The fallacy of independence testing and the use of the product rule.

Use of the product rule which implies the assumption of within and between locus independence, is still common, particularly in the United States of America. Whilst it may be considered by some to be an acceptable approximation it is not logical to suggest that independence testing somehow "validates" its use. This paper discusses the nature of this fallacy.

The New Zealand DNA databank: its development and significance as a crime solving tool.

The New Zealand DNA Databank was established following the introduction of legislation in August 1996. Using the Second Generation Multiplex (SGM), DNA profiles from over 13,000 convicted offenders and volunteer donors have been completed to the National DNA Database. Since June 1998, DNA profiles from over 1,400 unsolved crimes have been entered onto the Crime Sample Database. Of all unsolved crimes analysed, 33% are linked to individuals and 21% are linked to other unsolved crimes. Several high profile types of case including homicides, sexual offenses and burglaries are amongst those regularly solved.

The calculation of DNA match probabilities in mixed race populations.

A coherent method is offered to estimate likelihood ratios for DNA match probabilities from mixed racial populations that avoids the approach of reporting separate estimates for each race. The method is demonstrated for some cases involving profiles derived from several individuals and incorporates a correction for 'subpopulation' effects.

Applications and extensions of subpopulation theory: a caseworkers guide.

This paper extends the calculation of conditional probabilities from those given by Balding and Nichols to casework situations where a series of possible DNA types are possible. Such situations may occur when a sample is identified containing a mixture of DNA from two or more people or where extra information can be determined about the subpopulation under consideration by analysis of additional samples. Using this approach, the error in the estimated likelihood ratios is expected to reduce as the number of additional individuals typed from the subpopulation increases.

Penetration and ejaculation; forensic aspects of rape.

A study was made of 104 alleged rape cases submitted to the DSIR in Auckland by the police over a period of approximately two years. Data were collected relating to the victims' perceptions of penetration and ejaculation, and the forensic evidence for these events. Seminal fluid was detected in 69 (66%) of the cases. In 81 (78%) of the cases penetration was reported; seminal fluid was detected in 58 of these. Of the five (5%) cases where it was reported that penetration did not occur, seminal fluid was detected in two. Forty (38%) of the victims reported ejaculation and seminal fluid was detected by 34 of these. Ejaculation was reported not to have occurred in 16 (15%) of the cases; seminal fluid was detected in seven of these. The victims of alleged sexual assault may not remember accurately if penetration or ejaculation took place.

Lactate dehydrogenase isoenzyme patterns in vaginal injury of a 4-year-old girl.

Lactate dehydrogenase isoenzyme patterns indicative of vaginal injury were identified in extracts of a bloodstain on a pair of knickers belonging to a 4-year-old girl. No evidence of semen or saliva was detected in the stain. The result corroborated the clinical findings and provided strong evidence of child sexual abuse.

Nucleotide sequence of the geminivirus chloris striate mosaic virus.

The genome of chloris striate mosaic virus (CSMV) comprises a single circular DNA as determined by analyses on virion single-stranded (ss) DNA and virus-specific covalently closed circular (ccc) DNA isolated from infected plants. The nucleotide sequence of CSMV DNA was determined from cccDNA and the data were accommodated into one DNA circle of 2750 nucleotides. Comparison of the nucleotide sequence with those of maize streak virus (MSV), wheat dwarf virus (WDV), and digitaria streak virus (DSV) showed 49, 47, and 48% DNA homology, respectively. The sequence has four potential open reading frames for proteins of greater than 10,000 mol wt, two in the viral (+) sense and two in the complementary (-) sense. Three of these potential coding regions have homologous counterparts, by comparison of the amino acid sequences, among the open reading frames reported for MSV, WDV, and DSV. CSMV encapasidates primer molecules able to prime the synthesis in vitro of a complementary strand to virion DNA, initiating this reaction at one site on the genome. The CSMV primer comprising approximately 88 nucleotides was located within the smaller of two intergenic or noncoding regions.

Organization and interviral homologies of the coat protein gene of white clover mosaic virus.

The sequence of 1612 nucleotides of the 3'-terminal region of white clover mosaic virus (WCIMV) has been determined from cDNA clones. The viral sense RNA contains four open reading frames of Mr 20,684, Mr 7219, Mr 12,989, and at least Mr 17,000. The latter begins 5' to the sequence determined. The amino acid sequence of the open reading frame encoding the 20,684 polypeptide shows marked homology to the coat proteins of three other potexviruses. The putative coat protein gene was subcloned in a T7 transcription plasmid and RNAs produced by in vitro transcription were translated in the rabbit reticulocyte lysate system. The polypeptide products comigrated on SDS-polyacrylamide gels with one of those synthesized by the in vitro translation of viral RNA, and were immunoprecipitable with antiserum raised against WCIMV, confirming the location of the coat protein gene.

The complete nucleotide sequence of the potexvirus white clover mosaic virus.

The complete nucleotide sequence (5845 nucleotides) of the genomic RNA of the potexvirus white clover mosaic virus (WC1MV) has been determined from a set of overlapping cDNA clones. Forty of the most 5'-terminal nucleotides of WC1MV showed homology to the 5' sequences of other potexviruses. The genome contained five open reading frames which coded for proteins of Mr 147, 417, Mr 26,356, Mr 12,989, Mr 7,219 and Mr 20,684 (the coat protein). The Mr 147,417 protein had domains of amino acid sequence homology with putative polymerases of other RNA viruses. The Mr 26,356 and Mr 12,989 proteins had homology with proteins of the hordeivirus barley stripe mosaic virus RNA beta and the furovirus beet necrotic yellow vein virus (BNYVV) RNA-2. A portion of the Mr 26,356 protein was also conserved in the cylindrical inclusion proteins of two potyviruses. The Mr 7,219 protein had homology with the 25K putative fungal transmission factor of BNYVV RNA-3.

An encapsidated, subgenomic messenger RNA encodes the coat protein of carnation mottle virus.

The translation strategy of carnation mottle virus (CarMV) in vitro has been generally assumed to involve internal initiation events on full-length, genomic RNA (4.3 kb). We suggest that this is, at least in part, incorrect. Encapsidated RNA, fractionated on denaturing sucrose gradients, or total RNA from CarMV-infected leaves, fractionated under non-denaturing conditions, was translated in an mRNA-dependent rabbit reticulocyte cell-free system. Evidence for subgenomic RNAs which encode a polypeptide of Mr 38 000 was found. This product was shown to be related to authentic CarMV coat protein by partial proteolysis with alpha-chymotrypsin and SDS/polyacrylamide-gel electrophoresis.

Muscle growth in two genetically different lines of swine.

Skeletal muscle growth in two genetic lines of pigs that differed in total muscle content was studied at live weights of 23, 45, 68, 91, 104, 118 kg. Total physically separable muscle and cross-sectional area of the longissimus dorsi muscle were greater in the muscular than in the obese genetic lines. Above 45 kg, animals in the muscular genetic line had less total separable fat than animals in the obese line, but the two lines did not differ in total physically separable fat at 23 and 45 kg live weight. Hence, these two genetic lines may differ in weight at which maturity is reached as much as in inherent propensity for obesity. Longissimus muscle form the muscular line had more water, less protein, and less lipid than longissimus from the obese line. DNA and RNA concentration, total DNA and RNA content, and RNA/DNA ratio of the pituitary and liver did not differ between the two genetic lines. Above 68 kg, longissimus from the muscular line had higher DNA and RNA concentrations than longissimus from the obese line; this difference did not exist between 23 and 68 kg. RNA/DNA ratio of the longissimus muscle was greater and protein-to-DNA and protein-to-RNA ratios in longissimus were lower in the muscular than in the obese line. Total DNA content of physically separable muscle increased 2.0 (obese) to 2.7 (muscular)-fold between 23 and 118 kg; hence, number of muscle nuclei increases during growth. Total DNA content of physically separable muscle was greater in the muscular than in the obese line and was the measurement most highly related to total muscle content.