Extraction of high quality DNA from biological materials and calcified tissues

Calcified tissues, such as bone and tooth, and some other sample types, such as those containing adhesive, present a challenge to standard extraction protocols. We have developed a lysis reagent, BTA™ lysis buffer, which is designed for use with PrepFiler™ Kit reagents. The BTA™ lysis buffer disrupts calcified tissue matrices and achieves effective extraction of DNA from pulverized bone and tooth samples. In addition, the BTA™ lysis buffer mildly but efficiently extracts DNA from challenging substrates like tape, chewing gum, and cigarette butts and, as with bone and tooth, DNA from these lysates is purified using established PrepFiler™ reagent extraction protocols.We successfully extracted DNA from powdered human bone samples, chewed gum and smoked cigarettes using BTA™ lysis buffer. Extraction yields for bone, gum and cigarette samples tested were consistent and reproducible. This extraction method efficiently removed potential PCR inhibitors from all samples tested, and C_T values for the internal PCR control of Quantifiler^® Human DNA Quantification Kit were consistent and within the normal range. The DNA extracted from these samples also provided conclusive profiles that were free of PCR artifacts when amplified using the AmpF?STR^® Identifiler^® PCR Amplification Kit. The protocol is easily adapted for automation.     

Developmental validation of a Cannabis sativa STR multiplex system for forensic analysis

A developmental validation study based on recommendations of the Scientific Working Group on DNA Analysis Methods (SWGDAM) was conducted on a multiplex system of 10 Cannabis sativa short tandem repeat loci. Amplification of the loci in four multiplex reactions was tested across DNA from dried root, stem, and leaf sources, and DNA from fresh, frozen, and dried leaf tissue with a template DNA range of 10.0-0.01 ng. The loci were amplified and scored consistently for all DNA sources when DNA template was in the range of 10.0-1.0 ng. Some allelic dropout and PCR failure occurred in reactions with lower template DNA amounts. Overall, amplification was best using 10.0 ng of template DNA from dried leaf tissue indicating that this is the optimal source material. Cross species amplification was observed in Humulus lupulus for three loci but there was no allelic overlap. This is the first study following SWGDAM validation guidelines to validate short tandem repeat markers for forensic use in plants.     

Prostitute homicides: a descriptive study

It has been estimated that women involved in street prostitution are 60 to 100 times more likely to be murdered than are nonprostitute females. In addition, homicides of prostitutes are notoriously difficult to investigate and, as such, many cases remain unsolved. Despite this large risk factor, little literature exists on homicides of prostitutes, and there is a lack of basic statistics and knowledge regarding this very specific victim group that could possibly help investigators. The aim of the current study is to conduct an exploratory study to explore the key characteristics of this group and how they differ from other subgroups of homicide. Forty-six cases of U.K. prostitute homicides are analyzed and compared to 59 male offender-female victim nonsexual homicide cases and 17 male offender-female victim sexual homicide cases.     

Elevated morphine concentrations determined during infant death investigations: artifacts of withdrawal of care

Abstract:  Three cases are reported of elevated postmortem blood morphine concentrations (189–3036 ng/mL) that were observed during the course of death investigations involving three children ranging in age from 1 week to 2 years, all of whom underwent withdrawal of life support. In all three cases, the presence of opiates in postmortem blood was indicated by immunoassay (ELISA) and quantitative confirmatory analysis of free morphine concentrations in postmortem blood was performed by solid-phase extraction followed by gas chromatography/mass spectrometry (GC/MS) in the selected ion monitoring mode. While the practice of withdrawing life support from terminally ill patients, with the accompanying administration of narcotics/analgesics has been reported in the medical literature, it has not been adequately described in the forensic literature. The implications of this practice on the forensic toxicological interpretation of morphine findings are discussed. To our knowledge, this is the first report of postmortem morphine concentrations arising directly from administration in conjunction with withdrawal of care in pediatric patients.     

Utility of immunoassay in drug screening in skeletal tissues: sampling considerations in detection of ketamine exposure in femoral bone and bone marrow following acute administration using ELISA

Detection of ketamine exposure in skeletal tissues by automated enzyme-linked immunosorbent assay (ELISA) and gas chromatography with electron capture detection (GC-ECD) is described. Rats (n = 18) received 0, 15, 30, or 75 mg/kg ketamine hydrochloride acutely (i.p.), and were euthanized within 15 min or 1 h. Ketamine was extracted from ground femoral bone by methanolic incubation followed by liquid-liquid extraction (LLE), while marrow was homogenized in alkaline solution, and then underwent LLE. Extracts were analyzed by ELISA, and subsequently by GC-ECD following derivatization with trifluoroacetic acid anhydride. The effect of tissue type (i.e., diaphyseal bone vs. epiphyseal bone vs. bone marrow) on the immunoassay response was examined through determination of binary classification test sensitivity (S) and measurement of the relative decrease in absorbance (%DA, drug-positive tissues vs. drug-free controls) in each tissue type. The %DA varied significantly between different tissues examined under a given dose condition, and generally decreased in the order marrow > epiphyseal bone > diaphyseal bone, at all dose levels examined. Measured S values for marrow, epiphyseal bone, and diaphyseal bone were 100%, 77%, and 23%, respectively (75 mg/kg dose). These results suggest that the type of skeletal tissue sampled and position sampled within a given bone (diaphyses vs. epiphyses) are important parameters in drug screening of skeletal tissues.     

Differentiating Between Generally and Partner-Only Violent Subgroups: Lifetime Antisocial Behavior,Family of Origin Violence,and Impulsivity

The present study examined self-reported lifetime antisocial behavior, family of origin violence, and impulsivity/behavioral disinhibition of 73 men entering treatment for partner violence. Participants were designated as generally violent (GV) (n = 46) or partner only violent (n = 27), based on self-reported violence against non-intimate individuals during the year prior to intake. As hypothesized, GV men reported more conduct disorder/delinquent behaviors, lifetime antisocial behaviors, and family of origin violence. The GV men also reported more behavioral disinhibition, however, group differences on impulsivity only approached statistical significance. In addition, as hypothesized, GV men reported they were more psychologically abusive toward their intimate partners. However, contrary to expectations, the subgroups did not differ on reports of physical violence toward their partners. This study employed a fairly simple technique of dividing men into groups based on self-reports of violence over the past year, thereby producing subgroups that differed on a number of important characteristics that may have implications for treatment. An advantage of this technique is that it would be relatively easy for other treatment programs to apply.

Effects of Perpetrator and Victim Gender on Negative Outcomes of Family Violence

Growing up in a violent home predisposes children to a host of behavioral and emotional difficulties. This study examined whether perpetrator and victim gender have an impact on depressive symptoms and aggressive behavior for victims of child physical abuse (CPA) and also with regard to witnessing interparental violence (IPV). This study also examined whether witnessing siblings being abused would elicit high levels of depressive symptoms and aggressive behavior. College students (n = 675) were assessed for both exposure to IPV and child physical abuse prior to age 18. Participants completed measures of depression and aggression. With regard to victims of CPA, participants victimized by both parents and those victimized by mothers only had significantly higher levels of aggression. For depressive symptoms, females having both parents as perpetrators or fathers only had significantly higher depressive symptoms. With regard to witnessing IPV, being abused by both parents was associated with endorsement of more aggression and depressive symptoms. With regard to witnessing sibling violence, the results were similar to those found for victims of CPA.

Joint match probabilities for Y chromosomal and autosomal markers

Empirical tests of association between Y chromosome and autosomal markers are presented and a theoretical framework for determining a joint match probability is recommended. Statistical analyses of association were performed in 16 US populations between the autosomal genotypes from loci CSF1PO, FGA, THO1, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S512, D21S11 and Y chromosome haplotypes from loci DYS19, DYS385ab, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS438, and DYS439. The sample populations include individuals of European-, African-, Hispanic-, Native-, and Asian-American ancestry. The results are consistent with independence of Y and autosomal markers, although small amounts of dependence would likely have escaped our tests. Given the data in hand, we suggest it is appropriate to compute joint match probabilities by multiplying the Y haplotype frequency with the appropriately corrected autosomal frequency. In addition to correcting for autosomal frequency differences between groups, a further correction may be required. Since two individuals sharing the same Y haplotype are likely to be more recently related than two randomly chosen individuals, the autosomal frequencies have to be adjusted to account for this, akin to the theta correction used to account for population substructure. The structure imposed on the autosomal frequencies conditioned in a Y match is a function of the number of markers scored and their mutation rate. However, in most settings theta<0.01. When population structure is already present in the autosomes, the additional effect due to conditioning on the Y is small. For example, if the amount of structure in the population is theta=0.01 or 0.03 (the NRCII range), then the effect of conditioning on the Y results in only a trivial increase in theta to 0.02-0.04, respectively.     

Alaska public health law reform

The Turning Point Model State Public Health Act (Turning Point Act), published in September 2003, provides a comprehensive template for states seeking public health law modernization. This case study examines the political and policy efforts undertaken in Alaska following the development of the Turning Point Act. It is the first in a series of case studies to assess states' consideration of the Turning Point Act for the purpose of public health law reform. Through a comparative analysis of these case studies and ongoing legislative tracking in all fifty states, researchers can assess (1) how states codify the Turning Point Act into state law and (2) how these modernized state laws influence or change public health practice, leading to improved health outcomes.