An improved method for post-PCR purification for mtDNA sequence analysis

Mitochondrial DNA (mtDNA) analysis of forensic samples typically is performed when the quantity and quality of DNA are insufficient for nuclear DNA analysis or when maternal relatives may be the only reference source. Many of the steps required in the analytical process are both lengthy and labor intensive. Therefore, improvements in the process that reduce labor without compromising the quality of the data are desirable. The current procedure requires purification of the amplicons by centrifugal filtration after PCR and prior to cycle sequencing. Because this method requires several manipulations to perform, alternate cleanup procedures were investigated. These include the use of 1) Qiagen QlAquick PCR Purification columns, 2) Concert Rapid PCR Purification columns, and 3) ExoSAP-IT reagent. When the yield of purified amplicons, quality of the sequence profile, and ease of assay were evaluated, the use of ExoSAP-IT reagent for post-amplification purification was chosen to replace the filtration method.     

Residential setting and parent-adolescent relationships during the college years

The relationship of residential setting (living with parents vs. living away from home while attending college) and gender with late adolescents' perceptions of their relationships with parents was examined. Four hundred four undergraduates students (mean age=20 years, 4 months) from two midwestern universities completed surveys. Two hundred four subjects lived with their parents and commuted to school, and 200 lived away at college. Controlling for student's age, parents' education, and financial and family considerations as factors in the choice of a college, living away was associated with greater independence, support, and mutual respect between parents and adolescents. In contrast, students who lived at home felt parents underestimated their maturity, and reported more conflict and avoidance in their relationships with parents. Regardless of residential setting, women reported more mutuality and support in their relationships with parents than men. The results suggest the importance of considering contextual issues during the transition to adulthood.This research was supported by a Campus Grant to the first author from the University of Michigan-Dearborn.Received Ph.D. in developmental psychology from the University of Michigan. Research interests include family relationships, cross-cultural studies, and political development in adolescence and young adulthood.Received Ph.D. in human development and family studies from Penn State University. Research interests include competence during the transition to young adulthood, career development, drug and alcohol use.Research interests include adolescents' relationships with parents and peers and cross-cultural studies.     

Quantification of human mitochondrial DNA using synthesized DNA standards

Successful mitochondrial DNA (mtDNA) forensic analysis depends on sufficient quantity and quality of mtDNA. A real-time quantitative PCR assay was developed to assess such characteristics in a DNA sample, which utilizes a duplex, synthetic DNA to ensure optimal quality assurance and quality control. The assay's 105-base pair target sequence facilitates amplification of degraded DNA and is minimally homologous to nonhuman mtDNA. The primers and probe hybridize to a region that has relatively few sequence polymorphisms. The assay can also identify the presence of PCR inhibitors and thus indicate the need for sample repurification. The results show that the assay provides information down to 10 copies and provides a dynamic range spanning seven orders of magnitude. Additional experiments demonstrated that as few as 300 mtDNA copies resulted in successful hypervariable region amplification, information that permits sample conservation and optimized downstream PCR testing. The assay described is rapid, reliable, and robust.     

Effect of post-mortem changes on peripheral and central whole blood and tissue clozapine and norclozapine concentrations in the domestic pig (Sus scrofa)

Interpretation of the results of psychoactive or other drug measurements in post-mortem blood specimens may not be straightforward, in part because analyte concentrations in blood may change after death. There is also the issue of comparability of plasma (or serum) results to those obtained in whole blood. To investigate these problems with respect to clozapine, this drug (10mg/kg daily) was given orally to two pigs. Blood was collected 3h post-dose on day 7, the animals were sacrificed, and blood taken from central and peripheral veins for up to 48 h after death. Tissue samples were also collected immediately after death and at 48 h. Ante-mortem whole blood clozapine/N-desmethylclozapine (norclozapine) concentrations were 0.86/1.07 and 1.11/1.15 mg/l in pigs 1 and 2, respectively. Blood clozapine and norclozapine concentrations generally increased after death (central vein: clozapine up to 300%, norclozapine up to 460%; peripheral vein: clozapine up to 155%, norclozapine up to 185%). Initial blood and kidney clozapine and norclozapine concentrations were comparable in both animals, but were some two-fold higher in heart, liver and striated muscle in pig 2. In both animals, the heart and striated muscle clozapine and norclozapine concentrations had increased some two- to three-fold at 48 h, whilst the liver and kidney concentrations were essentially unchanged. The reason for the increase in heart and striated muscle concentrations at 48 h is unclear, but could be simple variation in sample site. The plasma:whole blood distribution of clozapine and norclozapine was studied in vitro. In human blood (one volunteer donor, haematocrit 0.50) the plots of plasma versus whole blood concentration were linear for both analytes across the range 0.1-1.5mg/l, although clozapine favoured plasma (plasma:whole blood ratio=1.12), whereas norclozapine favoured whole blood (ratio 0.68). In pig blood, the plots of plasma versus whole blood were non-linear in both cases, although clozapine favoured plasma to a greater extent than norclozapine. This may be due to lower plasma clozapine and norclozapine protein binding capacity in the pig as compared to man.     

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.

Development of a Validity Scale for the Dissociative Experience Scale-Revised: Atypicality,Structure, and Inconsistency

Psychological Injury and Law - The Dissociative Experiences Scale (DES), the most commonly used scale for assessing dissociation across settings, lacks a validity scale. In this study, six methods...     

A Study of the Background Levels of Male DNA on Underpants Worn by Females

This study is the first to examine the background level of male DNA on underpants worn by females in the absence of sexual contact. Here, we examined 103 samples from the inside front of underpants from 85 female volunteers. Samples were examined for the presence of male DNA using NGM SElect and PowerPlex Y23 kits. Only five samples gave a “complete” Y-STR profile, even though 83.5% of our volunteers cohabited with a male. In all cases where a partner reference sample was available, the Y-STR profile matched the cohabiting partner. We have demonstrated that a Y-STR profile is not expected on the inside front of underpants worn by females after social contact alone. The results of this study are informative for evaluating the significance of a Y-STR profile on underpants in cases of alleged sexual assault.