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.

Mitochondrial DNA (mtDNA) analysis of forensic samples typically is performed when the quantity and quality of DNA are insuf-ficient 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 de-sirable. 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 QIAquick PCR Purification columns, 2) Concert Rapid PCR Purification columns, and 3) ExoSAP-IT(tm) reagent. When the yield of purified amplicons, qual-ity of the sequence profile, and ease of assay were evaluated, the use of ExoSAP-IT(tm) reagent for post-amplification purification was chosen to re-place the filtration method.

The effects of long‐term chronic stress during prepubertal periods of growth and development on an organism's ability to release ACTH during future episodes of an acute novel stress and in response to exogenous CRH were examined. Following a 6‐week stress period, in which prepubertal male and female WKY rats were subjected to three different and randomly given stress paradigms (heat, noise and immobilization) at various times of the day (in order to prevent adaptation to stress), chronically stressed male rats were far less able to respond to CRH plus a novel ether stress than were their male controls or their female counterparts. Although baseline ACTH levels were similar in both male and female control and experimental rats, when subjected to a subsequent acute ether stress, the differences in ACTH response between controls and experimentals as well as between males and females were significant. ACTH response to stressors was significantly blunted in both male and female experimental rats compared to their controls, but the male response was significantly lower than that of the females. These results suggest that prepubertal chronic stress may permanently alter an organism's ability to release ACTH, even when subjected to a novel and traumatic ether stress, and that males may be much more susceptible than females to prepubertal stress. Long‐term stress, therefore, if experienced during critical developmental periods such as preadolescence, can permanently damage the stress response mechanism and cause other, more serious physiological disorders.