Validation of a 16-locus fluorescent multiplex system

STR multiplexes have been indispensable for the efficient genotyping of forensic samples. The PowerPlex 16 System contains the coreCODIS loci, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, THOI, TPOX, vWA, the sex determinant locus, amelogenin, and two pentanucleotide STR loci, Penta D and Penta E. This multiplex satisfies the locus requirements for most national databases and is the most efficient currently available system due to its single PCR amplification. To provide the groundwork for judicial acceptance, including the publication of primer sequences, and to evaluate laboratory-to-laboratory variation, a developmental validation for casework on this commercially available system was performed in 24 laboratories and produced the following conclusions. Amplification was reliable on a variety of thermal cyclers and product could be analyzed on either an ABI PRISM 310 Genetic Analyzer or an ABI PRISM 377 DNA Sequencer. Genotyping using single source samples was consistent between 0.25 and 2 ng of input DNA template with a few laboratories obtaining complete genotypes at 0.0625 ng. However, heterozygote allele imbalance (<60% peak height balance) caused by stochastic effects was observed at a rate of 13% with 0.125 ng DNA and 22% at 0.0625 ng DNA. Mixture analyses were done using a total of 1 ng of DNA template. Most alleles were detected in mixtures of 4 to 1 and some minor alleles were detected in mixtures of 19 to 1. Optimum amplification cycle number was dependent on the sensitivity of the detection instrument used and could also be adjusted to accommodate larger amounts of DNA on solid supports such as FTA paper. Reaction conditions including volume, annealing temperature, and concentrations of primer, AmpliTaq Gold, and magnesium were shown to be optimal yet robust enough to withstand moderate variations without affecting genotype analysis. Environmental, matrix and standard source analyses revealed an ability to obtain complete genotypes in all sample types except those exposed to 80 degrees C for 12-48 days. Finally, comparison of genotype results from the PowerPlex 16 System with other commercially available systems on non-probative reference and forensic samples showed consistent results.     

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.     

Medical aspects of capital punishment executions

Between 1976 and the middle of 2001, approximately 718 human executions occurred in the U.S. (a small segment of worldwide executions). Data regarding the medical aspects of these executions are not readily available. We searched all public domain data in the U.S. to obtain specific data for all persons executed in the U.S. since 1976. Of the five methods of execution used (lethal injection, lethal gas, electrocution, hanging, and firing squad), significant differences emerged as measured by rate of complications, duration of time spent by the condemned in the "death chamber," as well as duration of time from the onset of execution procedures to pronouncement of death. These data suggest that human executions are difficult to carry out. Human executions are associated with significant physical complications. These data may help inform future discussions on human executions.     

A method for the longitudinal sectioning of single hair samples

A simple technique is presented for the longitudinal sectioning of hair samples without the need for any embedding medium. The technique applies to single hair samples that are placed in a groove of a particular depth and can either be scraped or cut to the desired level. Planar sections are obtained that reveal the internal structure of the sample and are ideal for the application of surface analytical techniques for the study of internal transverse and longitudinal distributions of xenobiotics.     

Analysis of ionic ratios in myocardial tissue and their relation to cardiac damage

The authors evaluated the usefulness of the postmortem biochemical analysis of ionic ratios in different parts of the heart and their relation to cardiac damage caused by chest trauma, as observed by anatomopathologic study. Fifty-nine 59 cases were studied, selected from routine necropsies, and samples were taken from different sites of cardiac tissue. The cause of death was trauma in 40 cases and nontraumatic causes in 19 cases. The object of this study was to analyze the levels of Na+, K+, Ca+2, Mg+2, and Zn+2 in different zones of the heart, and the relationship between intracellular and extracellular ion ratios and the different causes of death and any anatomopathologic alterations observed. The biochemical tests revealed a possible relation between the ionic values and cause of death. Alterations in cell membrane permeability and corresponding modification of the ionic ratios were produced earlier than histologic alterations, which need longer to establish themselves whether or not they follow a traumatic process.     

Population genetics of nine short tandem repeat loci: allele frequency distribution in a Brazilian population sample

Gene and genotype frequencies in relation to the D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, and D7S820 loci were determined in a sample of 290 unrelated individuals (204 Caucasians and 86 mulattoes) living in the city of S?o Paulo, Brazil. The sex test Amelogenin was also performed in all subjects from our sample, revealing the expected sex in all instances. Allele frequency data obtained from the analysis of these samples were in the usual range of other population groups with similar racial background. In the sample of Caucasian individuals, panmictic proportions were ruled out in relation to TPOX and CSF1PO loci, but only in the latter was the overall frequency of heterozygotes significantly less than expected. In the sample of mulattoes, Hardy-Weinberg proportions were rejected in relation to FGA and CSF1PO loci, but in no instance were the overall numbers of heterozygotes different from the corresponding expected ones under panmixia. Taking into account all this and also the number of tests performed, the degree of genetic heterogeneity of Brazilian populations, and the critical level reached by the significant results (1% < alpha<5%), the departures from panmixia here observed can be considered to be negligible in altering significantly biologic relationship odds calculated under the assumption of random matings.