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.     

A more sensitive method for the quantitation of genomic DNA by Alu amplification

Current procedures for human DNA quantitation reach their limit at 150 pg DNA, which is above the limit of the PCR profiling range using Profiler-Plus (Applied Biosystems, CA). This study tested the potential for the use of primate specific Alu sequences in forensic science for the sensitive detection and quantitaion of DNA. A fluorescently labelled primer pair was designed enabling high efficiency amplification of the core Alu sequence within primate DNA. Quantitation was performed by measurement of fluorescence intensity and comparison to a series of standard template DNA amounts via the construction of a standard curve. The new Alu-based quantitation protocol developed has shown its feasibility in more sensitively quantitating (100-2.5 pg) unknown amounts of human DNA for forensic use. The method is compatible with the use and throughput of current forensic procedures.     

Evaluation of some oxygen, sulfur, and selenium substituted ninhydrin analogues, nitrophenylninhydrin and benzo[f]furoninhydrin

Six ninhydrin analogues containing oxygen, sulfur, and selenium substituents at the C-5 position, 5-(4-nitrophenyl)ninhydrin, and benzo[f]furoninhydrin were evaluated as fingerprint development reagents. The analogues all showed good fingerprint color development but were not superior to ninhydrin in this respect. The benzo[f]furoninhydrin complex was strongly luminescent at room temperature following zinc complexation, while the remaining analogues required cooling to -196 degrees C to produce optimum luminescence. The benzo[f]furo, nitrophenyl, and methyl selenide analogues showed the best potential as fingerprint reagents with the benzo[f]furo analogue comparing favorably with DFO.     

Brain acetaldehyde and ethanol: method of determination and diagnostic significance in ethanol poisoning

The content of ethanol and acetaldehyde in the limbic cortex and reticular formation of the brain was measured by gas-liquid chromatography in lethal ethanol poisoning. The content of acetaldehyde was significantly increased in the gyrus cinguli of the brain. Lethal poisonings occurred during any stage of ethanol intoxication. The data characterizing individual ethanol tolerance were obtained, which can be used for differential diagnosis of ethanol poisoning in practical forensic medicine.     

A method for group identification of hair infected with Microsporum

A method for identifying the group appurtenance of biological objects from subjects suffering from various diseases is developed. The method can be used in examination of putrefactive objects (blood, secretions, hair, etc.) and in cases when the group appurtenance cannot be identified by other methods.     

Antigenic differentiation of blood stains by the Lewis system in practical forensic medical expert evaluation

A modification of quantitative absorption and absorption elution tests with blood stain washing before the absorption phase is presented. Due to washing, the effect of carrier object on anti-Le(a) and anti-Le(b) sera is decreased and the sensitivity of the method is increased. Additional adsorption of the sera and elution into test erythrocytes treated with protease C is suggested for increasing the number of standard sera fit for the absorption-elution test. A new technology for preparing anti-Le(a) and anti-Le(b) immunoreagents is described.