Multiplex PCR and minisequencing of SNPs--a model with 35 Y chromosome SNPs

We have developed a robust single nucleotide polymorphism (SNPs) typing assay with co-amplification of 25 DNA-fragments and the detection of 35 human Y chromosome SNPs. The sizes of the PCR products ranged from 79 to 186 base pairs. PCR primers were designed to have a theoretical Tm of 60 +/- 5 degrees C at a salt concentration of 180 mM. The sizes of the primers ranged from 19 to 34 nucleotides. The concentration of amplification primers was adjusted to obtain balanced amounts of PCR products in 8mM MgCl2. For routine purposes, 1 ng of genomic DNA was amplified and the lower limit was approximately 100 pg DNA. The minisequencing reactions were performed simultaneously for all 35 SNPs with fluorescently labelled dideoxynucleotides. The size of the minisequencing primers ranged from 19 to 106 nucleotides. The minisequencing reactions were analysed by capillary electrophoresis and multicolour fluorescence detection. Female DNA did not influence the results of Y chromosome SNP typing when added in concentrations more than 300 times the concentrations of male DNA. The frequencies of the 35 SNPs were determined in 194 male Danes. The gene diversity of the SNPs ranged from 0.01 to 0.5.     

Results of the GEP-ISFG collaborative study on two Y-STRs tetraplexes: GEPY I (DYS461, GATA C4, DYS437 and DYS438) and GEPY II (DYS460, GATA A10, GATA H4 and DYS439)

A collaborative exercise was carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG) in order to evaluate the performance of two Y-chromosome STR PCR tetraplexes, which include the loci DYS461, GATA C4, DYS437 and DYS438 (GEPY I), and DYS460, GATA A10, GATA H4 and DYS439 (GEPY II). The participating laboratories were asked to type three samples for the eight markers, using a specific amplification protocol. In addition, two control samples, with known haplotypes, were provided. The results obtained by the 13 different participating laboratories were identical, except for two laboratories that failed to type correctly the same two samples for GATA C4. By sequence analyses, two different GATA C4 allele structures were found. One control sample (allele 21) and two questioned samples (allele 22, correctly typed by all the laboratories, and allele 25) presented the following repeat structure: (TCTA)4(TGTA)2(TCTA)2(TGTA)2(TCTA)n, but different from the one found for allele 26 in one sample included in this exercise, as well as in the second control sample (allele 23), namely (TCTA)4(TGTA)2(TCTA)2(TGTA)2(TCTA)2(TGTA)2(TCTA)n. The collaborative exercise results proved that both Y-tetraplexes produce good amplification results, with the advantage of being efficiently typed using different separation and detection methodologies. However, since GATA C4 repeat presents a complex structure, with alleles differing in sequence structure, efficient denaturing conditions should be followed in order to avoid typing errors due to sizing problems.     

Traumatic subarachnoid hemorrhage and extracranial vertebral artery injury: a case report and review of the literature

The case is presented of a 19-year-old man who was assaulted and died shortly afterward from a large traumatic basal subarachnoid hemorrhage (TBSAH) that arose from rupture of the left vertebral artery, proximal to the point at which the artery penetrated the dura. The literature regarding TBSAH and vertebral artery rupture is reviewed, and a number of points are highlighted: patients with TBSAH may remain conscious for a period of hours after injury, subcutaneous or muscular bruising may be contralateral to the ruptured vessel, fractures of the transverse processes of the cervical vertebrae and significant pathology of the vertebral artery are not typically associated with TBSAH, and rupture of the vertebral artery may be intracranial, junctional, or extracranial.     

Dosage of treatment to sexual offenders: are we overprescribing?

A sample of 337 offenders who received treatment in a variety of sex offender treatment programs in the Ontario region of Correctional Service Canada between 1993 and 1998 were divided based on the highest intensity sex offender programming that they received (low, moderate, and high). The three groups were compared with reference to a variety of actuarial risk assessment measures, criminogenic factors, and the number and type of treatment programs completed. It was hypothesized that the high-intensity group would have more criminogenic risk factors, higher actuarial scores, and participate in more treatment programs than both the moderate- and low-intensity groups. The results indicate that in general, the hypotheses were supported. Nonetheless, the results suggest that the low-intensity group may be receiving too much sex offender-specific treatment.     

A molecular genetic approach for forensic animal species identification

This study investigated potential markers within chromosomal, mitochondrial DNA (mtDNA) and ribosomal RNA (rRNA) with the aim of developing a DNA based method to allow differentiation between animal species. Such discrimination tests may have important applications in the forensic science, agriculture, quarantine and customs fields. DNA samples from five different animal individuals within the same species for 10 species of animal (including human) were analysed. DNA extraction and quantitation followed by PCR amplification and GeneScan visualisation formed the basis of the experimental analysis. Five gene markers from three different types of genes were investigated. These included genomic markers for the beta-actin and TP53 tumor suppressor gene. Mitochondrial DNA markers, designed by Bataille et al. [Forensic Sci. Int. 99 (1999) 165], examined the Cytochrome b gene and Hypervariable Displacement Loop (D-Loop) region. Finally, a ribosomal RNA marker for the 28S rRNA gene optimised by Naito et al. [J. Forensic Sci. 37 (1992) 396] was used as a possible marker for speciation. Results showed a difference of only several base pairs between all species for the beta-actin and 28S markers, with the exception of Sus scrofa (pig) beta-actin fragment length, which produced a significantly smaller fragment. Multiplexing of Cytochrome b and D-Loop markers gave limited species information, although positive discrimination of human DNA was evident. The most specific and discriminatory results were shown using the TP53 gene since this marker produced greatest fragment size differences between animal species studied. Sample differentiation for all species was possible following TP53 amplification, suggesting that this gene could be used as a potential animal species identifier.     

Smothering of children older than 1 year of age-diagnostic significance of morphological findings

A homicide by smothering may leave unobtrusive or even no injuries if the victim is not able to struggle. This is especially true for infants up to 12 months of age. The differentiation between the sudden infant death syndrome and smothering, by an autopsy alone may be impossible. To establish whether this is different in older children because of their rising capacity for defence six cases of smothering in children >1 year of age were re-examined. The age of the children ranged between 1.5 and 7 years (surviving child). The smothering was inflicted with hands in three cases (including the surviving child), with a pillow in two cases, and the way of smothering remained unknown in one case (no confession of the perpetrator). Depending on the tool used for smothering, abrasions on the facial skin (hands, lesser in cases of smothering by a pillow) and petechiae (pillow/hands) could be seen by external examination. The older the children were, the more injuries could be found. Together with circumstantial evidence all cases could have been established by a thorough autopsy. This is important to prevent further offences against (surviving) siblings.     

Genetic polymorphism revealed by 13 tetrameric and 2 pentameric STR loci in four Mongoloid tribal population

The short tandem repeat allelic profiles at to 15 autosomal polymorphic loci were analyzed in four tribal populations of Mizoram (India). The analysis was performed on 354 unrelated healthy individuals belonging to Mongoloid races. All the samples were subjected to sex test (Amelogenin marker) besides the STR typing and in all instances; it has shown no deviation from expectation. The allele frequencies for all the analyzed loci in the studied populations are within expected range in comparison to the populations from same racial background. No significant deviation from the Hardy-Weinberg Equilibrium was observed for all the populations. In no cases the observed heterozygosity is less than that of expected values and it varied from 0.978 (Penta E) to as low as 0.425 (THO1). The discriminatory power and exclusion probability values for all the analyzed markers are significantly high and thus reveal high forensic significance. There is no evidence for association of alleles among the 15 studied loci. This allele frequency data will be useful for human identity testing in Mizo population.     

Response of short tandem repeat systems to temperature and sizing methods

In capillary gel electrophoresis (CE), changing run conditions such as temperature can result in minor variations in the size determination of an allele. These effects are caused by secondary structure differences that can occur between the amplified sample and the internal standard. The type of method chosen to generate the sizing curve in STR analysis can influence the relationship between estimated allele size and temperature. To better understand the effects of temperature and sizing method on the reproducibility of DNA migration, two fluorescently labeled allelic ladders, CTTv and Y-PLEX 6 were analyzed using the ABI Prism 310 Genetic Analyzer. The default method on the Genetic Analyzer utilizes an electrophoretic temperature of 60 degrees C and a Local Southern method to generate a sizing curve from the fragment migration times of the internal lane standard. In this work, electrophoresis was conducted at 35-70 degrees C using the commercially available POP 4 buffer at pH 8 and two sizing methods, Global Southern and Local Southern, were compared. The slopes of the regression line between estimated allele size and temperature, using either sizing method, were measured in order to demonstrate the temperature sensitivity of migration time and the importance of the operator-chosen method. Our results indicate that the Global Southern method is a better choice in situations where temperature fluctuations can occur. In addition, the temperature dependence of the DNA size estimates using the POP 4 system were compared to results obtained using an experimental buffer consisting of 3% hydroxyethylcellulose at pH 11. These results demonstrate that secondary structure effects are minimized at an elevated pH, increasing the precision of size estimates obtained.