Evaluation of sieving polymers for fast, reproducible electrophoretic analysis of short tandem repeats (STR) in capillaries

Efficient capillary electrophoretic STR analysis requires rapid, reproducible and robust separation of DNA fragments with reasonable capillary longevity--this is currently accomplished using proprietary commercial polymeric sieving matrices specifically developed for this separation. These matrices, while effective, are costly and do not provide adequate resolution of STR DNA fragments in capillaries with shorter effective separation lengths, increasing the time required to accomplish the separation and minimizing the potential extrapolation to other miniaturized platforms. As the forensic community looks toward next generation microchip technology as a means of processing casework more rapidly, new sieving polymers need to be evaluated for utilization in this platform. The research presented here describes the assessment of commercially-available polymeric sieving matrices for STR analysis, with consideration given to feasibility of incorporation into a microdevice. Polymer composition, molecular weight, and concentration were evaluated, along with an assessment of the effects of buffer composition, separation temperature, and capillary length. These variables were evaluated individually or collectively on the ability to resolve STR DNA fragments and the reproducibility of the separations and the results compared to a proprietary commercial product. A 600,000 Da MW poly(ethylene oxide) (PEO) solution at a 3% (w/v) concentration was determined to be the most suitable matrix for these separations. This polymer, in coated capillaries, provided highly robust and reproducible separations, with near baseline resolution of fragments having single base differences. Reductions in the temperature of the separation, from 60 degrees C to 40 degrees C, and the urea concentration of the buffer, from 7 M to 3.5 M, provided increased longevity of the PEO polymer for repeated separations. Comparison of this polymer with currently specified commercial products used for STR analysis showed that the optimized PEO matrix provided superior separations under all conditions tested. In addition, PEO could be utilized in shorter capillary systems, with a concurrent decrease in analysis time, highlighting its potential for use in shortened capillary or microdevice systems.     

The effects of skeletal preparation techniques on DNA from human and non-human bone

The forensic pathologist increasingly relies on the forensic anthropologist to be the consulting expert in human identification. Likewise, if identification is not possible from visual inspection of skeletal remains, the forensic biologist may be called upon to conduct DNA analysis. The possibility of downstream DNA testing needs to be considered when skeletal preparation techniques are employed to deflesh human remains, as they have the potential to strongly impact genetic analyses and subsequent identification. In this study, three cleaning techniques, boiling bone in water, in bleach, and in powdered detergent/sodium carbonate, were tested for their effect on nuclear and mtDNA recovery from a variety of human and non-human bones. A statistically significant reduction in DNA yields occurred in non-human bones cleaned with bleach, and DNA degradation was apparent electrophoretically. The human bones also showed much lower yields from bleach cleaning, while the detergent/carbonate method allowed the largest segments of DNA to be amplified, indicating it may have a less degradative effect on bone DNA than either of the other cleaning processes.     

Personal identification by complex study of characteristics of the teeth and dentition structures

A multimodality dental examination was made and anatomomorphological features of teeth and tooth sequences were studied in 200 policemen using clinical examination, morphological investigation of plaster jaw models, analysis of x-rays and statistics, intraoral videocamera pictures, computer processing. The scheme of creation of dental data bank applicable in personal identification is offered.     

Criteria of personal identification by the hyoid bone

We studied 16 sizes of the hyoid bone (HB) from 158 corpses of known gender, Russians, who had died at the age over 15 years by the K. Miller et al program (1998) and 205 back projection electroroentgenograms of the neck of patients of the traumatological hospital in the town of Kirov. HB skeletization was made by the enzymatic drug enzyme. SPSS package and multidimentional statistics (regression and discriminant analyses) were used for data processing. It was established that HB is an informative object of forensic medical personal identification allowing determination of the body length and gender as well as anatomical, x-ray and osteometric individual features. Precision of human body length diagnosis by HB is 6.71-7.26 cm. Therefore, this method should be used in combination with the diagnosis by long tubular bones. Diagnosis of human gender by osteometric signs of HB based on one-dimentional discriminant analysis is feasible in 80-85%, based on canonical discriminant analysis--in 91.1% cases. The method of anatomic, osteometric and roentgenological assessment of HB individuality can be used in personal identification by intravital x-ray of cervical organs and in traumatic defects of HB. The methods may be applied in forensic medical practice in expert examination of body fragments or isolated bone fragments.     

Identification of exhumed remains of fire tragedy victims using conventional methods and autosomal/Y-chromosomal short tandem repeat DNA profiling

In a fire tragedy in Manila in December 1998, one of the worst tragic incidents which resulted in the reported death of 23 children, identity could not be established initially resulting in the burial of still unidentified bodies. Underscoring the importance of identifying each of the human remains, the bodies were exhumed 3 months after the tragedy. We describe here our work, which was the first national case handled by local laboratories wherein conventional and molecular-based techniques were successfully applied in forensic identification. The study reports analysis of DNA obtained from skeletal remains exposed to conditions of burning, burial, and exhumation. DNA typing methods using autosomal and Y-chromosomal short tandem repeat (Y-STR) markers reinforced postmortem examinations using conventional identification techniques. The strategy resulted in the identification of 18 out of the 21 human remains analyzed, overcoming challenges encountered due to the absence of established procedures for the recovery of mass disaster remains. There was incomplete antemortem information to match the postmortem data obtained from the remains of 3 female child victims. Two victims were readily identified due to the availability of antemortem tissues. In the absence of this biologic material, parentage testing was performed using reference blood samples collected from parents and relatives. Data on patrilineal lineage based on common Y-STR haplotypes augmented autosomal DNA typing, particularly in deficiency cases.     

Methodological development: structured outcome assessment and community risk monitoring (SORM)

This paper describes an effort to develop a clinical tool for the continuous monitoring of risk for violence in forensic mental health clients who have left their institutions and who are dwelling in the community on a conditional release basis. The model is called Structured Outcome Assessment and Community Risk Monitoring (SORM). The SORM consists of 30 dynamic factors and each factor in SORM is assessed in two ways: The current absence, presence or partial och intermittent presence of the factors, which is an actuarial (systematized and 'objective') assessment. Secondly, the risk effect, i.e. whether the presence/absence of factors currently increases, decreases or is perceived as unrelated to violence risk, is a clinical (or impressionistic) assessment. Thus, the factors considered via the SORM can be coded as risk factors or protective factors (or as factors unimportant to risk of violence) depending on circumstances that apply in the individual case. Further, the SORM has a built-in module for gathering idiographical information about risk-affecting contextual factors. The use of the SORM and its potential as a risk monitoring instrument is illustrated via preliminary data and case vignettes from an ongoing multicenter project. In this research project, patients leaving any of the 9 participating forensic hospitals in Sweden is assessed at release on a variety of static background factors, and the SORM is then administered every 30 days for 2 years.     

When Initial Interviews Are Delayed a Year: Effect on Children's 2-Year Recall

Three- to nine-year-old children were interviewed about a medical emergency (injury requiring hospital ER treatment) two years after it occurred. Half of the number of children had been interviewed shortly after injury as well as 6 and 12 months later, while the remaining children had had only one prior interview a year after injury. There was remarkably little long-term deterioration in memory by both groups. Having a delayed initial interview had two effects, and both were relevant only to the harder-to-remember hospital treatment event: (a) The late-interview group was less accurate, and (b) early-interview children had more extensive free recall, suggesting that multiple prior interviews teach children the “rules of the memory game' when they are asked open-ended questions. Forensic implications are discussed.