An evaluation of the polymerase chain reaction method for forensic applications

This paper describes the use of polymerase chain reaction (PCR) for amplifying small amounts of DNA obtained from samples of interest to the forensic scientist. A region of the HLA DQalpha (DQa) locus was amplified in DNA prepared from the following: hair roots, liquid blood, blood-stains, semen and vaginal swabs (semen free and semen contaminated). A population study was conducted using DNA from 78 unrelated individuals. The observed distribution of HLA DQa alleles varied from that reported for an American population but obeyed the Hardy-Weinberg equilibrium. Interpretation problems associated with the PCR technique are discussed.     

The polymerase chain reaction and post-mortem forensic identity testing: application of amplified D1S80 and HLA-DQ alpha loci to the identification of fire victims.

The application of DNA typing methods after amplification by the polymerase chain reaction (PCR) of DNA derived from body tissues from charred fire victims was investigated. A total of 26 different tissue specimens from ten extensively burned individuals were analyzed. The samples included femoral muscle, psoas muscle, bone marrow and blood. The post-mortem period varied from 38 to 183 h. After amplifying the DNA by PCR from the various tissues, the D1S80 locus was analyzed with a high resolution polyacrylamide gel electrophoresis technique followed by silver staining and the alleles of the HLA-DQ alpha locus were detected by using a reverse dot blot format. All samples could be typed for both loci and the genotypes were consistent in the various tissues from each individual. A parentage test was performed in two cases and Mendelian inheritance of the alleles for both loci was observed.     

Application of species-specific polymerase chain reaction in the forensic identification of tiger species

Globally, tigers are considered to be endangered, and are listed on Appendix I of CITES. A simple test, using a species-specific primer pair, was developed to identify tiger meat, faeces and dried skin, and provide forensic evidence of illegal wildlife trade. The specific fragment of mitochondrial cytochrome b gene was also successfully amplified from raw DNA products extracted from single tiger hairs. This PCR-based approach opens a new avenue to forensic identification of less-than-optimal samples.     

PCR-based detection of HLA-DQ alpha and polymarker loci and their application in forensic science

Studies were conducted to evaluate the forensic applicability of multiplex amplification of HLA-DQ alpha and PM loci. These loci could be simultaneously typed by reverse dot blot approach where allele specific oligonucleotide probes were immobilized on a nylon membrane strip. Allele and genotype frequencies for HLA-DQ alpha and PM loci were determined in Chinese Han population. The frequency data can be used in individual identification and paternity test.     

Sex identification of forensic specimens by polymerase chain reaction (PCR): two alternative methods

Sex identification of forensic samples (bloodstains and decomposed tissue) by polymerase chain reaction (PCR) was investigated. Amplification of a segment of the amelogenin gene using a pair of primers revealed both Y- and X-specific bands at the same time. The gene has counterparts in both the X and Y chromosomes and a small deletion in the former made it possible to distinguish them. Analysis of the X-specific band is the most reliable method for sex identification. THe locus includes a single copy gene so a sample of 250 ng/tube of deoxyribonucleic acid (DNA) is required for identification. Amplification of part of the DYZ1 locus was attempted as an alternative method for analysis of infinitesimal amounts of sample. Even DNA from putrefied tissue could be analyzed by PCR because the locus consists of thousands of copies of repeating units pHY10.     

DNA typing of forensic material with mixed genotypes using allele-specific enzymatic amplification (polymerase chain reaction).

Biological material in forensic casework frequently contains a mixture of genotypes, with a predominance of material from the victim and only trace amounts from the person committing the crime. Physical separation of the two genotypes or preferential lysis of different cell types may sometimes be possible. However, it is often difficult to achieve complete separation due to the lysis of cells or lack of material. We have developed an enzymatic amplification system for the HLA DQA1 locus, that will allow the presence of individual alleles in a sample with mixed genotypes to be determined, independent of their initial proportion in the sample. This system permits the identification of an allele representing less than 10(-4) of the background genotype. Use of polymerase chain reaction (PCR) with general primers allows only alleles representing more than about 1% to be detected, while the allele-specific amplification represents up to a 1000-fold increase in sensitivity. This method was applied to a rape case and a combined rape and murder case; in both cases the biological evidential materials contained a mixture of alleles from the victim and the rapist. Allele-specific PCR revealed the presence of alleles identical to those of the suspect using DNA from a vaginal swab taken after a rape incident, whereas by using general primers in the PCR only trace amounts of alleles other than those of the victim were found. Similarly, allele-specific amplification of DNA from vaginal swabs from the murder case revealed the presence of alleles identical to those of the suspect, while standard PCR only indicated the presence of genetic material from the victim.     

Real-time polymerase chain reaction quantification of canine DNA

The accurate quantification of target DNA is an important step in the short tandem repeat analysis of forensic biological samples. By utilizing quantification data to control the amount of template DNA in the polymerase chain reaction (PCR), forensic scientists can optimize testing and minimize the consumption of limited samples. The ability to identify and quantify target DNA in mixed-species samples is crucial when it may be overwhelmed by nontarget DNA, as in cases of dog attack. We evaluated two quantitative real-time PCR assays for dynamic range, species specificity, and inhibition by humic acid. While both assays proved to be highly sensitive and discriminating, the Melanocortin-1 Receptor (MC1R) gene Taqman assay had the advantages of a shorter run time, greater efficiency, and safer reagents. In its application to forensic casework, the MC1R assay has been advantageous for quantifying dog DNA in a variety of mixed-species samples and facilitating the successful profiling of individual dogs.     

The detection of RhD antigen of the rhesus system by the real time polymerase chain reaction

A method for the detection of RhD using a DNA preparation and the newly-developed test-system based on the real time polymerase chain reaction is described. The study including a large variety of specimens has demonstrated the applicability of the novel system to forensic medical examination.     

The effect of luminol on presumptive tests and DNA analysis using the polymerase chain reaction.

This study was designed to test the following factors involved with processing luminol treated bloodstained evidence: 1) The reactivity of other presumptive chemical color tests, phenolphthalin (PT) and tetramethylbenzidine (TMB), following the application of the light emitting luminol presumptive test. 2) The effect of different cleanings of various bloody substrates on the luminol test. 3) The effect of different cleanings of various bloody substrates on the ability to obtain DNA suitable for PCR testing. 4) The ability to extract DNA from luminol treated bloodstained substrates using three extraction techniques. 5) The effect of spraying washed and unwashed bloodstains on various substrates with luminol on the ability to correctly type the DNA using PCR. Our findings indicated that luminol did not adversely effect the PCR testing and did not interfere with the PT and TMB presumptive tests for blood. It was determined that the substrate and the method of cleaning were the major factors affecting DNA yield and the ability to type the bloodstains using PCR based technologies.     

An analysis of single and multi-copy methods for DNA quantitation by real-time polymerase chain reaction

The goal of this paper was to examine and compare two different commercially available approaches to the determination of the relative quantities of autosomal and Y chromosomal DNA using real-time PCR. One, Quantifiler^® Duo, utilizes a TaqMan^® assay with single copy probes for both autosomal human and Y quantification. The other method, Plexor HY^® utilizes a primer quenching assay with multi-copy probes for its quantification of autosomal human and Y chromosomal DNA. To test these approaches we have utilized the NIST Human DNA Quantitation Standard Reference Material 2372, a set of three different NIST human DNA quantification standards, to examine the precision, accuracy and sensitivity of the real-time PCR assays. We also examined data from both systems utilizing casework samples. The results show that both systems produced linear estimates for DNA quantity over a broad range of input DNA. However we did observe some apparent copy number effects when comparing the three different NIST standards which we attributed to issues with sequence variations in the different standards. Overall, the single copy approach provided better accuracy while the multi-copy approach produced better sensitivity. Thus the choice of which system to use should depend upon the goals of the user.     

Fixation of cells for analysis by laser microdissection--comparative studies in forensic trace material

This paper is focused on the preparation of samples for laser microdissection (LM) in forensic casework. In forensic genetics, it is essential to preserve and separate cellular traces during sample preparation, as they are usually gathered in very small amounts and are often contaminated with undesired cells. This is made possible by laser microdissection, a technique developed to cut cells or tissue of a certain type from a microscopical specimen by UV laser and catapult them directly into a PCR reactor. This method minimizes the risk of getting inconclusive, mixed DNA profiles due to contamination by foreign DNA and also supplies information about the cellular origin of a DNA profile. A method for optimized fixation and staining of spermatozoa for laser microdissection was established. Four different fixation methods combined with two staining methods were tested on two different microscope slides. Moreover, the effect of a blocker pen to contain the specimen on the slide was investigated.     

Analysis with the Combur-Test--special aspects in forensic trace examination

The Combur Test is a ready-made and easy-to-use pretest for blood. It is based on the oxidation of tetramethylbenzidine (TMB), which is catalysed by haemoglobin and its derivatives. Despite its high sensitivity, there are many known substances which are responsible for false positive and false negative test results. On the basis of experiments of our own, case reports and the pertinent literature special aspects of the application of the Combur Test in the forensic routine case work are discussed.     

Use of radiographs in the forensic autopsy

The X-ray examination of corpses is a most useful tool in the field of medicolegal diagnostics. Sometimes the cause of death can already be seen before autopsy. The following modes of application are reviewed: search for foreign bodies, identification of poison, air-embolism, pneumothorax, air-filled lungs and gastro-intestinal organs in new-borns, diagnosis of tuberculosis in corpses, visibility of hidden fractures, structure of bones as a factor of biomechanical load capacity, identification and determination of age and experimental research with corpses.     

Detection of species of graft in xenotransplants using arbitrary primed polymerase chain reaction.

Because of a shortage in the availability of human organs, xenografts have been attempted in humans with cardiac, renal, and hepatic failure, despite limited success. Use of xenografts, however, is regulated under law in various countries. In xenotransplant cases related to violation of transplantation law, determination of species of the source of tissue and organ(s) becomes highly essential. Random amplified polymorphic DNA (RAPD) protocols using six sets of arbitrary short-sequenced primers have been standardized for verifying claims of porcine cardiac and renal grafts in human transplantation cases. Six arbitrary primers used were found to generate unique amplicon patterns at 36 degrees C annealing temperature. Among the selected primers, a single primer set having the sequence 5'- GGTGCGGGAA -3' is found to be the most informative in discerning porcine tissue contamination in humans. The patterns obtained were consistent for a particular genome. The grafted organs in the studied case were analyzed to be of porcine origin.     

Use of forensic investigations in anti-doping

The fight against doping is mainly focused on direct detection, using analytical methods for the detection of doping agents in biological samples. However, the World Anti-Doping Code also defines doping as possession, administration or attempted administration of prohibited substances or methods, trafficking or attempted trafficking in any prohibited substance or methods. As these issues correspond to criminal investigation, a forensic approach can help assessing potential violation of these rules. In the context of a rowing competition, genetic analyses were conducted on biological samples collected in infusion apparatus, bags and tubing in order to obtain DNA profiles. As no database of athletes' DNA profiles was available, the use of information from the location detection as well as contextual information were key to determine a population of suspected athletes and to obtain reference DNA profiles for comparison. Analysis of samples from infusion systems provided 8 different DNA profiles. The comparison between these profiles and 8 reference profiles from suspected athletes could not be distinguished. This case-study is one of the first where a forensic approach was applied for anti-doping purposes. Based on this investigation, the International Rowing Federation authorities decided to ban not only the incriminated athletes, but also the coaches and officials for 2 years.     

Use of orthopantomographs in forensic identification

Radiographs of the head region were used for identification of 17 victims during an 11-year period in the Department of Forensic Medicine, University of Turku, Finland. Examinations resulted in positive identification of 10 victims. Proof of identity of four people was based on exclusion identifications. In one case, comparable information supporting the identity was achieved. Due to insufficient ante- and postmortem material, two individuals remained unidentified by radiological methods. The use of orthopantomography in identification is recommended because it enables visualization of the structures of the jaws and related areas on a single radiograph.