Use In Forensic Casework Research Articles

Seven SNPs and YAP demonstrate that the chromosome Y lineages of Basques are different from Georgians and Berbers

The Y chromosome is an important tool for discriminating related males and populations. The short tandem repeat (STR) markers of the Y chromosome are highly variable and can therefore discriminate between chromosomes of different paternal lineages, which is of great usefulness in forensic casework. The power of discrimination of each single nucleotide polymorphism (SNP) marker is lower than that of STR markers, although a broad combination of them also provides high discrimination capacity. SNP markers provide an additional advantage over STRs because they have more capacity for distinguishing human population groups and within specific populations. Thus, they are a useful tool for evaluating similarities between Basque, Georgian and Berber populations. The relationships found to date among these populations are based, principally, on linguistic data [1,4,6]. Previous genetic data have only related Basque and Berber populations [2]. Given the evidence reported so far, a more detailed study appeared necessary to evaluate the genetic relationships between Basque, Georgian and Berber populations. Accordingly, we decided to use SNP markers of the Y chromosome as the best tool for discriminating among populations.

High resolution Y chromosome typing: 19 STRs amplified in three multiplex reactions

Nineteen Y-specific short tandem repeat (STR) loci have been amplified in 768 samples from the Iberian Peninsula in order to evaluate their usefulness in forensic casework. Two previously published multiplex reactions by Thomas et al. [Hum. Genet. 6 (1999) 577] (MS1, modified here: DYS19, DYS388, DYS390, DYS391, DYS392 and DYS393) and by Ayub et al. [Nucl. Acids Res. 28 (2000) e8] (CTS: DYS434, DYS435, DYS436, DYS437, DYS438 and DYS439) plus a novel one reported here (EBF: DYS385, DYS389, DYS460, DYS461, DYS462 and amelogenin) have been used. DYS385, DYS439 and DYS391 were the most informative loci with allele diversities of 0.7997, 0.6683 and 0.5940, respectively. A total of 635 different haplotypes were observed, of which 573 (90.24%) were found in single individuals. The overall haplotype diversity was 0.9988 and that obtained by each multiplex system was 0.9812 for EBF, 0.9292 for MS1 and 0.9089 for CTS.

Validation of the AmpFℓSTR™ Profiler Plus PCR Amplification Kit for Use in Forensic Casework

According to TWGDAM guideline 4.5 (1), prior to implementing a new DNA analysis procedure or an existing DNA analysis procedure developed by another laboratory, the forensic laboratory must first demonstrate reliability of the procedure in-house. Seven phases were designed to validate the use of the AmpFlSTR Profiler Plus PCR Amplification Kit, as well as the PE Applied Biosystems 310 Genetic Analyzer. This report summarizes the results obtained for each of the seven phases of the validation study which included the following evaluations: polymer, reproducibility, sensitivity, stutter, precision, mixtures and nonprobative casework.

Validation of the AMP FlSTR ® SGM Plus™ system for use in forensic casework

The AMP FlSTR ® SGM Plus™ system is a commercially available STR multiplex produced by Applied Biosystems, a division of Perkin Elmer, Foster City, California, USA that supersedes SGM. The multiplex contains the six SGM loci, amelogenin and four additional loci. These additional loci are D3S1358, D19S433, D16S539 and D2S1338. Consequently, the match probability is significantly improved (conservatively quoted as 1 in 10 9 for reporting a full profile match). The system was subjected to validation. For example, ageing and degradation studies demonstrated semen stains to be the most stable evidence type, whereas buccal scrapes were the least stable. An apparent rise in the sensitivity increases the chance of obtaining successful results from the more difficult samples submitted for analysis. Two of the new loci (D3S1358 and D19S433) are low molecular weight (between 100 and 150 base pairs); this improved the success rates of the degraded samples where high molecular weight loci may drop out. Of 26 non-primates tested, four gave results that appeared as single peaks and were unlikely to cause interpretation problems. None of the 19 micro-organisms tested gave discernible results. Extensive casework and simulated casework studies demonstrated that SGM and SGM plus results were comparable. There was one example of a null allele (primer binding site mutation) recorded at the HUMFIBRA locus (in both systems). However, a concordance study of 1000 samples using both SGM and SGM plus did not demonstrate any differences in typing.

Validation of the PowerPlex 1.1™ Loci for Use in Human Identification

STR typing is now the favored method of DNA analysis for the purposes of human identification in the forensic community. The Forensic Services Division of the Detroit Police Department has completed its validation of the PowerPlex 1.1 loci (CSF1PO, TPOX, THO1, vWA, D16S539, D7S820, D13S317, and D5S818) for use in forensic casework. Detroit Metro Area Red Cross samples were typed from each of five racial/ethnic groups--the Hispanic, Caucasian, African American, Asian, and American Indian populations--and allele and genotype frequencies were calculated. A rare off-ladder variant (9.1 allele at D7S820) was identified among the database samples. A number of validation studies were performed. DNA was extracted from different substrates and typed as expected, except for the DNA extracted from leather (signal absent from the D16S539, D7S820, D13S317, CSF1PO, and TPOX loci) and from dirt (no PCR product generated). The minor contributor in the mixture study (250 pg input DNA) was facile to discern. The Concordance study, the variety of fluids from the same individual, and NIST standards studies all produced the expected results. Finally, STR data confirmed previous DNA typing results from adjudicated casework samples.

Validation of mitochondrial DNA minisequencing for forensic casework.

The validation of multiplex solid-phase fluorescent minisequencing of mitochondrial DNA (mtDNA) for use in forensic casework is presented. Validation included testing of the reliability and species specificity of the technique, analysis of mixed body fluid samples, analysis of samples and substrate controls from previous cases and somatic stability of mtDNA. Animal, bacterial and fungal species extracts were examined and the test did not show cross-reactivity with other species. Hair, blood, saliva, faeces and semen or vaginal samples were tested from five male and five female individuals. For all the samples tested, heteroplasmy was observed only at position 302/309.1. Body fluid mixtures (blood:saliva, semen:saliva, faeces:semen, vaginal:semen) and DNA:DNA mixtures were examined. In total, 189 mixtures were analysed of which one resulted in a hybrid profile consisting of peaks from each of the two donors. The semen fraction of the semen:saliva and vaginal:semen mixtures appeared to be concentrated in the supernatant fraction of the extract thus highlighting the need to extract both the pellet and supernatant fractions of a stain. Control samples, crime stains and their substrate controls from previous cases were examined. Of the 12 loci typed by minisequencing, 11 could be verified by comparison to results from the sequencing method currently in use for casework and no discrepancies were observed between the two. MtDNA minisequencing was found to be a reliable and reproducible technique and its rapid and discriminating nature make it particularly suitable as a screening technique.

A Bayesian approach to validating STR multiplex databases for use in forensic casework.

A previous paper in this journal has described the conventional statistical analysis of three databases (Caucasian, Afro-Caribbean and Asians from the Indian subcontinent) where individuals are typed at six short tandem repeat (STR) loci. This paper presents a Bayesian analysis of the same data and the approach is centred on the concept of estimating coancestry coefficients from mixed databases. Posterior distributions for the three databases are presented and the discussed and the consequences of implementing bootstrap estimation procedures are also shown.

Allele frequency distributions of 13 PCR-based systems in a population from North-East Spain.

Population data studies were carried out on a Caucasian population from North-East Spain (n = 129-292 individuals) for 13 PCR-based polymorphic DNA loci: six short tandem repeat loci (HumTH01, HumTPOX, HumCSF1PO, HumF13A01, HumFES/FPS, HumvWFA31), the six PM loci (HLA-DQ alpha, LDLR, GYPA, HBGG, D7S8, GC) and one variable number tandem repeat locus (D1S80). The genotypes distributions were in accordance with Hardy-Weinberg expectations. The combined use of the 13 polymorphic systems provides a high power of discrimination and power of exclusion for use in forensic casework and paternity testing.

The validation of a 7-locus multiplex STR test for use in forensic casework. (I). Mixtures, ageing, degradation and species studies.

We have evaluated a multiplex STR system for routine forensic use, which co-amplifies six short tandem repeat (STR) loci; HUMTH01, D21S11, D18S51, D8S1179, HUMVWF31/A and HUMFIBRA (FGA), in conjunction with the X-Y homologous gene Amelogenin. Analysis of PCR products employs denaturing polyacrylamide gels coupled with fluorescent labelled primers and detection is undertaken on ABD 373A automated sequencers. The technique was shown to be robust and reproducible when samples were analysed under conditions consistent with those encountered in a forensic environment. The system was demonstrated to be human specific and is suitable for use with both aged and degraded material. Somatic stability was proven with a wide range of tissue types and we were able to detect mixtures at ratios between 1:10 and 10:1. During this study no incidence of sample mis-typing due to allelic or locus drop-out was observed. Furthermore, although additional artefact bands were occasionally encountered these did not interfere with the interpretation of results. The performance of the system with poor quality samples demonstrated its suitability as a powerful tool in forensic investigation.

The validation of a 7-locus multiplex STR test for use in forensic casework. (II), Artefacts, casework studies and success rates.

PCR-based DNA typing of biological evidence is now widely used in forensic analyses due to the obvious advantages of enhanced sensitivity, the ability to distinguish discrete alleles and efficacy with degraded samples. A multiplex short tandem repeat (STR) system has been previously developed which successfully co-amplifies six STR loci HUMTH01, D21S11, D18S51, D8S1179, HUMVWF31/A and HUMFIBRA (FGA) in conjunction with the X-Y homologous gene Amelogenin. This is known as the second generation multiplex system (SGM). Detection of the PCR products is undertaken on ABD 373A or 377 automated sequencers using denaturing polyacrylamide gels coupled with fluorescent-based technology. We have evaluated this system for routine forensic use and demonstrated that the technique is robust and reproducible under conditions consistent with those encountered in a forensic environment. A total of 132 stains from simulated and actual casework were analysed, together with relevant control areas and reference samples. The success rate was high with 76% of stains giving full profiles; we were also able to successfully detect and interpret mixtures. No mistyping was observed. A detailed examination of each of these profiles has assisted in the development of guidelines for casework interpretation. Although artefacts, stutter peaks and undenatured DNA were occasionally observed, these did not interfere with the accuracy of interpretation. In addition 38 samples, previously examined using the quadruplex system, were analysed with the SGM to enable a direct comparison to be made between the systems. The performance of the system with poor quality samples demonstrated its use as a rapid and powerful technique for individual identification.

Chemiluminescent Detection of RFLP Patterns in Forensic DNA Analysis

DNA testing by restriction fragment length polymorphism (RFLP) analysis is an extremely important technique used in forensic science laboratories. While RFLP testing is a highly informative method, it traditionally has had several disadvantages. It is time consuming and involves work with radioactive phosphorous. A detection method that is faster and safer than isotopic detection is presented. Various membranes, fixation methods and transfer procedures were evaluated for DNA retention and sensitivity using alkaline phosphatase conjugated oligonucleotide probes and a chemiluminescent substrate. Blood samples and evidentiary material from forensic casework were analyzed by both chemiluminescent and isotopic detection. Results of each method were compared for pattern appearance, band size, and composite profile frequency. The chemiluminescent system had very good sensitivity, detecting 3-25 ng K562 DNA. Most patterns developed by both methods appeared the same. The variation observed between band sizes and frequency estimates generated by each method was as expected for an inter-gel comparison. The chemiluminescent detection procedure described here is suitable for use in forensic casework.

A study of independence between STR and conventional blood type loci

Blood samples from approximately 200 Scottish Caucasian individuals were typed at conventional loci (PGM, Gc and EAP) and also with a four locus STR multiplex. Tests of the data are described which demonstrate that the assumptions of between locus independence are robust for use in forensic casework.

Further validation of a multiplex STR system for use in routine forensic identity testing

A polymerase chain reaction- (PCR) based short tandem repeat (STR) system has recently been developed for use in routine forensic identity testing [1]. The methodology involves the simultaneous amplification of alleles at four loci on different chromosomes, followed by the fluorescent detection of products using an automated DNA sequencer. The adoption of this technology into operational casework offers several advantages over systems currently in use, particularly the ability to obtain results from very old or small samples, reduced operator time when compared with conventional DNA (single locus probe) analysis and the potential for automation. Validation studies were incorporated into the development work on this system [2,11]. The scope of these studies has been extended by further investigation carried out in this laboratory to test the reliability of the system under normal operational procedures. It was demonstrated that the precision of size determination was sufficient for the discrimination of alleles and size windows for allelic designation were established. A collaborative exercise carried out in conjunction with two independent laboratories demonstrated the robustness of allelic designation. Having tested both the DNA quantification and amplification techniques against DNA samples from a wide range of animal and microbial species, it was confirmed that results are only obtained from higher primate DNA. The PCR methodology was tested with both simulated and real casework samples (over 250 in total). Reportable results were obtained from most items yielding extracted DNA. Approximately 20% of the casework items from which no grouping (ABO, PGM) nor SLP results were obtained, gave reportable STR results. A method for the routine purification of DNA extracts which failed to amplify was established and validated for use in forensic casework. The STR multiplex system developed by Kimpton et al. [1] proved robust and reliable when tested under the operational procedures in place in this laboratory.

Validation of the AmpliFLP™ D1S80 PCR Amplification Kit for Forensic Casework Analysis According to TWGDAM Guidelines

The validation of the AmpliFLP D1S80 PCR Amplification Kit for use in forensic casework was accomplished by performing all the relevant experiments outlined in the TWGDAM guidelines. Standard specimen and reproducibility studies were performed using organic and rapid DNA extraction techniques on both stain and liquid samples (blood, semen and saliva). Over 300 samples from three different populations (US Caucasians, African Americans and US Hispanics) were analyzed to determine allele and genotype frequencies. Purified DNA was mixed in defined ratios (ranging from unmixed DNA samples to 1:9 mixtures of 2 different DNA samples) prior to amplification to demonstrate that samples containing DNA from more than one individual can be detected and, in many cases, that the genotypes contributing to the mixture can be identified. Since casework samples frequently are exposed to environmental insults that can result in DNA degradation, purified DNA was degraded in the laboratory to analyze the effect of DNA fragment length on D1S80 amplification. It is crucial in the validation process to examine actual casework evidentiary material. This D1S80 kit can be used successfully by forensic scientists to amplify and type nonprobative evidentiary material, including bloodstains collected from crime scenes and rape kit materials collected for sexual assault cases. The D1S80 kit is specific to human DNA, and the D1S80 alleles are inherited according to the laws of Mendel. The sensitivity of the novel gel electrophoresis gel matrix allowed the PCR cycle number to be reduced to 29 cycles and the D1S80 kit sensitivity to be increased to 2.5 ng from the previous D1S80 Reagent Set specifications of 30 cycles and 5 ng, respectively.

The validation of short tandem repeat (STR) loci for use in forensic casework.

A quadruplex reaction has been developed which amplifies the short tandem repeat (STR) loci HUM-VWA31/A, HUMTHO1, HUMF13A1 and HUMFES/FPS. Detection of the PCR products employs denaturing polyacrylamide gels coupled with fluorescent-based technology. This system has been evaluated for use in routine forensic casework and has been shown to be both robust and reproducible. The quadruplex reaction is as sensitive as the commercially available HLA DQ alpha Amplitype typing system and can be used on both degraded and aged material. The problems of environmental contamination have been shown to be limited provided strict procedural practices are followed-i.e. physical separation of sample extraction and amplified products; the use of dedicated equipment such as pipettes; the separation of amplification preparation area. The ability of the system to detect mixtures and the successful analysis of case stains has shown that this system is well suited as a tool for forensic investigation.

Discriminant function analysis of the central portion of the innominate.

Terry Collection innominates of 260 American whites and blacks (65 males and 65 females of each race) were analyzed by discriminant function analysis for sex assessment, race known, and for simultaneous race and sex assessment. Measurements from the preservationally favored central portion of the innominate were chosen for their potential usefulness in forensic casework and to objectify long-recognized "formal" differences between the sexes in the greater sciatic notch. These included acetabular diameter, greater sciatic notch height, and position of greatest notch depth. Accuracy of sex prediction when race is known is roughly 90% in both the base sample of 260 and a test sample of 200 (50 additional individuals for each group). For simultaneous race and sex assessment, accuracy of prediction is roughly 60% for each group in both samples. This represents about a 45% reduction in error over random assignment by race and sex.

The Release of Basic Drugs by the Enzymic Digestion of Tissues in Cases of Poisoning

The proteolytic enzyme subtilisin Carlsberg has been used to digest human livers containing a variety of basic drugs. Results of subsequent drug analysis showed that greatly increased levels of these drugs were released compared with degradation using conventional acid hydrolysis procedures. An analytical procedure is proposed for use in forensic case work for the isolation of basic drugs, unchanged benzodiazepines and drug metabolites in high yield from a single liver digest.