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.

We have examined the performance and reproducibility of an automated DNA profiling system which is based on the multiplex amplification of 4 tetrameric STR loci-HUMVWFA31/A. HUMTH01, HUMF13A1 and HUMFES/FPS. The system was able to type 100 pg of purified, undegraded, genomic DNA. At lower concentrations of DNA (below 100 pg), allelec drop-out occurred due to stochastic differences in allele copy number. Minor variation of individual PCR reagent concentrations or cycling temperatures did not result in a significant effect on the efficiency of amplification of any of the 4 loci in the quadruplex system. More substantial variation of reagent concentrations or cycling temperatures outside the optimum range of the system resulted in a reduction or complete loss of signal for one or more loci. This was also observed at high ionic strength or extreme pH. However, under all reagent concentrations and conditions studied, no artefact bands that could potentially result in the mistyping of a sample were apparent within the read region (130-240 bases) of the gel. Evaluation of both native and denaturing polyacrylamide gels revealed that, although native gels displayed faster run times, the sizing precision of such gels for certain STR loci was lower than that of denaturing gels. Also, artefact bands may be present within the read region of native gels. In conclusion the quadruplex amplification system described, coupled with automated fluorescence-based detection on denaturing polyacrylamide gels, appeared to be a robust and reliable system for individual identification.

Many highly polymorphic minisatellite loci can be detected simultaneously in the human genome by hybridisation to probes consisting of tandem repeats of the 'core' sequence. The resulting DNA fingerprints produced by Southern blot hybridisation are comprised of multiple hypervariable DNA fragments, show somatic and germline stability and are completely specific to an individual. DNA of high molecular weight can be isolated from blood and semen stains up to 4 years old. Sperm nuclei can be separated from vaginal debris and the sperm DNA examined in isolation, allowing the positive identification of rapists. Correspondingly, vaginal DNA can be isolated from extracts ofpenile swabs. DNA can also beisolatedfromextractsofbuccal swabs. A blind trial involving 4 3 blood samples, 11 bloodstains and 11 semen stains has been successfully carried out. It is envisaged that genetic fingerprinting will shortly form an important part of casework procedure in forensic science laboratories. Materials and methodsDNA fingerprints were produced as described by Jeffreys et al. [ I , 21. DNA was extracted from whole blood, semen, blood stains, semen stains, hair roots, buccal swabs, and semen-free vaginal swabs by overnight incubation in a sodium dodecyl sulphate (SDS) proteinase K/dithiothreitol (DTT) mixture. Approximately 10 pg of vaginal DNA can be obtained from semen-free vaginal swabs. This quantity of DNA will obscure the presence of sperm DNA from semen-contaminated vaginal swabs; hence it is essential to separate sperm from vaginal cells as previously described [31. Vaginal cells from semen-contaminated swabs were preferentially lysed bv a Dreliminarv incubation in the absence of DTT for 30 Abbreviations: SDS, sodium dodecyl sulphate; Dm, dithiothreitol; kb, kilobase min using the lysG misture. Sperm nuclei were pelleted by centrifugation, washed once with the same mixture, repelleted 0 VCH Verlagsgesellschaft mbH, D-

Populations of white Caucasians, Afro-Caribbeans and Asians residing within the UK have been analysed at 4 different hypervariable loci. A computerised system was used to store and to analyse the data. Simulation experiments were carried out in order to determine whether there was any evidence for population stratification, which would lead to non-independence of allelic distributions.

The history of DNA profiling in the Home Office Forensic Science Service began with the introduction of multilocus probes into casework in 1986. The use of single-locus probes was introduced in 1990, supported by databases of three ethnic groups; interpretation is backed up using a Bayesian approach. Databases were compiled using an image analysis computing system. Quality control systems are described, detailing requirements before a sample can be included in the database.

Allele and genotype frequencies for British Caucasian, Afro-Caribbean and Asian populations were determined for a total of over 600 unrelated individuals at the HLA-DQ alpha locus. These were analysed by polymerase chain reaction amplification of the DNA followed by hybridisation to allele specific oligonucleotide probes in a reversed dot-blot test. Six different alleles were detected and the allele distributions for the 3 populations analysed displayed significant differences. However, the British Caucasian genotypes were statistically very similar to previously published data from US Caucasians as were British Afro-Caribbean genotype frequencies with US Black data. In Caucasians the allele frequencies ranged from 5.2% to 26.9% with a power of discrimination of 0.93. DQ alpha genotype frequencies of Caucasian and Afro-Caribbean populations do not deviate from Hardy-Weinberg equilibrium. However, the Asian data displayed significant deviation due to excess homozygotes.

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