Swiss Caucasian population data for the STR loci D2S1338 and D19S433 using the AmpFISTR SGM plus PCR amplification kit.

Allele and genotype frequencies for the ten STR loci D3S1358, VWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, TH01, FGA were determined in a Swiss Caucasian population sample (n=206) using the AmpFISTR SGM Plus Amplification kit. Electrophoresis was carried out on an ABI PRISM CE 310 Genetic Analyzer instrument. Previously, allele frequencies were published for the 13 STR loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, THO1, TPOX, CSF1PO and D16S539 for the same samples (n=206) amplified with the AmpFISTR Profiler Plus and Cofiler PCR Amplification kits. Since the results for the eight loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, THO1, D16S539 shared between the AmpFISTR SGM Plus, Profiler Plus and Cofiler PCR Amplification kits already are published, only the allele frequencies for the two STR loci D2S1338 and D19S433 are reported in this paper. The two loci meet Hardy-Weinberg expectations. In addition, there is little evidence for association of alleles among the 15 loci (amplified with the Profiler, Cofiler, and SGM Plus amplification kits). The allelic frequency data can be used in forensic analyses to estimate the frequency of a multiple STR locus DNA profile in the Swiss population.

Swiss Caucasian population data for 13 STR loci using AmpFISTR profiler plus and cofiler PCR amplification kits.

Allele and genotype frequencies for the 13 core STR loci (D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, THO1, TPOX, CSF1PO, and D16S539) were determined in a Swiss Caucasian population sample (n = 206) using two commercially available multiplex PCR kits (AmpFISTR Profiler Plus and AmpFISTR Cofiler) and subsequent electrophoresis on an ABI PRISM CE 310 Genetic Analyzer instrument. All loci meet Hardy-Weinberg expectations. In addition, there is little evidence for association of alleles among the 13 loci. The allelic frequency data can be used in forensic analyses and paternity tests to estimate the frequency of a multiple STR locus DNA profile in the Swiss population.

Evaluation of prostate-specific antigen (PSA) membrane test assays for the forensic identification of seminal fluid.

Prostate specific antigen (PSA, also known as p30), a glycoprotein produced by the prostatic gland and secreted into seminal plasma, is a marker used for demonstrating the presence of seminal fluid. Methods for the detection of PSA include Ouchterlony double diffusion, crossover electrophoresis, rocket immuno-electrophoresis, radial immunodiffusion, and ELISA. The extremely sensitive ELISA technique can detect PSA in concentrations as low as approximately 4 ng/mL. However, all these techniques are cumbersome and time consuming to perform in forensic laboratories, especially when only a few samples per week are processed. Various membrane tests are currently used in clinical settings to screen a patient's serum for the presence of PSA at levels greater than 4 ng/mL. In this study we evaluated three immunochromatographic PSA membrane tests by analyzing semen stains stored at room temperature for up to 30 years, post-coital vaginal swabs taken at different time after intercourse, semen-free vaginal swabs, and various female and male body fluids, including urine. The data demonstrate that PSA membrane test assays offer the same sensitivity as ELISA-based tests and provide a rapid approach for the forensic identification of seminal fluid. Furthermore, when the supernatant from a DNA extraction is used for the assay, there is essentially no DNA consumption for determining the presence of PSA in a forensic sample.

Effects of toluidine blue and destaining reagents used in sexual assault examinations on the ability to obtain DNA profiles from postcoital vaginal swabs.

Toluidine blue is an important tool to detect and document genital and perianal injuries following sexual assault. Application of toluidine blue dye and its subsequent removal from unstained areas by means of a destaining reagent, such as diluted acetic acid or a lubricant has been shown to increase the detection rate of posterior fourchette lacerations from 16% to 40% in adult rape victims. Currently, limited information on toluidine blue positive findings in sexually active control groups imposes some limitation on the interpretation of these injuries. Because injuries could otherwise be attributed to improper handling of an examination speculum or the improper insertion of the examining finger, the toluidine blue test should be performed prior to any digital or speculum examination and thus prior to the collection of forensic evidence. For forensic DNA identity testing, it becomes pertinent to determine whether toluidine blue and the destaining reagents used in a sexual assault examination have an adverse effect on the recovery of high molecular weight DNA from postcoital vaginal swabs and thereby have an impact on restriction fragment length polymorphism (RFLP) analysis or PCR-based tests. It is known that some of the lubricants used can have a destructive effect on sperm motility. In order to investigate the potential effects, postcoital vaginal swabs were taken 6 h after sexual intercourse and exposed directly to 1% toluidine blue in aqueous solution, 1-10% acetic acid, and various surgical and vaginal lubricants. Subsequently, the DNA was isolated and DNA identity typing (RFLP and PCR-based) was performed. The results demonstrate, that these reagents have no negative effect on the ability to obtain DNA profiles, either RFLP or PCR-based, from shallow and deep vaginal swabs. The quantity and quality of extractable high molecular weight DNA obtained was comparable with that from uncontaminated postcoital vaginal swabs. RFLP patterns and PCR-based typing results on the D1S80, HUMTH01, TPOX, and CSF1PO loci were consistent with the uncontaminated control swabs and the corresponding whole blood samples of the donors. Therefore, evidentiary material inadvertently contaminated with these reagents can be successfully typed.

[A foldable cardboard box for drying and storage of by cotton swab collected biological samples]. / Eine faltbare Kartonbox zur Trocknung und Aufbewahrung von mittels Wattetupfern gesicherten biologischen Spuren.

The ability to perform successful DNA analysis on biological evidence obtained at a crime scene or during a sexual assault examination depends very much on the first step--how specimens are collected and preserved. Body fluids and their wet or dry stains, are often recovered using dry cotton swabs or cotton swabs moistened with sterile water or saline. In order to prevent decomposition and deterioration of a specimen, resulting in degradation or loss of DNA, it is recommended to either air dry or freeze these swabs as soon as possible after collection. We designed a simple, foldable cardboard box, which is suitable for the drying and storage of biological evidence collected on cotton swabs. Immediately after collection swabs are placed into the drying racks within the cardboard box, which is subsequently folded, labeled, sealed and initialed. At room temperature swabs completely air dry within the sealed box within 6-9 hours. In this box the evidence is properly packed, labelled and sealed, thus preventing cross contamination, degradation and sample switch. It is a valuable device for the collection of biological evidence at a crime scene, during sexual assault examinations, and for the collection of buccal swabs for PCR-based databasing and paternity testing.

Using multiplex PCR amplification and typing kits for the analysis of DNA evidence in a serial killer case.

Analysis of DNA evidence in a serial killer case was performed using the AmpliType HLA-DQ alpha-, AmpliType PM-, and the GenePrint STR Multiplex System PCR Amplification Kits. In addition, a sex typing procedure using the X-Y homologous gene amelogenin was carried out. DNA profiles from a single hair with attached sheath material, recovered from underneath the seat cover of the suspect's car seat were compared with DNA profiles derived from reference head hairs from a homicide victim. From the evidentiary sample only 9 ng of human DNA could be recovered. In a sample, where the quantity of DNA becomes a critical issue a powerful route is the simultaneous amplification of several loci (multiplex PCR). This is the first report where commercially available multiplex PCR amplification and typing kits have been introduced for the analysis of DNA evidence in a serial killer case and the analysis has been admitted in court.

A method for the purification and recovery of genomic DNA from an HLA DQA1 amplification product and its subsequent amplification and typing with the AmpliType PM PCR Amplification and Typing Kit.

DNA from plucked single hairs from ten individuals was extracted by two different methods and subsequently amplified and typed using the AmpliType HLA DQ alpha Forensic DNA Amplification and Typing Kit. The remaining untyped portions of the DQA1 amplification products were stored refrigerated or frozen for two weeks and subsequently purified using Centricon 100 microconcentrators. Genomic DNA was recovered from the DQA1 amplification PCR and used again as a template for a subsequent multiplex PCR. Twenty microL of each retentate were amplified and typed with the AmpliType PM PCR Amplification and Typing Kit. All typing results were consistent with DQA1 and PM results of control hairs and reference blood samples from the donors and all results were consistent with those obtained when the samples were typed solely for PM. The DQA1-Centricon 100-PM approach is useful when the genomic DNA from an evidentiary sample has been used completely for HLA-DQA1 typing, so that only the amplified product is remaining. The typing of five more genetic markers can be achieved from a HLA-DQA1 sample, so additional information for identification purposes could be provided. However, genomic DNA as well as the DQA1 product are recovered and the latter will also serve as a template in the subsequent PM amplifications. Therefore there will be more DQA1 product after the PM amplification than would be expected when only genomic DNA was used as a template. Thus certain practices should be considered when reading the types from PM probe strips if this DQA1-Centricon 100-PM approach is used.

Confirmation of the identity of human skeletal remains using Multiplex PCR Amplification and Typing Kits.

The identify of human skeletal remains found in a wooded area approximately one year after the person was reported missing was provisionally established by routine methods and circumstantial evidence. Multiplex PCR systems--the AmpliType PM PCR Amplification and Typing Kit and the GenePrint STR Triplex Amplification and Typing Kit--were used to confirm the identification. DNA profiles from femur bone from the remains were compared with profiles derived from head hairs from a hairbrush recovered in the missing woman's apartment. In addition, a sex typing procedure using the X-Y homologous gene amelogenin was carried out. This is the first report of a case using commercially available multiplex PCR amplification and typing kits to confirm the identity of skeletal remains.

Swiss population data and forensic efficiency values on 3 tetrameric short tandem repeat loci-HUMTH01, TPOX, and CSF1PO-derived using a STR multiplex system.

Allele and genotype frequencies for 3 tetrameric short tandem repeat loci were determined in a Swiss population sample (n = 100) using the GenePrint STR Multiplex System, electrophoresis of the PCR products in DNA sequencing gels and subsequent detection of allelic fragments by silver staining. The loci are HUMTH01, TPOX, and CSF1PO. The observed heterozygosities are 83.0%, 60.0%, and 72.0%, respectively. The discrimination power determined for the individual loci is 0.914, 0.780, and 0.860, respectively, and the combined discrimination power for the triplex is 0.997. All loci meet Hardy-Weinberg expectations and after Bonferroni correction there was no evidence that the population sample deviates from expectations of independence. Moreover, independence of alleles at these STR loci with other PCR-based loci derived from the same Swiss population sample, previously reported, were considered. These loci were DQA1, LDLR, GYPA, HBGG, D7S8, GC and D1S80. Again, after Bonferroni correction there was no evidence that the population sample deviates from expectations of independence among alleles at the 10 different PCR-based loci. Thus, the allelic frequency data can be used in human identity testing to estimate the frequency of a multiple PCR-based DNA profile in the Swiss population.

Swiss population data on the loci HLA-DQ alpha, LDLR, GYPA, HBGG, D7S8, Gc and D1S80.

Allele and genotype frequencies for seven polymerase chain reaction-based DNA genetic markers were determined in a Swiss sample population. The loci are D1S80, HLA-DQ alpha, low density lipoprotein receptor, glycophorin A, hemoglobin G gammaglobin, D7S8, and group-specific component. All loci meet Hardy-Weinberg expectations. In addition, there is no detectable association of alleles between loci. The frequency data can be used in forensic analyses and paternity tests to estimate the frequency of a DNA profile in the Swiss population.

Swiss population data on three tetrameric short tandem repeat loci--VWA, HUMTHO1, and F13A1--derived using multiplex PCR and laser fluorescence detection.

Allele and genotype frequencies for 3 tetrameric short tandem repeat loci VWA, HUMTHO1, and F13A1 were determined in a Swiss population sample using multiplex PCR and subsequent electrophoresis in DNA sequencing gels processed by automated laser fluorescence detection. The technique allows single base pair resolution and rapid typing, with a concomitant reduction in the potential for human transcriptional typing errors. All loci meet Hardy-Weinberg expectations. In addition, there is little evidence for association of alleles among the 3 loci. The allelic frequency data can be used in forensic analyses and paternity tests to estimate the frequency of a multiple STR locus DNA profile in the Swiss population.

[Preliminary tests with Hemastix and Sangur test strips and Phosphatesmo KM test paper do not modify DNA typing of blood and semen stains]. / Vorproben mit Hemastix-und Sangur Testastäbchen sowie Phosphatesmo KM-Testpapier beeinflussen die DNA-Typisierung von Blut- und Spermaspuren nicht.

Some of the commonly used presumptive test reagents for identification of blood and semen could affect the recovery or the analysis of high-molecular-weight DNA from evidentiary samples. The study presents data on restriction fragment length polymorphisms (RFLP) and polymerase chain reaction (PCR) analyses from blood and semen stains treated with Hemastix (Bayer Diagnostics), Sangur (Boehringer) and Phosphatesmo-KM-paper (Macherey-Nagel). The results demonstrate that the three tests examined do not have any effect on quality and quantity of the DNA nor on DNA typing by RFLP and PCR analyses. In conclusion, presumptive testing of blood and semen stains using Hemastix, Sangur, or Phosphatesmo paper can principally be carried out prior to submission to DNA analysis. In spite of these findings, it is recommended to continue the prudent practice of testing only small portions of an evidentiary stain in order to prevent contamination.

Effects of nonoxinol-9 on the ability to obtain DNA profiles from postcoital vaginal swabs.

Nonoxinol-9, the active ingredient of many spermicide foams and creams, has been shown to inactivate effectively high titres of HIV in vitro. Therefore the early administration of nonoxinol-9, perhaps by a rape victim herself, has been suggested as a potential prophylactic therapy for prevention of a possible HIV infection. For forensic DNA identity testing, it becomes pertinent to determine whether nonoxinol-9 could have an adverse effect on the recovery of high molecular weight DNA from postcoital vaginal swabs and thereby have an impact on restriction fragment length polymorphism (RFLP) analysis. If high molecular weight DNA can not be recovered, it may still be possible to proceed with analyses using PCR-based tests. In order to investigate the potential effects of nonoxinol-9, inserts, gels, or sponges containing nonoxinol-9 were applied either 15 min pre- or 15 to 60 min post coitus. Postcoital vaginal swabs were taken one and six h after sexual intercourse, the DNA was isolated and DNA identity typing was performed. The results demonstrate that nonoxinol-9 has no negative effect on the ability to obtain DNA profiles, either RFLP or PCR-based, from postcoital vaginal swabs. The quantity of extractable high molecular weight DNA obtained (as determined by slot-blot analysis) was comparable with that from uncontaminated postcoital vaginal swabs. RFLP patterns and PCR-based typing results at the HLA-DQ alpha and D1S80 loci from the nonoxinol-9 treated swabs were consistent with the uncontaminated control swabs and the corresponding whole blood samples of the donors. Therefore an early prophylactic administration of the topical anti-HIV agent nonoxinol-9 is not an impedient for obtaining DNA profiles from evidentiary material.

[Population genetic Hae III/RFLP and HLA-DQ-alpha data of a Caucasian population sample in Switzerland]. / Populationsgenetische Hae III/RFLP- und HLA-DQ-alpha-Daten einer kaukasischen Bevölkerungsstichprobe der Schweiz.

DNA from unrelated individuals (Caucasians; n = 200-271) from Switzerland were digested with Hae III and successively hybridized to the DNA probes D1S7, D2S44, D4S139, D10S28, D14S13, D17S26 and D17S79. An allele frequency distribution was determined for each locus. Furthermore, from the same individuals (n = 200) after amplification of DNA the allele and genotype frequencies at the HLA-DQ alpha locus were determined. The allele frequency distribution in Swiss Caucasians is statistically similar to American Caucasian population samples. In criminal cases a DNA-profile derived from four single-locus probes always leads to a very high value of discrimination and in paternity testing the probability of paternity always exceeds 99.9% regardless to the reference population used for biostatistical evaluation. Therefore for use in forensic analysis and paternity testing pooled caucasian databases might be used for the determination of the frequency of occurrence of a DNA-profile.

[Conversion of forensic paternity determination to DNA analysis]. / Umstellung der Abstammungsbegutachtung auf DNA-Analyse.

For many years, the resolution of disputed paternity cases by genetic means relied on laboratory blood tests of red cell antigens and red cell and serum protein electrophoresis. These systems are generally characterized as lacking polymorphism and having low powers of exclusion, therefore it was necessary to use a panel of up to 23 marker systems. Over the past five years, a variety of DNA restriction fragment length polymorphism (RFLP) systems have been developed that characterize individuals at the DNA level. In this paper we describe the implementation of a Hae III RFLP system utilized in our laboratory for the resolution of disputed parentage cases as well as forensic casework. Since the analysis of highly polymorphic VNTR loci has proven to be the most powerful method to date for the determination of biological relatedness the utilization of the conventional methodologies should be completely replaced.

[Effects of nonoxinol 9 on RFLP typing of postcoital vaginal smears]. / Auswirkungen von Nonoxinol 9 auf die RFLP-Typisierung postkoitaler Vaginalabstriche.

Nonoxinol 9 effectively inactivates high titres of HIV in vitro, which suggest its use for reducing HIV-transmission via sexual intercourse. Therefore, the suggestion has been made for the treatment of sexual assault victims with a topical anti-HIV agent such as nonoxinol 9 as soon as possible after a sexual assault has occurred. From the forensic point of view it becomes pertinent to determine whether or not nonoxinol 9 would have an adverse effect on the high-molecular weight deoxyribonucleic acid (DNA) in vaginal swabs and thereby impact RFLP results. The study demonstrates that nonoxinol 9 does not have a negative effect on the ability to produce RFLP patterns. Therefore, the early administration of the topical anti-HIV agent nonoxinol 9 has to be considered as an important step in the medical treatment of sexual assault victims.

Typing of deoxyribonucleic acid (DNA) extracted from compact bone from human remains.

The application of deoxyribonucleic acid (DNA) typing methods for the potential identification of unknown human remains was investigated. DNA was isolated from compact bone tissue from badly decomposed bodies and from known and unknown human remains, using a decalcification and ion wash procedure. Restriction fragment length polymorphism (RFLP) analysis of variable number of tandem repeats (VNTR) loci yielded results in some cases, but more often the DNA was too degraded to produce RFLP patterns. No RFLP profiles could be obtained from putrefied soft tissues. However, DNA extracted from compact bone tissue of human remains up to eleven years old was successfully amplified using the polymerase chain reaction (PCR) for the VNTR loci D1S80, D17S5, COL2A1, and APO B, as well as the HLA-DQ alpha locus. This is especially significant, since PCR results were obtained from those samples whose DNA had been degraded substantially and had yielded no RFLP patterns. All DNA types determined from the compact bone tissue from decomposed bodies whose identification had been established first by other means (and whose parents or offspring were available for typing) demonstrated mendelian inheritance of the alleles of the loci analyzed. These results suggest that amplification and typing of DNA extracted from compact bone of human remains could be useful in establishing the identity of a person, as well as in excluding possible false identifications.

PCR-based typing of DNA extracted from cigarette butts.

Limited genetic marker information can be obtained from saliva by typing by conventional serological means. Thus, the application of PCR-based DNA typing methods was investigated as a potential approach for typing genetic markers in saliva. DNA was isolated from 200 cigarettes smoked by 10 different individuals (20 cigarettes per individual) and from 3 cigarette butts recovered from 2 crime scenes (adjudicated cases) using a Chelex 100 extraction procedure. The amount of recovered human DNA was quantified by slot-blot analysis and ranged from approximately less than 2-160 ng DNA per cigarette butt for the 200 samples, and 8 ng, 50 ng, and 100 ng for the cigarette butts from the adjudicated cases. The DNA was successfully amplified by the polymerase chain reaction (PCR) for the HLA-DQ alpha locus (99 out of 100 samples) as well as for the variable number of tandem repeat (VNTR) locus D1S80 (99 out of 100 samples). Amplification and typing of DNA was successful on all samples recovered from the crime scenes. The results suggest that PCR-based typing of DNA offers a potential method for genetically characterizing traces of saliva on cigarette butts.

[DNA diagnostics in hemophilia A and B]. / DNA-Diagnostik bei Hämophilie A und B.

Carrier detection and prenatal diagnosis of hemophilia A and B are possible with cloned factor-VIII:C- and factor-IX-gene-specific or linked probes which detect restriction fragment length polymorphisms (RFLPs). In this study, 12 hemophilia-A- and 5 hemophilia-B-families were studied to identify carriers and provide adequate genetic counselling to women who were heterozygous for one or more of the intragenic or linked DNA probes with respect to future pregnancies.