Differential DNA extraction of challenging simulated sexual-assault samples: a Swiss collaborative study
Séverine VuichardU. V. BorerMichel BottinelliChristian CossuNaseem MalikVerena MeierChristian GehrigAndrea SulzerM.-L. MorerodVincent Castella
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Abstract:
In sexual-assault cases, autosomal DNA analysis of gynecological swabs is a challenge, as the presence of a large quantity of female material may prevent detection of the male DNA. A solution to this problem is differential DNA extraction, but there is no established best practice for this. We decided to test the efficacy of a number of different protocols on simulated casework samples. Four difficult samples were sent to the nine Swiss laboratories active in forensic genetics. In each laboratory, staff used their routine protocols to separate the epithelial-cell fraction, enriched with the non-sperm DNA, from the sperm fraction. DNA extracts were then sent to the organizing laboratory for analysis. Estimates of male:female DNA ratio without differential DNA extraction ranged from 1:38 to 1:339, depending on the semen used to prepare the samples. After differential DNA extraction, most of the ratios ranged from 1:12 to 9:1, allowing detection of the male DNA. Compared with direct DNA extraction, cell separation resulted in losses of 94-98% of the male DNA. As expected, more male DNA was generally present in the sperm than in the epithelial-cell fraction. However, for about 30% of the samples, the reverse trend was seen. The recovery of male and female DNA was highly variable, depending on the laboratory involved. An experimental design similar to the one used in this study may be of assistance for local protocol testing and improvement.Keywords:
Sexual assault
STR analysis
STR analysis
Multiplex
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Minisatellite
STR multiplex system
STR analysis
DNA profiling
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STR multiplex system
STR analysis
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Y-short tandem repeats (STRs) are located in the nonrecombining part of the Y-chromosome. Its genetic characteristics are helpful to paternity testing. The male lineage can be pursued over several generations. Since an overwhelming majority of rape or sexual assault cases involve male suspects, these markers proved to be very useful with mixed stains. Routine casework performed in our laboratory has demonstrated that Y-STRs analysis can detect minimal amounts of male DNA in a stain. Analysis of Y-STRs should be conducted even when preliminary tests for the presence of sperms are negative or when the analysis of autosomal STRs shows no male component in the mixed stains.
Y-STR
Sexual assault
STR analysis
Stain
DNA profiling
Lineage (genetic)
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Science Crime Investigation (SCI) is a criminal investigation based on scientific proves. The utilization of DNA analysis are proven to be more efficient and accurate. DNA typing method was used to identify human based on DNA profile. The technique consisted of DNA extraction, DNA quantification, DNA amplification, and fragment analysis. DNA extraction from the forensic evidence was the crucial step to determine the success rate of the DNA analysis. The aimed of this research was to analyze the effect of different DNA extraction methods that commonly used in the forensic investigation in Indonesia. The DNA extraction methods were: (A) silica based method using QIAamp® DNA Investigator kit (Qiagen), (B) magnet beads based method using Prepfiler® BTA forensic kit (Applied Biosystem), and (C) resin based method Chelex® 100. Biological evidence used in this research were saliva, blood, hair, bone, and touch DNA. The indicators were the number of DNA concentration, inhibitor, and the percentage of amplified allele on 24 loci Short Tandem Repeat (STR). Three indicators were used to compare the effectiveness of DNA extraction methods in particular biological samples. Based on the result, silica-based method using QIAamp® DNA Investigator extraction method was effectively used on saliva, blood, and bone samples. Prepfiler® BTA Forensic kit extraction method was effective on saliva, blood, hair, and semen samples. Chelex®100 extraction methods were effectively used on saliva, blood, and hair samples.
STR analysis
Blood Stains
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The replication error phenotype, revealed by the observation of widespread microsatellite instability (MIN), has been identified as a new mechanism of cancer susceptibility, and the comparison of the allele sizes of polymorphic microsatellite repeats between normal and tumor DNA is now frequently undertaken in colorectal and other human neoplasias. The lack of precise characterization of the electrophoretic profiles of microsatellites is one of the main sources of discord between the rate of MIN reported for the same type of tumor by different investigators. The recent introduction of fluorescent-based semiautomated microsatellite analysis allows a more accurate size comparison, but one or more artificial peaks, generated during polymerase chain reaction (PCR) and/or electrophoresis, are frequently detected along with the true allele peaks. The aim of this study was to characterize the most frequent artificial extra peaks in the short tandem repeats (STRs) used by us to assess MIN in human cancers. We analyzed eight microsatellite loci in 113 primary brain tumors. HumFibra/FGA exhibited the most frequent extra peak formation. For each microsatellite there is a characteristic pattern of artifact formation which must be recognized to avoid a false-positive diagnosis of MIN.
Microsatellite Instability
STR analysis
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The PrepFiler Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 microL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA paper), and touch evidence-type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol-chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.
genomic DNA
Blood Stains
STR analysis
DNA profiling
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An improved method of quantifying the donor:host blood cell ratio after haematological stem cell transplantation (PBSCT) using a variable number of tandem repeat (VNTR) markers is presented. The post-transplant DNA is extracted from the patient's blood and amplified by semiquantitative polymerase chain reaction (PCR) without prior mock transplant and plotting of a standard curve. The amplification products are then analysed by ABI PRISM 310 capillary electrophoresis apparatus. The resultant peak areas are correlated with the corresponding microsatellite allele by GeneScan software and the donor:host cell ratio is calculated. Improvements to PCR amplification conditions are essential for the outcome of the quantification since preferential amplification of alleles in the PCR process can account for the marked deviation found between the results gained by measurement of different microsatellite loci. To assess the accuracy of the method, the post-transplant blood samples of 6 patients who had undergone either myeloablative or non-myeloablative transplantation regimens were analysed retrospectively (median observation time 298 days). By analysing 3 or 4 microsatellite loci we were able to detect full engraftment or mixed chimaerism after transplant with a measurement precision of < or = 4.5 (standard deviation). Sensitivity for different primers ranges from 2% to 5%. The results of the microsatellite analysis correlated well with the corresponding clinical findings. We conclude that post-transplant analysis of microsatellite loci using semiquantitative PCR without standard is suitable for clinical purposes.
STR analysis
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Background: A paternity disagreement analyzed with 15 autosomal microsatellite markers indicated allele sharing between the mother, questioned child and the alleged father generating an inconclusive paternity result.
STR analysis
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The replication error phenotype, revealed by the observation of widespread microsatellite instability (MIN), has been identified as a new mechanism of cancer susceptibility, and the comparison of the allele sizes of polymorphic microsatellite repeats between normal and tumor DNA is now frequently undertaken in colorectal and other human neoplasias. The lack of precise characterization of the electrophoretic profiles of microsatellites is one of the main sources of discord between the rate of MIN reported for the same type of tumor by different investigators. The recent introduction of fluorescent-based semiautomated microsatellite analysis allows a more accurate size comparison, but one or more artificial peaks, generated during polymerase chain reaction (PCR) and/or electrophoresis, are frequently detected along with the true allele peaks. The aim of this study was to characterize the most frequent artificial extra peaks in the short tandem repeats (STRs) used by us to assess MIN in human cancers. We analyzed eight microsatellite loci in 113 primary brain tumors. HumFibra/FGA exhibited the most frequent extra peak formation. For each microsatellite there is a characteristic pattern of artifact formation which must be recognized to avoid a false-positive diagnosis of MIN.
Microsatellite Instability
STR analysis
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Citations (8)