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1.
Laser microdissection can be used in forensic casework to isolate specific cell types from mixtures of biological samples. Extraction of DNA from selected cells is still required prior to STR amplification. Because of the relatively pristine nature of the recovered cells, laser microdissection is more sensitive than more traditional methods of DNA analysis, theoretically resulting in DNA profiles from less cellular material. A one-tube extraction and amplification method minimises loss of DNA through liquid transfers and reduces the potential for contamination events occurring. In this paper, the development of a one-tube method for the effective extraction of DNA from laser microdissected sperm and epithelial cells is described. The performance of the in-house method was compared to that of a commercial DNA extraction kit for extraction of DNA from sperm and the downstream compatibility with STR amplification was determined for both sperm and epithelial samples. Full Identifiler? profiles after 28 amplification cycles were obtained from as few as 15 epithelial cells and 30 sperm.  相似文献   

2.
A 55-year-old male nurse was accused of having introduced his fingers by force into the anus of a 20-year-old female patient. Debris from the fingernails of the suspect recovered 2 days after the incident was analysed with the VNTR locus D1S80, the triplex PCR system AmpFlSTR Blue kit, the AmpFlSTR Profiler kit and the pentaplex system genRES MPX. The D1S80 singleplex reaction revealed indications of DNA from the victim in the fingernail debris of the left hand. Using the AmpFlSTR Blue kit and AmpFlSTR Profiler, DNA alleles of the victim were found at four additional loci, while allelic drop-out was observed at five other loci. Only the pentaplex kit genRES MPX revealed alleles at all loci which could be assigned to the victim. Calculation of likelihood ratios resulted in a value of 1.4 × 105 using the combination of the multiplex systems AmpFlSTR Blue kit and AmpFlSTR Profiler and 2.8 × 108 for the genRES MPX kit. This case demonstrates the high sensitivity of the new genRES MPX kit and that DNA profiling of fingernail debris is possible despite a time lapse of 2 days between the incident and recovery of the evidential material. Received: 30 March 2000 / Accepted: 19 September 2000  相似文献   

3.
Sex-specific isolation of cells from mixtures would greatly facilitate forensic casework. Thus, male and female cell mixtures were marked with a fluorescent X/Y-probe CEP X SpectrumOrange/Y SpectrumGreen DNA probe kit for fluorescence in situ hybridization, and single cells were isolated via laser microdissection (LMD). DNA profiling of LMD isolated, hybridized cells showed usable short tandem repeat profiles for at least 20 cells, which are comparable with results from other studies. To simulate casework samples, the method was also optimized for air-dried samples.  相似文献   

4.
Laser microdissection (LMD) is a tool used in forensic laboratories for the analysis of DNA from specifically targeted cells. In our laboratory, LMD analysis is applied to case samples where it has been shown (or it is anticipated) that there are small numbers of target cells present, such as sperm. In this paper, we extend previous LMD technology developed in this laboratory1. A method for improved recovery and visualisation of cells from substrates for downstream laser microdissection and DNA analysis is discussed and the optimal numbers of LMD-recovered sperm and epithelial cells for Low Copy Number (LCN) DNA analysis is determined.  相似文献   

5.
We describe the forensic validation of Promega’s PowerPlex® European Standard Investigator 16 (ESI 16) multiplex kit and compare results generated with the AmpFlSTR® SGM Plus® (SGM+) multiplex. ESI 16 combines the loci contained within the SGM+ multiplex with five additional loci: D2S441, D10S1248, D22S1045, D1S1656, and D12S391. A relative reduction in amplicon size of the SGM+ loci facilitates an increased robustness and amplification success of these amplicons with degraded DNA samples. Tests performed herein supplement ESI 16 data published previously with sensitivity, profile quality, mock casework, inhibitor and mixture study data collected in our laboratories in alignment with our internal technical and quality guidelines and those issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), the DNA Advisory Board (DAB) and the DNA working group (DNAWG) of the European Network of Forensic Science Institutes (ENFSI). Full profiles were routinely generated from a fully heterozygous single source DNA template using 62.5 pg for ESI 16 and 500 pg for SGM+. This increase in sensitivity has a consequent effect on mixture analyses and the detection of minor mixture components. The improved PCR chemistry confers enhanced tolerance to high levels of laboratory prepared inhibitors compared with SGM+ results. In summary, our results demonstrate that the ESI 16 multiplex kit is more robust and sensitive compared with SGM+ and will be a suitable replacement system for the analysis of forensic DNA samples providing compliance with the European standard set of STR loci.  相似文献   

6.
The Investigator® Quantiplex HYres kit was evaluated as a potential replacement for dual DNA quantification of casework samples. This kit was determined to be highly sensitive with a limit of quantification and limit of detection of 0.0049 ng/μL and 0.0003 ng/μL, respectively, for both human and male DNA, using full or half reaction volumes. It was also accurate in assessing the amount of male DNA present in 96 mock and actual casework male:female mixtures (various ratios) processed in this exercise. The close correlation between the male/human DNA ratios expressed in percentages derived from the Investigator® Quantiplex HYres quantification results and the male DNA proportion calculated in mixed AmpFlSTR® Profiler® Plus or AmpFlSTR® Identifiler® Plus profiles, using the Amelogenin Y peak and STR loci, allowed guidelines to be developed to facilitate decisions regarding when to submit samples to Y-STR rather than autosomal STR profiling. The internal control (IC) target was shown to be more sensitive to inhibitors compared to the human and male DNA targets included in the Investigator® Quantiplex HYres kit serving as a good quality assessor of DNA extracts. The new kit met our criteria of enhanced sensitivity, accuracy, consistency, reliability and robustness for casework DNA quantification.  相似文献   

7.
Allele frequencies for the nine tetrameric STR loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820 were determined in a population sample of 155 unrelated Bavarians using the AmpFlSTR Profiler Plus PCR amplification kit. No deviations from the Hardy-Weinberg equilibrium were observed. The influence of the PCR cycle number as well as the template DNA concentration on the performance of the kit was studied. DNA concentrations lower than 75 pg DNA per 25 μl reaction volume resulted in allelic drop-out. Received: 19 April 1999 / Accepted: 27 September 1999  相似文献   

8.
Casework evidence samples are likely to be placed under diverse and harsh environments as compared to quantified DNA samples including serial-diluted standard DNA samples. Internal validation of a novel STR kit using casework evidence sample, which is conducted according to various conditions such as DNA contamination and degradation, is crucial before being used as a forensic application. Therefore, this study aimed to elucidate the reliability of the Investigator® 24plex QS kit through DNA derived from casework evidence and to assess whether it is applicable to STR analysis together with PowerPlex® Fusion System and GlobalFiler™ PCR Amplification Kit. DNA was extracted from 189 casework evidence samples in a total of 77 cases. The mismatch of the allelic size of this kit through allelic sizing precision test, was suitable according to ENFSI guidelines. All heterozygous balance of the three kits were above 0.6 recommended value of ENFSI guideline. The number of allele drop-in was most frequent in the GlobalFiler™ PCR Amplification Kit. In addition, the number of allele drop-out was most frequent in the Investigator® 24plex QS kit. The cutoff concentration of DNA detected in three kits of one complete STR was approximately 45 pg/μL on average. Despite of several limitations, the Investigator® 24plex QS kit is considered to have the capability to be used for STR analysis of casework evidence samples.  相似文献   

9.
In response to the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe, Promega® developed a suite of four new DNA profiling kits. This paper describes the developmental validation study performed on the PowerPlex® ESI 16 (European Standard Investigator 16) and the PowerPlex® ESI 17 Systems. The PowerPlex® ESI 16 System combines the 11 loci compatible with the UK National DNA Database®, contained within the AmpFlSTR® SGM Plus® PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to reduce the amplicon size of the loci found in the AmpFlSTR® SGM Plus® kit. This design facilitates increased robustness and amplification success for the loci used in the national DNA databases created in many countries, when analyzing degraded DNA samples. The PowerPlex® ESI 17 System amplifies the same loci as the PowerPlex® ESI 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR® SGM Plus® kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex® ESI 16 and ESI 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5 pg of a fully heterozygous single source DNA template. This high level of sensitivity was found to impact on mixture analyses, where 54–86% of unique minor contributor alleles were routinely observed in a 1:19 mixture ratio. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of data obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.  相似文献   

10.
The AmpFSTR SEfiler kit co-amplifies 11 short tandem repeat loci including SE33 in a single multiplex. After establishing the optimum in primer titration studies, the primer concentrations of all loci in the multiplex were chosen such that the heterozygote peak height ratios of each of the loci were balanced. The combined primer set was then tested to determine the robustness of the multiplex under various conditions. Different MgCl2 concentrations were evaluated to establish the optimum concentration for the multiplex. The amplification of the various loci in the multiplex was tested at several annealing temperatures (55–63°C). Additionally, DNA from primates, non-primates and microorganisms were amplified to investigate the specificity of the kit. The stability of the AmpFSTR SEfiler kit was determined by addition of hematin, to simulate inhibition, and the use of degraded DNA. Population studies revealed a probability of identity of 6.47×10–15 for African Americans and 7.46×10–14 for US Caucasians. To assess the ability of the multiplex to analyze forensic samples, testing on blood, oral swabs and mixtures was performed. Based on the various studies, it was determined that the AmpFSTR SEfiler PCR amplification kit can be used to successfully analyze a variety of forensic, databasing and paternity samples.For research, forensic or paternity use only. Not for use in diagnostic procedures.The PCR process is covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd.Applied Biosystems, ABI PRISM, AmpFSTR, GeneScan, Genotyper, LIZ, MicroAmp, SGM Plus, and VIC are registered trademarks and FAM, Hi-Di, Identifiler, NED, PET, POP, POP-4, and ROX are trademarks of Applera Corporation or its subsidiaries in the US and certain other countries.AmpliTaq Gold, GeneAmp, and QuantiBlot are registered trademarks of Roche Molecular Systems, Inc.  相似文献   

11.
DNA collected from crime scenes may have experienced different levels of degradation. This is mainly due to sample exposure to different environmental factors. The impact of DNA degradation on short tandem repeat (STR) profiling can lead to partial or null information and in some cases, the identification of the trace may fail. The availability of a system enabling the assessment not only of the quantity of the DNA but also of its quality in terms of degradation would result in shorter time for sample processing, more reliable identifications and cost reduction by predicting the quality of the DNA profiles prior to STR analysis. We report here a study on 181 selected degraded DNA samples extracted from real crime scene evidence. The selected samples were processed by combining the use of a new commercial quantification kit (Quantifiler® Trio) with a new 24 marker multiplex PCR amplification kit (Globalfiler® Kit). Applying different statistical analyses we investigated the reliability of the Degradation Index provided by the Quantifiler® Trio in determining the level of DNA degradation in a forensic sample. This useful information can be used to predict the quality of the profile obtained after STR amplification. The combination of such a quantification kit with different PCR protocols allowed us to define practical guidelines for processing degraded forensic DNA samples with a simplified and comprehensive approach.  相似文献   

12.
Fired cartridge cases are a common type of evidence found at crime scenes. However, due to the high chamber temperatures and touch nature of this evidence, DNA testing is not commonly sought because it is believed DNA is only present in low levels, whether it is due to initial low levels of DNA and/or DNA degradation from the heat or inhibition of the PCR reaction. Moreover, very few laboratories report STR typing success with fired cases. This study focused on obtaining STR profiles from fired cartridge cases using the AmpFℓSTR® MiniFiler™ kit, which is designed to amplify DNA from low level, inhibited, and degraded samples. Comparisons to other STR amplification kits were also conducted. In attempt to simulate casework, random individuals loaded cartridges into a firearm. DNA was recovered from the fired cartridge cases using the double swab technique and extracted using an automated large volume DNA IQ™ method. Initially, testing focused on known shedders handling cartridges for 30 s prior to firing. A significantly greater number of alleles was obtained following amplification with the MiniFiler™ kit versus the PowerPlex® 16 BIO kit. No alleles were observed using the Identifiler® kit. In an attempt to better simulate casework, a random selection of laboratory personnel handled shotshells for as long as needed to load and fire the weapon. In this mock sample study, the MiniFiler™ kit successfully amplified an average of 22% of expected alleles from DNA recovered from shotshell cases versus the PowerPlex® 16 BIO kit where an average of 7% of alleles were observed. However, the total number of alleles obtained from the two kits was not significantly different. The quality of the DNA obtained from fired cases was studied with evidence of inhibition in at least 11% of shotshell case samples. After swabbing the head and the hull of three shotshell cases separately, a significantly greater number of alleles was obtained from the hull as opposed to the head of the fired shotshell case. In addition, after firing, various internal firearm surfaces were swabbed, including the chamber of barrel, ejection port, and breechface, in an attempt to obtain amplifiable DNA. DNA was obtained from the chamber of the barrel and was amplifiable using the MiniFiler™ kit, although mixtures were obtained with extensive drop-in and drop-out making this analysis unlikely to aid an investigation.  相似文献   

13.
The 13 short tandem repeat (STR) loci D3S1358, vWA, FGA, D16S539, TH01, TPOX, CSF1PO, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820 as well as the amelogenin locus, contained in AmpFlSTR Profiler Plus and/or AmpFlSTR Cofiler and/or AmpFlSTR Green I PCR amplification kits, were studied in four populations from the Iberian Peninsula, Basques, Catalans, Andalusians and Portuguese and two North African populations (Moroccan Arabs and Berbers). The aim of the study was to obtain accurate allele frequency data and other genetic parameters of forensic interest on the main representative human groups living in Iberia and Morocco using an automated method and commercial amplification kits. Received: 15 March 1999 / Received in revised form: 25 June 1999  相似文献   

14.
Flip-open style cell phones were investigated for the potential to produce quality genetic profiles that could be used in forensic casework. Swabs were taken of the outside/back and the inside ear speaker of ten flip-phones on two occasions – prior to and seven days after cleaning with 95% ethanol. Buccal swabs were collected as exemplars. The samples were amplified using the AmpFlSTR ProfilerPlus PCR Kit for 35 cycles and STR profiles were generated using an ABI Prism 310 Genetic Analyzer and GeneMapper ID analysis software v3.2. The phone profiles were compared to the references and to each other, to assess the quality of the profiles. The completeness of the profiles varied greatly, even within an experimental condition. There was no significant difference in the percentage correct alleles or in the number of drop-in alleles in the DNA profiles for the outside/inside locations or for the pre/post-cleaning times. The findings of this study demonstrate the need for collecting multiple samples from discrete locations on a cell phone if such evidence is encountered in forensic cases.  相似文献   

15.
In the last few years the cost and ease of massively parallel sequencing (MPS) has reduced dramatically to the point that it can now be considered as a tool for use in forensic case work. An important consideration for the implementation of any new forensic technology is the ability to remain compatible with previous technology. During this study we sequenced the amplicons of two commercial forensic short tandem repeat (STR) multiplexes AmpFlSTR Identifiler and PowerPlex Y using the Illumina MiSeq and Ion PGM Sequencer (Life Technologies) and characterised the sequence data from a forensic perspective. Using the MPS data from both platforms we determined the STR genotypes of forensic samples and found previously undocumented sequence variation in seven STR alleles. By characterising features of the DNA sequence profiles, such as stutter and locus imbalance we identified areas for future development that will be needed prior to casework implementation. The rapid development of this technology has meant many in the forensic community have been ‘left behind’. We also provide an explanation, for forensic scientists, of what is happening at the different stages of the MPS workflow, from library preparation through to bioinformatics, and how this may affect the results.  相似文献   

16.
ABSTRACT

Since 2007, the Institute of Environmental and Science Research (ESR) has been using the AmpF/STR Identifiler profiling kit as the standard DNA test. Whilst this approach works well for most samples with a DNA input range of 400 pg to 2 ng, samples containing less DNA often have limited to no profiling success. If required, such samples can undergo testing using a more sensitive technique, such as mini-STR analysis or Low Copy Number (LCN) DNA analysis. The introduction of a more sensitive standard DNA test into the laboratory would likely prove to be greatly beneficial. Profiling success should increase, particularly for samples containing trace DNA concentrations, and the number of samples requiring rework and further analysis using an ultra-sensitive technique would potentially reduce. A number of new generation multiplex kits with increased sensitivity are now commercially available. One such kit is the AmpF/STR Identifiler Plus profiling kit, which has enhanced buffer formulation and an optimized PCR cycling protocol. This study investigates the profiling results from trace DNA samples using Identifiler at 28 cycles and Identifiler Plus, at 28 and 29 cycles, analysed using the 3500xL Applied BioSystems Genetic Analyzer. Any further benefits from including a post-PCR clean-up step and enhanced capillary electrophoresis conditions are investigated and discussed.  相似文献   

17.
Polymerase chain reaction (PCR) plays an important role in forensic DNA analysis. However, the amplification of low-template DNA (LTDNA) samples usually encounters unsatisfactory results for the limited efficiency of PCR, which would interfere with the subsequent profile interpretation. Polymerase chain displacement reaction (PCDR) is a highly-efficient technique characterized by combining PCR and strand displacement reaction into a single PCDR cycle. This study explored the feasibility of PCDR for improving forensic LTDNA analysis. STR markers commonly used in forensic genetics were subjected to PCDR amplification and capillary electrophoresis detection. The results of singleplex reactions indicated that PCDR surpassed original PCR in efficiency for STR amplification. The average peak height of alleles in PCDR profiles was linearly correlated to the number of outer primers adopted for initiating the strand displacement process. Further, we assessed the multiplexing potential of PCDR by incorporating 17 STRs included in the expanded CODIS core loci and Amelogenin gene into a multiplex PCDR system. For pristine DNA templates ranged from 200 pg to 12.5 pg, the multiplex PCDR system consistently exhibited higher allele peak height as well as less allele dropout compared to the multiplex PCR references. Meanwhile, a significant reduction of stutter ratio was extensively observed in PCDR profiles. We also tested mock casework samples to verify the practical ability of multiplex PCDR for LTDNA detection. With DNA input varying from 48.1 pg to 6.6 pg, the multiplex PCDR system consistently obtained more allelic information than multiplex PCR methods. Our data collectively suggested that it is feasible to apply PCDR in forensic LTDNA analysis.  相似文献   

18.
Three new mini-STR primer sets are suggested for three conventional STRs, CSF1P0, D8S1179 and D13S317, included in multiplex PCR kits commercially available and commonly used for DNA typing in forensic applications. The primer pairs for the three loci were redesigned in order to reduce or eliminate the flanking regions of the polymorphism obtaining amplification products, which have dimensions less than 120bp in size. A comparison of results for typing carried out with the newly designed primers on DNA extracted from 100 blood samples provided by healthy donors, previously typed with conventional STRs, showed no genotype difference underlining their precision and reproducibility. The forensic usefulness of the new mini-STR primers was evaluated on highly degraded DNA from casework samples (e.g. archival post-mortem Bouin's fluid-fixed paraffin-embedded tissue specimens) for which commercial STR kit had proven inefficient.  相似文献   

19.
Mini-STRs     
The use of the short-tandem repeat (STR) as the DNA marker of choice in forensic profiling has lead to the construction of criminal intelligence databases that now contain millions of profiles used in the detection and linking of suspects and scenes. The incredible size and success rate of current systems have ensured that efforts to move away from STR profiling and toward the use of alternate DNA markers are practically impossible, mainly because of the financial implications involved in such a move. Problems are routinely encountered when template DNA is of suboptimal condition, as is commonly the case in the forensic laboratory, whereby full profile generation of degraded samples is not possible. A redesigned amplification protocol that results in the generation of shorter polymerase chain reaction products yet is fully compatible with current STR databases has been introduced: the MiniPlex.  相似文献   

20.
In humans, the amelogenin gene is present on both the X and the Y chromosomes. However, there are size differences in this gene between these chromosomes, which have been utilised for sexing in forensic casework and prenatal diagnosis. Our study using the AmpFl STR Profiler Plus kit, showed a deletion of Y chromosome-specific amelogenin in five Indian males (1.85%). We propose to call them “deleted-amelogenin males” (DAMs), who but for the detection of the presence of other Y-specific markers (e.g. SRY, STR and 50f2) would have been identified as females. Considering the consequences of the result obtained only using the amelogenin marker, we suggest the use of additional Y chromosome markers for unambiguous gender identification. Received: 17 May 2001 / Accepted: 24 July 2001  相似文献   

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