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James P Landers - One of the best experts on this subject based on the ideXlab platform.
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analytical approaches to Differential Extraction for sexual assault evidence
Analytica Chimica Acta, 2021Co-Authors: Charles P Clark, Rachelle Turiello, Robin Cotton, James P LandersAbstract:Many forensic laboratories face growing demands for the processing of DNA evidence from sexual assault investigations. In these cases, evidence collected from the crime scene or from the victim in the form of a Sexual Assault and Evidence Collection Kit (SAECK) typically contains a mixture of cells from at least two donors. Isolation of DNA contributions to link a sample to an alleged offender requires precise chemical treatment of each sample with the goal of separating epithelial cells from non-sperm cells. Currently, the vast majority of laboratories employ Differential chemical lysis methods that require lengthy incubations and several manual steps, preventing complete automation. Numerous alternative methods for the Differential Extraction (DE) of sexual assault evidence have been developed to provide a solution to the growing backlog of samples observed in the US and other countries. Here, we will discuss the predominant methodology for the DE of DNA from sexual assault samples and review alternative approaches from literature. We illustrate three criteria that provide a measure of success in performing these types of chemical separations and examine all methods based upon these expectations. We conclude by providing some general insight into the application of DE techniques in forensic laboratories and discuss the potential future directions of alternative technologies.
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Isolation of a Low Number of Sperm Cells from Female DNA in a Glass–PDMS–Glass Microchip via Bead-Assisted Acoustic Differential Extraction
2019Co-Authors: Charles P Clark, Johan Nilsson, Thomas Laurell, Brian L. Poe, Jenny A. Lounsbury, James P LandersAbstract:We report an improved separation method for the isolation of sperm cells from dilute, “large volume” samples containing female DNA using bead-assisted acoustic trapping. In an enclosed glass–PDMS–glass (GPG) resonator, we exploit a three-layer microfluidic architecture to generate “trapping nodes” in ultrasonic standing waves. We investigate the dependence of trapping efficiency on particle concentration for both sperm cells and polymeric beads. After determination of the critical concentration of polymeric beads required to seed the trapping event, sperm cells in dilute solution are trapped as a result of the enhanced secondary radiation force (SRF). Sperm-cell-containing samples with volumes up to 300 μL and cell concentrations as low as ∼10 cells/μL are amenable to effective trapping in the presence of an abundance of female DNA in solution. Complete processing of samples is accomplished with separation of the female and male fractions within 15 min. We demonstrate that the collected fractions are amenable to subsequent DNA Extraction, short tandem repeat PCR, and the generation of STR profiles for the isolated sperm cells
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acoustic Differential Extraction for forensic analysis of sexual assault evidence
Analytical Chemistry, 2009Co-Authors: Jessica V Norris, Mikael Evander, Katie M Horsmanhall, Johan Nilsson, Thomas Laurell, James P LandersAbstract:Forensic DNA analysis of samples obtained from sexual assault evidence relies on separation of male and female components of the recovered genetic material. The conventional separation method used by crime laboratories, Differential Extraction (DE), is one of the most time-consuming sample preparation steps, requires extensive sample handling, is difficult to automate, and often results in inefficient separation of female DNA from the male sample components. To circumvent conventional DE, acoustic Differential Extraction (ADE) analysis was developed on a microfluidic device. The ADE method relies on acoustic trapping of sperm cells in the presence of epithelial cell lysate (which is unretained), and laminar flow valving to direct the male and female fractions to separate outlets. Following the separation of sperm from epithelial cell lysate, DNA Extraction, quantitation, amplification, and separation were performed using conventional laboratory methods. The results show that highly purified male and femal...
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acoustic Differential Extraction for forensic analysis of sexual assault evidence
Analytical Chemistry, 2009Co-Authors: Jessica V Norris, Mikael Evander, Katie M Horsmanhall, Johan Nilsson, Thomas Laurell, James P LandersAbstract:Forensic DNA analysis of samples obtained from sexual assault evidence relies on separation of male and female components of the recovered genetic material. The conventional separation method used by crime laboratories, Differential Extraction (DE), is one of the most time-consuming sample preparation steps, requires extensive sample handling, is difficult to automate, and often results in inefficient separation of female DNA from the male sample components. To circumvent conventional DE, acoustic Differential Extraction (ADE) analysis was developed on a microfluidic device. The ADE method relies on acoustic trapping of sperm cells in the presence of epithelial cell lysate (which is unretained), and laminar flow valving to direct the male and female fractions to separate outlets. Following the separation of sperm from epithelial cell lysate, DNA Extraction, quantitation, amplification, and separation were performed using conventional laboratory methods. The results show that highly purified male and female fractions can be obtained with the ADE microdevice from mock sexual assault samples in 14 min. ADE analysis provides the potential to significantly alter the means by which sexual assault evidence is processed in crime laboratories.
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separation of sperm and epithelial cells in a microfabricated device potential application to forensic analysis of sexual assault evidence
Analytical Chemistry, 2005Co-Authors: Katie M Horsman, Susan L R Barker, Jerome P Ferrance, Kymberly A Forrest, Katherine A Koen, James P LandersAbstract:Forensic DNA analysis of sexual assault evidence requires separation of DNA from epithelial (victim) and sperm (perpetrator) cells. The conventional method used by crime laboratories, which is termed “Differential Extraction”, is a time-consuming process. To supplant the conventional process, separation of sperm from a biological mixture containing epithelial cells has been demonstrated on a microfluidic device. This separation utilizes the Differential physical properties of the cells that result in settling of the epithelial cells to the bottom of the inlet reservoir and subsequent adherence to the glass substrate. As a result, low flow rates can be used to separate the sperm cells from the epithelial cell-containing biological mixture. Following cell separation on the microdevice, DNA Extraction, amplification, and separation were performed using conventional laboratory methods, showing that the cell separation product in the outlet reservoir was of male origin. The reported cell separation has the pot...
Gregory J Wilson - One of the best experts on this subject based on the ideXlab platform.
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development of a pericardial acellular matrix biomaterial biochemical and mechanical effects of cell Extraction
Journal of Biomedical Materials Research, 1994Co-Authors: David W Courtman, C A Pereira, V Kashef, D Mccomb, J M Lee, Gregory J WilsonAbstract:There is evidence to suggest that the cellular components of homografts and bioprosthetic xenografts may contribute to calcification or immunogenic reactions. A four-step detergent and enzymatic Extraction process has been developed to remove cellular components from bovine pericardial tissue. The process results in an acellular matrix material consisting primarily of elastin, insoluble collagen, and tightly bound glycosaminoglycans. Light and electron microscopy confirmed that nearly all cellular constituents are removed without ultrastructural evidence of damage to fibrous components. Collagen denaturation temperatures remained unaltered. Biochemical analysis confirmed the retention of collagen and elastin and some Differential Extraction of glycosaminoglycans. Low strain rate fracture testing and high strain rate viscoelastic characterization showed that, with the exception of slightly increased stress relaxation, the mechanical properties of the fresh tissue were preserved in the pericardial acellular matrix. Crosslinking of the material in glutaraldehyde or poly(glycidyl ether) produced mechanical changes consistent with the same treatments of fresh tissue. The pericardial acellular matrix is a promising approach to the production of biomaterials for heart valve or cardiovascular patching applications.
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development of a pericardial acellular matrix biomaterial biochemical and mechanical effects of cell Extraction
Journal of Biomedical Materials Research, 1994Co-Authors: David W Courtman, C A Pereira, V Kashef, D Mccomb, Michael J Lee, Gregory J WilsonAbstract:There is evidence to suggest that the cellular components of homografts and bioprosthetic xenografts may contribute to calcification or immunogenic reactions. A fourstep detergent and enzymatic Extraction process has been developed to remove cellular components from bovine pericardial tissue. The process results in an acellular matrix material consisting primarily of elastin, insoluble collagen, and tightly bound glycosaminoglycans. Light and electron microscopy confirmed that nearly all cellular constituents are removed without ultrastructural evidence of damage to fibrous components. Collagen denaturation temperatures remained unaltered. Biochemical analysis confirmed the retention of collagen and elastin and some Differential Extraction of glycosaminoglycans. Low strain rate fracture testing and high strain rate viscoelastic characterization showed that, with the exception of slightly increased stress relaxation, the mechanical properties of the fresh tissue were preserved in the pericardial acellular matrix. Crosslinking of the material in glutaraldehyde or poly(glycidyl ether) produced mechanical changes consistent with the same treatments of fresh tissue. The pericardial acellular matrix is a promising approach to the production of biomaterials for heart valve or cardiovascular patching applications. © 1994 John Wiley & Sons, Inc.
Anne E. Cowan - One of the best experts on this subject based on the ideXlab platform.
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systematic characterization of nuclear proteome during apoptosis a quantitative proteomic study by Differential Extraction and stable isotope labeling
Molecular & Cellular Proteomics, 2006Co-Authors: Sunil Hwang, Viveka Mayya, Karim Rezaul, Deborah H Lundgren, Anne E. CowanAbstract:Identification and characterization of the nuclear proteome is important for detailed understanding of multiple signaling events in eukaryotic cells. Toward this goal, we extensively characterized the nuclear proteome of human T leukemia cells by sequential Extraction of nuclear proteins with different physicochemical properties using three buffer conditions. This large scale proteomic study also tested the feasibility and technical challenges associated with stable isotope labeling by amino acids in cell culture (SILAC) to uncover quantitative changes during apoptosis. Analyzing proteins from three nuclear fractions extracted from naive and apoptotic cells generated 780,530 MS/MS spectra that were used for database searching using the SEQUEST algorithm. This analysis resulted in the identification and quantification of 1,174 putative nuclear proteins. A number of known nuclear proteins involved in apoptosis as well as novel proteins not known to be part of the nuclear apoptotic machinery were identified and quantified. Consistent with SILACbased quantifications, immunofluorescence staining of nucleus, mitochondria, and some associated proteins from both organelles revealed a dynamic recruitment of mitochondria into nuclear invaginations during apoptosis. Molecular & Cellular Proteomics 5:1131–1145, 2006.
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systematic characterization of nuclear proteome during apoptosis a quantitative proteomic study by Differential Extraction and stable isotope labeling
Molecular & Cellular Proteomics, 2006Co-Authors: Sunil Hwang, Viveka Mayya, Karim Rezaul, Deborah H Lundgren, Anne E. Cowan, Jimmy K Eng, David K HanAbstract:Identification and characterization of the nuclear proteome is important for detailed understanding of multiple signaling events in eukaryotic cells. Toward this goal, we extensively characterized the nuclear proteome of human T leukemia cells by sequential Extraction of nuclear proteins with different physicochemical properties using three buffer conditions. This large scale proteomic study also tested the feasibility and technical challenges associated with stable isotope labeling by amino acids in cell culture (SILAC) to uncover quantitative changes during apoptosis. Analyzing proteins from three nuclear fractions extracted from naive and apoptotic cells generated 780,530 MS/MS spectra that were used for database searching using the SEQUEST algorithm. This analysis resulted in the identification and quantification of 1,174 putative nuclear proteins. A number of known nuclear proteins involved in apoptosis as well as novel proteins not known to be part of the nuclear apoptotic machinery were identified and quantified. Consistent with SILAC-based quantifications, immunofluorescence staining of nucleus, mitochondria, and some associated proteins from both organelles revealed a dynamic recruitment of mitochondria into nuclear invaginations during apoptosis.
Martin R Buoncristiani - One of the best experts on this subject based on the ideXlab platform.
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evaluating the efficacy of dna Differential Extraction methods for sexual assault evidence
Forensic Science International-genetics, 2017Co-Authors: Sonja B Klein, Martin R BuoncristianiAbstract:Analysis of sexual assault evidence, often a mixture of spermatozoa and victim epithelial cells, represents a significant portion of a forensic DNA laboratory's case load. Successful genotyping of sperm DNA from these mixed cell samples, particularly with low amounts of sperm, depends on maximizing sperm DNA recovery and minimizing non-sperm DNA carryover. For evaluating the efficacy of the Differential Extraction, we present a method which uses a Separation Potential Ratio (SPRED) to consider both sperm DNA recovery and non-sperm DNA removal as variables for determining separation efficiency. In addition, we describe how the ratio of male-to-female DNA in the sperm fraction may be estimated by using the SPRED of the Differential Extraction method in conjunction with the estimated ratio of male-to-female DNA initially present on the mixed swab. This approach may be useful for evaluating or modifying Differential Extraction methods, as we demonstrate by comparing experimental results obtained from the traditional Differential Extraction and the Erase Sperm Isolation Kit (PTC©) procedures.
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development of a rapid 96 well alkaline based Differential dna Extraction method for sexual assault evidence
Forensic Science International-genetics, 2012Co-Authors: William R Hudlow, Martin R BuoncristianiAbstract:Abstract We present a rapid alkaline lysis procedure for the Extraction of DNA from sexual assault evidence that generates purified sperm fraction extracts that yield STR typing results similar to those obtained from the traditional organic/dithiothreitol Differential Extraction. Specifically, a sodium hydroxide based Differential Extraction method has been developed in a single-tube format and further optimized in a 96-well format. The method yields purified extracts from a small sample set (∼2–6 swabs) in approximately 2 h and from a larger sample set (up to 96 swabs) in approximately 4 h. While conventional Differential Extraction methods require vigorous sample manipulation to remove the spermatozoa from the substrate, the method described here exploits the propensity of sperm to adhere to a substrate and does not require any manipulation of the substrate after it is sampled. For swabs, sample handling is minimized by employing a process where the tip of the swab, including the shaft, is transferred to the appropriate vessel eliminating the need for potentially hazardous scalpels to separate the swab material from the shaft. The absence of multiple handling steps allows the process to be semi-automated, however the procedure as described here does not require use of a robotic system. This method may provide forensic laboratories a cost-effective tool for the eradication of backlogs of sexual assault evidence, and more timely service to their client agencies. In addition, we have demonstrated that a modification of the procedure can be used to retrieve residual sperm-cell DNA from previously extracted swabs.
Johan Nilsson - One of the best experts on this subject based on the ideXlab platform.
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Isolation of a Low Number of Sperm Cells from Female DNA in a Glass–PDMS–Glass Microchip via Bead-Assisted Acoustic Differential Extraction
2019Co-Authors: Charles P Clark, Johan Nilsson, Thomas Laurell, Brian L. Poe, Jenny A. Lounsbury, James P LandersAbstract:We report an improved separation method for the isolation of sperm cells from dilute, “large volume” samples containing female DNA using bead-assisted acoustic trapping. In an enclosed glass–PDMS–glass (GPG) resonator, we exploit a three-layer microfluidic architecture to generate “trapping nodes” in ultrasonic standing waves. We investigate the dependence of trapping efficiency on particle concentration for both sperm cells and polymeric beads. After determination of the critical concentration of polymeric beads required to seed the trapping event, sperm cells in dilute solution are trapped as a result of the enhanced secondary radiation force (SRF). Sperm-cell-containing samples with volumes up to 300 μL and cell concentrations as low as ∼10 cells/μL are amenable to effective trapping in the presence of an abundance of female DNA in solution. Complete processing of samples is accomplished with separation of the female and male fractions within 15 min. We demonstrate that the collected fractions are amenable to subsequent DNA Extraction, short tandem repeat PCR, and the generation of STR profiles for the isolated sperm cells
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acoustic Differential Extraction for forensic analysis of sexual assault evidence
Analytical Chemistry, 2009Co-Authors: Jessica V Norris, Mikael Evander, Katie M Horsmanhall, Johan Nilsson, Thomas Laurell, James P LandersAbstract:Forensic DNA analysis of samples obtained from sexual assault evidence relies on separation of male and female components of the recovered genetic material. The conventional separation method used by crime laboratories, Differential Extraction (DE), is one of the most time-consuming sample preparation steps, requires extensive sample handling, is difficult to automate, and often results in inefficient separation of female DNA from the male sample components. To circumvent conventional DE, acoustic Differential Extraction (ADE) analysis was developed on a microfluidic device. The ADE method relies on acoustic trapping of sperm cells in the presence of epithelial cell lysate (which is unretained), and laminar flow valving to direct the male and female fractions to separate outlets. Following the separation of sperm from epithelial cell lysate, DNA Extraction, quantitation, amplification, and separation were performed using conventional laboratory methods. The results show that highly purified male and femal...
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acoustic Differential Extraction for forensic analysis of sexual assault evidence
Analytical Chemistry, 2009Co-Authors: Jessica V Norris, Mikael Evander, Katie M Horsmanhall, Johan Nilsson, Thomas Laurell, James P LandersAbstract:Forensic DNA analysis of samples obtained from sexual assault evidence relies on separation of male and female components of the recovered genetic material. The conventional separation method used by crime laboratories, Differential Extraction (DE), is one of the most time-consuming sample preparation steps, requires extensive sample handling, is difficult to automate, and often results in inefficient separation of female DNA from the male sample components. To circumvent conventional DE, acoustic Differential Extraction (ADE) analysis was developed on a microfluidic device. The ADE method relies on acoustic trapping of sperm cells in the presence of epithelial cell lysate (which is unretained), and laminar flow valving to direct the male and female fractions to separate outlets. Following the separation of sperm from epithelial cell lysate, DNA Extraction, quantitation, amplification, and separation were performed using conventional laboratory methods. The results show that highly purified male and female fractions can be obtained with the ADE microdevice from mock sexual assault samples in 14 min. ADE analysis provides the potential to significantly alter the means by which sexual assault evidence is processed in crime laboratories.