Development of a rapid, 96-well alkaline based differential DNA extraction method for sexual assault evidence

Hudlow, William R.; Buoncristiani, Martin R.

doi:10.1016/j.fsigen.2010.12.015

Cited by 33 publications

(22 citation statements)

References 19 publications

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“…Protein unfolding occurs due to ionization of amino acid residues in the presence of strong alkaline conditions resulting in disruption of the cell structure [72]. The alkaline lysis has been expanded to extract DNA from forensic evidence such as bloodstains and semen stains [64].…”

Section: B6 Alkaline Lysismentioning

confidence: 99%

“…Because of the harsh conditions that can eventually denature the DNA molecule, it is important to optimize the exposure time to alkaline pH [64].…”

Section: B6 Alkaline Lysismentioning

confidence: 99%

“…Differential extraction and its modified versions are still used today to separate different cell fractions in a mixture [53,90,125]. Although a gold standard for mixture analysis, this method is time-consuming, technique-dependent, difficult to automate, and can result in relatively inefficient separations of female DNA from the male component [34,64,92]. Moreover, if the perpetrator is vasectomized or is azoospermic, differential extraction does not aid in mixture resolution due to the lack of sperm cells [32].…”

Section: B7 Differential Extractionmentioning

confidence: 99%

“…As a result we evaluated tris (2-carboxyethyl) phosphine (TCEP), a stronger reducing agent, under the assumption that the key issue for selective extraction was the disruption of the disulfide bonds in the sperm cells [78]. [64,78,125]. Voorhees et al demonstrated a slight increase in cell recovery by using cellulase enzyme to digest the cellulose fibers of the cotton swab [125].…”

Section: C2 Determination Of Optimum Pressure Parametersmentioning

confidence: 99%

“…Alkaline lysis Hudlow et al developed an alkaline-based differential extraction method that when combined with DNase digestion generated a purified sperm fraction [64]. Upon applying this method and quantifying the extracted DNA, it was observed that there was a significant loss of sperm DNA prior to DNase digestion.…”

Section: B2 Dna Extractionmentioning

confidence: 99%

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A Novel Method for Rapid and Selective Extraction of Male DNA from Rape Kits using Alkaline Lysis and Pressure Cycling Technology (PCT)

Nori¹

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Section: B6 Alkaline Lysismentioning

confidence: 99%

“…Because of the harsh conditions that can eventually denature the DNA molecule, it is important to optimize the exposure time to alkaline pH [64].…”

Section: B6 Alkaline Lysismentioning

confidence: 99%

Section: B7 Differential Extractionmentioning

confidence: 99%

Section: C2 Determination Of Optimum Pressure Parametersmentioning

confidence: 99%

Section: B2 Dna Extractionmentioning

confidence: 99%

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A Novel Method for Rapid and Selective Extraction of Male DNA from Rape Kits using Alkaline Lysis and Pressure Cycling Technology (PCT)

Nori¹

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DNA Extraction Methods in Forensic Analysis

Lee

Shewale

2017

Encyclopedia of Analytical Chemistry

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Forensic biological evidence encompasses a diverse conglomerate of samples containing genetic material found in a variety of biological fluids and tissues such as human blood, semen, saliva, epithelial cells, hair, bone, teeth, fingernails, and putrefied tissues. In addition to human samples, nonhuman samples from plant, animal, bacteria, and fungi may also need to be processed. The samples and body fluids may be present as dried stains on an assortment of substrates, often mixed with PCR (polymerase chain reaction) inhibitors, exposed to environmental conditions and uncontrolled degradation, and are often present in limited quantities. In addition to the intrinsic characteristics of samples that include the type of body fluid (e.g. blood, saliva, semen), nature of the tissues (e.g. buccal cells, hair, bone, tooth), quantity [e.g. trace DNA (deoxyribonucleic acid), low copy number], and source (e.g. reference, evidence), they can also be grouped by their extrinsic characteristics such as the substrate (e.g. swab, clothing) and nature of the crime (e.g. sexual assault, burglary, homicide) from which they are derived. These samples need to be processed using the most effective methods of nucleic acid extraction and purification for downstream quantification and genetic profiling by PCR. Compositionally, there are an unlimited number of combinations of sample and substrate types including the quantity and quality of the sample, substrate and conditions encountered, and contaminant and inhibitor levels. Some factors may be observed during screening, whereas many of these remain transparent to the analyst. This renders the choice of the most efficient DNA extraction methods, one of the most unpredictable steps in forensic DNA profiling. Research has led to significant enhancements, testing, and validation of advanced extraction and amplification chemistries along with new instrumentation platforms providing systems capable of automated, sensitive, rapid, and robust extraction and genotyping and/or sequencing of short tandem repeats (STR), single‐nucleotide polymorphisms (SNPs), and mitochondrial DNA. All three genetic marker classes have been accepted in the court systems worldwide and this along with legislation that has expanded both the law and funding for forensic DNA profiling has led to a high and increasing demand for forensic DNA testing. The increased demand and new expanded capabilities have opened the door to the analysis of forensic samples that are extremely compromised in both quality and quantity. Over the past 10 years, new DNA extraction methods have been developed to reproducibly extract DNA and remove or mitigate PCR inhibitors from the majority of sample types with speed and efficiency, resulting in high‐yield template free of PCR inhibition. In addition, automated processing has been tested and validated to meet the demand for high‐throughput processing. This article summarizes the importance of DNA extraction, provides an update on methods for single source, compromised evidence, sexual assault evidence, automation, differential extraction, and an overview of the most widely adopted methods and the best practices for DNA isolation from forensic biological samples.

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Deoxyribonucleic Acid Extraction Methods in Forensic Analysis

Fernandez-Tejero¹,

Ghemrawi²,

McCord³

et al. 2022

Encyclopedia of Analytical Chemistry

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Forensic biological evidence encompasses a diverse conglomerate of samples containing genetic material found in a variety of biological fluids and tissues such as human blood, semen, saliva, epithelial cells, hair, bone, teeth, fingernails, and putrefied tissues. In addition to human samples, nonhuman samples from plant, animal, bacteria, and fungi may also need to be processed. The samples and body fluids may be present as dried stains on an assortment of substrates, often mixed with polymerase chain reaction (PCR) inhibitors, exposed to environmental conditions and uncontrolled degradation, and are often present in limited quantities. In addition to the intrinsic characteristics of samples that include the type of body fluid (e.g. blood, saliva, semen), nature of the tissues (e.g. buccal cells, hair, bone, tooth), quantity [e.g. trace DNA (deoxyribonucleic acid), low copy number], and source (e.g. reference, evidence), they can also be grouped by their extrinsic characteristics such as the substrate (e.g. swab, clothing) and nature of the crime (e.g. sexual assault, burglary, homicide) from which they are derived. These samples need to be processed using the most effective methods of nucleic acid extraction and purification for downstream quantification and genetic profiling by PCR. Compositionally, there are an unlimited number of combinations of sample and substrate types including the quantity and quality of the sample, substrate and conditions encountered, and contaminant and inhibitor levels. Some factors may be observed during screening, whereas many of these remain transparent to the analyst. This renders the choice of the most efficient DNA extraction methods, one of the most unpredictable steps in forensic DNA profiling. Research has led to significant enhancements, testing, and validation of advanced extraction and amplification chemistries along with new instrumentation platforms providing systems capable of automated, sensitive, rapid, and robust extraction and genotyping and/or sequencing of short tandem repeats (STR), single‐nucleotide polymorphisms (SNPs), and mitochondrial DNA. All three genetic marker classes have been accepted in the court systems worldwide and this along with legislation that has expanded both the law and funding for forensic DNA profiling has led to a high and increasing demand for forensic DNA testing. The increased demand and newly expanded capabilities have opened the door to the analysis of forensic samples that are extremely compromised in both quality and quantity. Over the past 10 years, new DNA extraction methods have been developed to reproducibly extract DNA and remove or mitigate PCR inhibitors from the majority of sample types with speed and efficiency, resulting in high‐yield template free of PCR inhibition. In addition, automated processing has been tested and validated to meet the demand for high‐throughput processing. This article summarizes the importance of DNA extraction, provides an update on methods for single source, compromised evidence, sexual assault evidence, automation, differential extraction, and an overview of the most widely adopted methods and the best practices for DNA isolation from forensic biological samples.

show abstract

Development of a rapid, 96-well alkaline based differential DNA extraction method for sexual assault evidence

Cited by 33 publications

References 19 publications

A Novel Method for Rapid and Selective Extraction of Male DNA from Rape Kits using Alkaline Lysis and Pressure Cycling Technology (PCT)

A Novel Method for Rapid and Selective Extraction of Male DNA from Rape Kits using Alkaline Lysis and Pressure Cycling Technology (PCT)

DNA Extraction Methods in Forensic Analysis

Deoxyribonucleic Acid Extraction Methods in Forensic Analysis

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