DNA Extraction Methods in Forensic Analysis

Abstract

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.

Recent trends and developments in forensic DNA extraction

“…Technically, direct PCR amplification is a broad representation of two methods—sample‐to‐PCR and lysate‐to‐PCR amplifications (Lee & Shewale, 2017). The former involves directly using a sample, such as a minute cutting of the collection swab head, for PCR amplification (Linacre, Pekarek, Swaran, & Tobe, 2010; Verheij, Harteveld, & Sijen, 2012).…”

“…The forensic applications and limitations of direct PCR amplification have already been reviewed (Cavanaugh & Bathrick, 2018; Lee & Shewale, 2017; Lynch & Fleming, 2019). As such, this section focuses mainly on trends, new developments, and challenges in direct PCR amplification that have not been addressed previously.…”

Methods for the analysis of mitochondrial DNA

“…The DNA extraction methods validated by forensic laboratories have been developed historically for the isolation of nuclear DNA (Holland & Parsons, 1999; Lee & Shewale, 2017). These extraction processes generally isolate all DNA present in the sample, including mtDNA.…”

“…This is important for forensic investigations as the biological material analyzed is often limited in DNA quantity and quality. Examples of extraction techniques which can be used for the extraction of mtDNA analysis include: organic extraction methods (phenol chloroform); chelating resin methods (Chelex 100); silica based methods; and magnetic bead methods (Lee & Shewale, 2017).…”

“…Such samples may also contain DNA inhibitors. Therefore the extraction method used to isolate DNA for analysis must isolate as much DNA as possible without introducing further damage, as well as minimizing the coextraction of inhibitors (Lee & Shewale, 2017).…”