Crime scene investigation is an important step in the entire criminal investigation process because this is where evidence is gathered. Blood from the perpetrator or victim of a crime can be left at crime scenes or transferred to other materials such as clothing, knives and guns. Most often, this body fluid is contaminated with soil at outdoor crime scenes but this might be the only or the most important evidence in solving a crime. This study aimed at identifying the most appropriate method of extracting quality DNA from soil contaminated blood using three commercial DNA extraction kits (PrepFiler Forensic DNA Extraction kit, Promega DNA IQ Kit, Blood Miniprep kit) that have been claimed by the manufacturers to be effective in extracting quality DNA from soil contaminated samples. Human blood was mixed with soil and stored at room temperature/25℃ for a 12 week period. The PrepFiler kit and DNA IQ kit were successful at removing possible PCR inhibitors from the soil during DNA extraction with no significant difference (p=0.887). The Blood Miniprep kit performed poor in terms of removing possible PCR inhibitors.
Table of Contents
1. INTRODUCTION
1.1 Background
1.2 Promega DNA IQ Extraction kit
1.3 PrepFiler Forensic DNA Extraction kit
1.4 Blood Genomic DNA Miniprep kit
1.5 QuantiFiler Trio Kit
2. MATERIALS AND METHODS
2.1 Ethical clearance
2.2 Blood sample
2.3 Soil sample
2.4 Storage facility
2.5 Reagents and Instruments for DNA Profiling
2.6 Study time
2.7 Sample preparation and storage
2.8 Sample preparation prior to analysis
2.9 Tests controls for the study
2.10 DNA extractions
2.11 DNA Quality Assessment
3. RESULTS
3.1 Assessing PCR Inhibition among the three extraction methods
3.2 Controls
4. DISCUSSION
5. CONCLUSION
Research Objectives and Key Topics
This study aims to evaluate and compare the effectiveness of three commercial DNA extraction kits—PrepFiler Forensic DNA Extraction kit, Promega DNA IQ Kit, and Blood Genomic DNA Miniprep kit—in successfully removing PCR inhibitors from blood samples contaminated with soil. The research seeks to identify which method provides the highest quality DNA suitable for forensic profiling when biological evidence is recovered from outdoor crime scenes.
- Forensic DNA extraction techniques
- Impact of soil-based PCR inhibitors on DNA amplification
- Comparative performance analysis of commercial extraction kits
- Optimization of forensic evidence recovery from contaminated samples
Excerpt from the Book
Background
Over the past 20 years, advances in some forensic science disciplines, especially the use of DNA techniques, have shown that some branches of forensic science have great additional potential to help investigators identify criminals and exonerate the innocent. A lot of crimes that may have gone unsolved are now being solved because forensic science is aiding in the identification of the perpetrators (National Academy of Sciences, 2009).
Sometimes, some foreign substances remain in a DNA sample after extraction thereby preventing successful amplification (Akane et al., 1994; Rådström et al., 2004; Butler, 2011). These substances are called inhibitors and can be present in biological evidence collected from crime scenes. Blood and semen on soil can be present at outdoor crime scenes and may contain inhibitors which may remain with the DNA after extraction (Bessetti, 2007; Schrader et al., 2012). Humic acid is a major PCR inhibitor found in soil and the major organic component of soil. When microbes degrade plant and animal materials, humic acid is formed (Zipper et al., 2003; Kasu and Shires, 2015). The chemical properties of humic acid is similar to that of double-stranded DNA (Buckwalter et al., 2014; Kasu and Shires, 2015). Eventually, traditional isolation methods, such as phenol-chloroform, and detergent and protease treatments are not able to remove this humic contaminant, thereby remaining in the final DNA elute (Bessetti, 2007; Lakay et al., 2007; Sutlovic et al., 2007; Shahzad et al., 2009). An ideal forensic DNA extraction process should prevent further degradation of the DNA (Butler, 2011) and also remove inhibitors that prevent or interfere with polymerase chain reaction (Bessetti, 2007; Butler, 2011). In forensic DNA, humic substances are major cause of amplification failure because they chelate the magnesium ions needed by the DNA polymerase (Harry et al., 1999; Fortin et al., 2004; Lakay et al., 2007; Buckwalter et al., 2014; Lasota, 2014; Kasu and Shires, 2015). The main idea behind DNA extraction is to get a high molecular weight DNA devoid of contaminants (Sambrook et al., 1989; Kirby, 1992).
Summary of Chapters
INTRODUCTION: Provides context on forensic DNA profiling and the challenges posed by environmental inhibitors like humic acid found in soil-contaminated evidence.
MATERIALS AND METHODS: Details the experimental design, including sample collection, soil-blood mixing procedures, storage conditions over 12 weeks, and the specific protocols used for the three DNA extraction kits.
RESULTS: Presents the quantification data and the performance of internal positive controls, showing amplification success or failure for each extraction kit.
DISCUSSION: Analyzes the efficacy of each kit in removing inhibitors, noting that PrepFiler and DNA IQ performed significantly better than the Blood Miniprep kit.
CONCLUSION: Summarizes that PrepFiler and DNA IQ are suitable for soil-contaminated forensic samples, while the Blood Miniprep kit is ineffective in this specific context.
Keywords
Forensic Science, DNA Extraction, PCR Inhibitors, Humic Acid, Soil Contamination, PrepFiler, Promega DNA IQ, Blood Miniprep, DNA Profiling, Amplification, Forensic Evidence, Internal Positive Control, Quality Assessment, Forensic Biology, Extraction Efficiency
Frequently Asked Questions
What is the primary focus of this research?
The research focuses on evaluating the capability of three specific DNA extraction kits to remove environmental PCR inhibitors from blood samples that have been mixed with soil.
What are the central themes of the work?
The central themes are forensic DNA isolation, the identification and impact of humic acid as a PCR inhibitor, and the comparative efficacy of commercial extraction technologies.
What is the main research question?
The study asks which of the three tested commercial DNA extraction kits is most effective at yielding high-quality DNA suitable for profiling from soil-contaminated blood samples.
Which scientific methods were employed?
The researchers used a combination of standard laboratory DNA extraction protocols, real-time PCR (7500 Real-time PCR machine), and quantification kits to assess DNA quality and inhibitor presence.
What topics are covered in the main section?
The main sections cover background literature, a detailed methodology for sample preparation and storage, experimental results, and a critical discussion of the kit performances.
Which keywords characterize this study?
Key terms include Forensic Science, PCR Inhibitors, Humic Acid, DNA Extraction, Soil Contamination, and Amplification Efficiency.
How does soil contamination typically affect DNA analysis?
Soil contains humic acid, which mimics DNA properties and interferes with the polymerase chain reaction, often preventing successful amplification and identification of the perpetrator.
Why did the Blood Miniprep kit perform poorly compared to the others?
The study concludes that the Blood Miniprep kit was unable to successfully remove the concentration of PCR inhibitors introduced by the soil, leading to consistent amplification failure across all samples.
- Citation du texte
- Alexander Badu-Boateng (Auteur), 2018, Evaluation of the Ability of Three Commercial DNA Extraction Kits to Remove PCR Inhibitors from Soil, Munich, GRIN Verlag, https://www.grin.com/document/436868
