Evaluation of the Ability of Three Commercial DNA Extraction Kits to Remove PCR Inhibitors from Soil

Table of Contents

ABSTRACT

INTRODUCTION
Background
Promega DNA IQ Extraction kit
PrepFiler Forensic DNA Extraction kit
Blood Genomic DNA Miniprep kit
QuantiFiler Trio Kit

MATERIALS AND METHODS
Ethical clearance
Blood sample
Soil sample
Storage facility
Reagents and Instruments for DNA Profiling
Study time
Sample preparation and storage
Sample preparation prior to analysis
Tests controls for the study

DNA extractions

DNA Quality Assessment

RESULTS
Assessing PCR Inhibition among the three extraction methods

Controls

DISCUSSION

CONCLUSION

REFERENCE

EVALUATION OF THE ABILITY OF THREE DNA EXTRACTION KITS TO REMOVE PCR INHIBITORS FROM SOIL

ABSTRACT

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.

INTRODUCTION

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). Many attempts in the extraction of DNA from forensic evidence have been undertaken over the years to eliminate soil inhibitors. Some of these methods or techniques have been incorporated into forensic human DNA isolation kits (Kasu and Shires, 2015).

Bloodstains are often encountered on weapons, clothing and other materials and sometimes on the body of the victim. When these materials are discarded at outdoor locations, they may come into contact with soil. Blood-mixed soil evidence may be of great value in crime investigation due to its potential presence at all outdoor crime scenes (Rohatgi and Kapoor, 2014). DNA obtained from blood, saliva or semen mixed with soil at an outdoor crime scene may become the main evidence to lead the investigation of a crime when a body is absent. However, biological sample mixed with soil is often contaminated with materials that pose a threat to the DNA profiling process (Kasu and Shires, 2015). Inhibitors found in soil can interfere with the cell lysis process, interfere by nucleic acid degradation and also inhibit polymerase activity thus preventing enzymatic amplification of the template DNA (Wilson, 1997; Gryson, 2010; Butler, 2011). According to Zhou et al., 1996 and Sutlovic et al., 2007, isolation of DNA from soils always ends in the co-isolation of humic materials which interfere with DNA detection and measurement.

Extraction of DNA from soil-mixed sample will co-extract DNA from microbes in the soil, organic matters as well as presence of PCR inhibitors in the final elute, and thus, there is the need to investigate the best and appropriate method for the extraction of DNA from soil-mixed sample.

DNA IQ, Blood Miniprep Kit and Prepfiler Forensic DNA kit have been claimed by the manufacturers to be effective in extracting quality DNA from samples in the presence of inhibitors and contaminants (Bessetti , 2007; Brevnov et al., 2009; Kasu and Shires, 2015; http://www.biopioneerinc.com/2016/molecular-biology/genomic-dna-isolation-kits/blood-genomic-dna-miniprep-kit-100-preps/ accessed 8th April, 2018).

Promega DNA IQ Extraction kit

The IQ means isolation and quantification. This is a quick extraction commercial kit which can deal with a lot of challenged forensic samples and remove inhibitors (Bessetti, 2007; Kasu and Shires, 2015). Amount of DNA is bound to a tube using paramagnetic resin. The resin has a limit for bound DNA and binds only a certain quantity of DNA. DNA quantity from this extraction method is stable within one sample type but changes from different samples (Promega, 2016).

PrepFiler Forensic DNA Extraction kit

This extraction method works on magnetic particle technique similar to Promega’s DNA IQ (Applied Biosystems, 2008; Butler, 2011). This kit has magnetic particles which binds DNA whiles PCR inhibitors are removed by washing the bound DNA, resulting in pure genomic DNA (Applied Biosystems, 2008; Brevnov et al., 2009).

Blood Genomic DNA Miniprep kit

This kit is meant for quick extraction of genomic DNA from blood samples and other body fluids. A small starting material is sufficient to obtain about 30 µg of genomic DNA devoid of contaminants (http://www.biopioneerinc.com/2016/molecular-biology/genomic-dna-isolation-kits/blood-genomic-dna-miniprep-kit-100-preps/ accessed 31st August, 2016).

QuantiFiler Trio Kit

The QuantiFiler Trio Kit quantifies total human DNA and total human male DNA at the same time.

MATERIALS AND METHODS

Ethical clearance

Ethical clearance for this study was obtained from the Committee of Human Research, Publications and Ethics of the Komfo Anokye Teaching Hospital and the School of Medical Sciences, Kwame Nkrumah University of Science and Technology (KNUST).

Blood sample

Fresh adult male whole blood sample from a single person in a tube with Ethylenediaminetetraacetic acid (EDTA) anticoagulant was obtained from Jubilee Hospital, an accredited private Hospital at Akim-Oda, Eastern Region of Ghana and transported on ice to the Forensic lab, Accra. Identity of donor isn’t known and sample collection was done by a staff of the facility.

Soil sample

Dried black soil was collected in paper envelope from a garden at the compound of the Ghana Police Forensic Lab and brought to the lab.

Storage facility

Room with an air condition of 25 °C temperature served as the storage condition for this study.

Reagents and Instruments for DNA Profiling

PrepFiler Forensic DNA extraction kit, Promega DNA IQ Extraction kit and Blood Miniprep kit were used for the DNA extractions. QuantiFiler trio DNA quantification kit and 7500 real-time PCR machine manufactured by Applied Biosystem’s were used for DNA Quantification.

Study time

The study was conducted at Jubilee Hospital, Akim-Oda and the Ghana Police Forensic Science Laboratory, Accra, over a period of 12 weeks .

Sample preparation and storage

1 g of soil was mixed with 1 ml of whole blood sample containing Ethylenediami-netetraacetic acid (EDTA). In all, six soil-blood mixed samples were stored at Room temperature/25 °C. Soil-blood mixed samples were stored for 2, 4, 6, 8, 10 and 12 week periods. The samples were stored in dried state on paper druggist folds at room temperature/25 °C.

Sample preparation prior to analysis

Soil-blood mixed samples were placed in appropriate tubes and 4 ml of DNAse/RNAse free water added to them. They were allowed to stand for 30 minutes at room temperature for the blood to come into solution after which they were vortexed thoroughly to mix soil particles and blood. In order to minimize the amount of lysis buffer used for the extraction, it was important to get the blood cells into solution and separate them from the large soil particles. This also ensured most cells were lysed for maximum DNA yield. The tubes were then centrifuged with Eppendorf centrifuge at 14000 rpm for 30 seconds. The supernatants were pippeted and 500 µl volume used for each extraction method.

Tests controls for the study

Prior to soil-blood mixed samples storage, clean blood and soil-blood mixed samples were extracted using all the three different extraction methods (PrepFiler Forensic method, Promega DNA IQ method, Blood Miniprep method). Tests controls were done following kits’ manufacturers’ protocols after going through the same sample preparation method described above.

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