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Comparative Study of DNA Extraction Potency of Three Commercial Kits on Soil-Blood Mixed Sample

Academic Paper 2018 18 Pages

Biology - Miscellaneous

Excerpt

Content

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION AND CONCLUSION

REFERENCES

ABSTRACT

This study aimed at identifying the best DNA extraction method in terms of yield for soil-blood mixed sample. 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 DNA from soil contaminated samples were used for the DNA extractions. Human blood was mixed with soil and stored at Room temperature for a 12 week period. The PrepFiler kit yielded more DNA than the DNA IQ and Blood Miniprep kits with no significant difference between PrepFiler and DNA IQ (P=0.603).

INTRODUCTION

Background

All incidence, be it a crime, accident, natural disaster, armed conflict, leaves traces of physical evidence at the scene ( UNODC, 2009). Physical evidence can take any form from clearly visible objects to microscopic objects, generated when a crime is committed and retrieved at the scene or at other locations ( UNODC, 2009). Blood is the most common body fluid found at crime scenes (Tobe et al., 2007; Legg, 2013; Vandewoestyne et al., 2015) and hence bloodstains present at a crime scene help investigators to know if a connection exists amongst a suspect, victim and crime scene (Sharma et al., 2011). Advances in molecular genetics techniques have had a beneficial impact in the field of forensic science (Legg, 2013), such as exonerating the innocent and identifying the perpetrator of a crime. At outdoor violent-related crime scenes, many situations may cause human biological evidence from the suspect or victim to be deposited in the soil (Kasu and Shires, 2015). This evidence mixed with soil at outdoor crime scenes might be the only source of evidence available to the investigator. This evidence can be analyzed using DNA to link the suspect or victim to the crime scene. Sometimes, soil mixed biological evidence are often not collected at crime scenes because they are limited in quantity, are difficult to work with and have high failure rate. This is as a result of the samples been heavily degraded due to environmental factors. This can lead to loss of important evidence that can lead to the identification of victim or suspect. It is therefore important that the most appropriate method of isolation is chosen to ensure that the most information is acquired from each precious forensic sample. In the case of soil-mixed samples, this information is currently lacking (Kasu and Shires, 2015).

Problem Statement and Justification

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). 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 resulting in low DNA elute (Butler, 2011). 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 which can all affect the final concentration of human DNA. There is thus the need to investigate the best and appropriate method for the extraction of DNA from soil-mixed sample.

Objective

The objective of this work was to do a comparative evaluation of three DNA extraction methods on soil-blood mixed samples found at crime scenes in terms of their DNA extraction potency.

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.

Storage facilities

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

Reagents and Instruments for DNA Extractions

PrepFiler Forensic DNA extraction kit, Promega DNA IQ Extraction kit and Blood Miniprep kit were used for the DNA extractions.

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).

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 Ethylenediaminetetraacetic 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 on paper druggist folds.

Sample preparation prior to analysis

The soil-blood mixed sample was placed in appropriate tube and 4 ml of DNAse/RNAse free water added to it. It was allowed to stand for 30 minutes at room temperature for the blood to come into solution after which it was 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 tube was then centrifuged with Eppendorf centrifuge at 14000 rpm for 30 seconds. The supernatant was pippeted and 500 µl volume used for each extraction method.

DNA tests controls for the study

Prior to storage, soil-blood mixed samples were extracted using all the three different extraction methods (PrepFiler Forensic method, Promega DNA IQ method, Blood Miniprep method) and these served as controls for this study. DNA extractions were done following the kits’ manufacturers’ protocols.

DNA extractions

Five hundred microliters (500 µl) of liquid supernatant from soil-blood mixed sample was pippeted and used for each DNA extraction after going through sample preparation method described. DNA Extractions were done using the kits manufacturers’ protocols.

Sample identification numbers for the entire study is found in Table 1 below. The first number(s) represent the storage duration in weeks, followed by the extraction method and the last letter represent the storage condition.

Table 1: Samples identification numbers

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Controls

Prep. Clean blood- PCB; Q. Clean blood- QCB; B. Clean blood- BCB

Prep soil-blood mixed- PSB; Q. Soil-blood mixed – QSB; B. Soil-blood mixed- BSB

P = PrepFiler Forensic kit; Q =Promega DNA IQ extraction kit; B = Blood Miniprep kit

For the sample names, the first number(s) represent the storage duration in weeks, followed by the extraction method and the last letter represent the storage condition; where R is for room temperature.

DNA Concentration determination

Total human DNA in the extracted samples was measured by Applied Biosystem’s QuantiFiler trio kit using 7500 Real-time PCR and following the manufacturer’s protocol. The Small Autosomal target (SA) determined the actual DNA concentration.

QuantiFiler Trio Kit

The QuantiFiler Trio Kit quantifies total human DNA and total human male DNA at the same time. Results from real time PCR with QuantiFiler trio can assist the analyst to know:

- If there is sufficient human DNA and/or human male DNA for subsequent STR analysis
- The quantity of sample to use for PCR amplification
- Ratio of male to female in mixed samples especially sexual assault samples.
- The quality of DNA (degradation and inhibition).

There are three targets of amplification in the QuantiFiler trio kit; small autosomal, large autosomal and Y target. The Small Autosomal and Y targets have short amplicons (75 to 80 bases) in order to maximize the chance of detecting degraded samples. The Large Autosomal has relatively longer amplicon (>200 bases) to aid an analyst know if the sample is degraded. The small autosomal quantity actually gives the concentration of the sample.

There is also an internal positive control which contains a synthetic template DNA. By assessing the internal positive control, an analyst can determine if a sample has zero concentration or contains inhibitors.

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Figure 1: Real-time PCR Amplification plot for male sample using QuantiFiler trio kit (Source: Ghana Police Forensic Science Lab)

ΔRn means Rn minus the baseline. Rn means the fluorescence of the reporter dye divided by the fluorescence of a passive reference dye; thus, Rn is the reporter signal normalized to the fluorescence signal of Applied Biosystems ROX dye.

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Figure 2: Real-time PCR amplification plot for female sample using QuantiFiler trio kit (Source: Applied Biosystems, 2016)

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Figure 3: Real-time PCR amplification plot for no template (NTC) sample using QuantiFiler trio kit (Source: Ghana Police Forensic Science Lab)

Threshold cycle is where an amplification curve and a threshold line meet. When the Threshold cycle (CT) value of a target is less than 40, then positive amplification has occurred. The Internal positive control (IPC) CT value is relatively the same in normal reactions but presence of inhibitors in the sample and/or DNA quantities can increase the IPC CT value compared to the average IPC CT value of the standards on the same reaction plate. When PCR inhibitors are in large concentration in a sample such that subsequent analysis could be affected, the IPC CT flag is triggered for that particular sample. Large Autosomal target may be affected by the increasing inhibitor concentration before the Small Autosomal target and before the IPC CT flag is triggered. Small rise in value of degradation index may be as a result of degradation and/or presence of inhibitors (ThermoFisher Scientific, 2016).

RESULTS

Real-time PCR DNA quantification results

From the summary of the entire experiment, the internal positive control (IPC) of seven samples (BSB, 2BR, 4BR, 6BR, 8BR, 10BR, 12BR) were flagged or not amplified. The Negative template control included in the quantification showed amplification for IPC but no amplification for other targets. This means all the RT-PCR assays worked well and that the sample preparation procedure was devoid of contamination. The positive control amplified and showed detectable DNA for all human and Y targets. This indicates good amplification and good formulation of reagents. Slopes of -3.291, -3.364 and -3.231 were obtained from standard curve for the Y-target, Large autosomal and Small autosomal, respectively; an indication of 99.73% amplification efficiency. The CT values for all amplified targets were less than 40 which suggest positive amplification in these samples.

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Figure 4: Standard curve for Y target

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Figure 5: Standard curve for Large autosomal target

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Figure 6: Standard curve for small autosomal target

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Figure 7: Amplification plot for the Negative template control (NTC)

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Figure 8: Amplification plot for Positive control showing amplification for all targets and IPC

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Figure 9: Amplification plot for sample 2BR showing no amplifications for targets and IPC

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Figure 10: Amplification plot for sample PCB (Prepfiler clean blood) showing amplification for all targets and IPC

Comparing DNA extraction potency among the three extraction kits

Assessment of the control samples from show PrepFiler Forensic kit yielding more DNA than DNA IQ kit for both clean blood and soil-blood mixed samples. DNA IQ also performed better than Blood Miniprep kit. PrepFiler Forensic kit yielded more DNA at the three storage conditions than DNA IQ with no significant difference (P=0.603) in concentrations. There was no significant difference (P=0.469) in DNA concentrations of PrepFiler and DNA IQ extractions of room temperature stored samples. There was also no significant difference (P=0.693) in DNA concentrations of PrepFiler and DNA IQ extractions of 4℃ stored samples. However, difference in DNA concentrations of PrepFiler and DNA IQ extractions from -20℃ stored samples was significant (P=0.000)

Table 2: DNA concentrations (ng/µl) from soil-blood mixed samples at 3 storage conditions

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Controls

PrepFiler clean blood (PCB)- 28.65ng/µl

DNA IQ clean blood (QCB)- 24.07ng/µl

PrepFiler soil-blood mixed (PSB)- 11.72ng/µl

DNA IQ soil-blood mixed (QSB)- 6.84ng/µl

Blood Miniprep clean blood (BCB)- 9.21ng/µl

Blood Miniprep soil-blood mixed (BSB)- No amplification

Prep. =PrepFiler Forensic kit; IQ. =Promega DNA IQ extraction kit; BM. =Blood Miniprep kit; N=No amplification

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Figure 11: DNA concentrations of soil-blood mixed samples over a 12 week period from the 3 extraction methods

DISCUSSION AND CONCLUSION

Some Blood Miniprep extracted soil-blood mixed samples had no amplification for IPC and all targets, suggesting that there was total amplification failure in this sample as a result of possible presence of high concentration of PCR inhibitors. These inhibitors possibly came from the soil as observed in work by Braid et al., in 2003, because the Blood Miniprep extraction for clean blood showed amplification for all targets and IPC.

PrepFiler extracted samples recorded the highest DNA concentrations compared to DNA IQ and Blood miniprep kits. PrepFiler forensic kit was the most potent in terms of ability to extract more DNA followed by DNA IQ Kit. Though Blood Miniprep kit extractions were mostly inhibited, the control that amplified confirms that this kit was the worst in terms of DNA extraction potency.

REFERENCES

Braid, M. D., Daniels, L. M. and Kitts, C. L. (2003). Removal of PCR inhibitors from soil DNA by chemical flocculation. Journal of Microbiological Methods, 52(3), 389-393.

Butler, J. M. (2011). Advanced topics in forensic DNA typing: methodology. Academic Press.

Kasu, M., and Shires, K. (2015). The validation of forensic DNA extraction systems to utilize soil contaminated biological evidence. Legal Medicine, 17(4), 232-238.

Legg, K.M. (2013). Development and Testing of a Rapid Multiplex Assay for the Identification of Biological Stains, Doctoral thesis, Faculty of Natural Science and Mathematics, University of Denver.

Rohatgi, R., and Kapoor, A. K. (2014). Effect of Different Types of Soil and Time Intervals on Isolation and Quantification of DNA: A Forensic Management Technology Perspective., International Journal of Emerging Research in Management &Technology ISSN: 2278-9359 Volume-3 Issue-7.

Sharma, M., Khajja B.S., Godara, D.R., and Mathur, G.K. (2011). Case Study: Theft and Murder Unraveled by Forensic Investigation http://www.forensicmag.com/article/2011/06/case-study-theft-and-murder-unraveled-forensic-investigation, accessed 10th June, 2016.

Tobe, S. S., Watson, N., and Daeid, N. N. (2007). Evaluation of six presumptive tests for blood, their specificity, sensitivity, and effect on high molecular weight DNA. Journal of forensic sciences, 52(1), 102-109.

UNODC (United Nation’s office on drugs and crime). (2009). Crime scene and physical evidence awareness for non-forensic personnel, United Nations publication Sales No. E.09.IV.5, ISBN 978-92-1-130273-8.

Vandewoestyne, M., Lepez, T., Van Hoofstat, D., and Deforce, D. (2015). Evaluation of a visualization assay for blood on forensic evidence. Journal of forensic sciences, 60(3), 707-711.

Details

Pages
18
Year
2018
File size
1.5 MB
Language
English
Catalog Number
v433986
Institution / College
Kwame Nkrumah University of Science and Technology
Grade
A
Tags
DNA DNA Extraction Blood DNA Comparative study DNA Quantification Forensic DNA Real-Time PCR Soil DNA PrepFiler Forensic DNA Kit DNA IQ Kit Blood Miniprep Kit DNA extraction from blood Alexander Badu Boateng

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Title: Comparative Study of DNA Extraction Potency of Three Commercial Kits on Soil-Blood Mixed Sample