DNA extraction with Qiagen bio-robot EZ1 DNA investigator kit, forensicGEM Sex Crime/Universal kit and Qiagen QIAamp investigator kit: a comparison and optimization study of DNA percent recovery on body fluids for forensic applications
Hanley, Alexandra E.
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The first step in forensic deoxyribonucleic acid (DNA) analysis process is extraction. The extraction process lyses cells and isolates the DNA from the rest of the cellular components. There are many different extraction methods that forensic laboratories can implement within their department, so it is important to determine which extraction method performs the best in regards to DNA recovery, cost, time and ease of use. The percent of DNA recovered can demonstrate how well the extraction mechanism works. Little research has been done to compare different extraction methods based on their percent DNA recovery alone. This project compares different extraction methods based on their percent DNA recovery. The three extraction methods that were investigated and compared were: the Qiagen BioRobot EZ1® DNA Investigator kit (EZ1 method) used on the Qiagen BioRobot EZ1®, the forensicGEM™ Sex Crime/Universal kit™ (ZyGEM/Acrosolv) method and the manual Qiagen QIAamp® Investigator kit. Two different percent recovery calculations were described in this study (method 1 and method 2) but only method 1 was used for analysis purposes. Only method 1 was used because it was determined to be the most reliable method for comparison. This demonstrates how important it is to calculate and report the percent recovery consistently because the results could differ depending on how the conclusions are reported. This project demonstrated that the ZyGEM/Acrosolv extraction method outperformed the other two methods when percent recovery was being investigated with two different biological fluids (semen and saliva). The percent recovery with sperm and epithelial cells (e-cells) with the ZyGEM/Acrosolv method was 109.4% and 103.9% respectively. With the EZ1 method, the sperm and e-cell DNA percent recovery was 92.3% and 55.7% respectively and the manual Qiagen had a 39.6% recovery with e-cell DNA and a 17.3% recovery with sperm cell DNA. A study was also performed to determine the optimum working conditions for the EZ1 method. An elution volume and incubation time study with the EZ1 method was performed and it was determined that the three elution volumes (50 µL, 100 µL and 200 µL) tested did not affect the percent recovery adversely. The different incubation times tested (3, 5 and 10 hours) did not affect the percent recovery of e-cells significantly, however, there was a downward trend in recovery as the incubation times increased. A digest volume study was also performed with the ZyGEM method which resulted in higher percent e-cell recoveries generated for the 100 µL digest volume when compared to 20 µL. All three extraction methods generated similar results in a refined dilution study which showed that when lower concentrations of DNA were extracted, the percent recovery was higher in comparison to higher concentrations of DNA being extracted. This aspect is very important as most forensic DNA samples are low in concentration which makes it important that these extraction methods are able to extract very low concentrations of DNA efficiently. The cost, ease of use and analysis time was also evaluated for all three methods and it was concluded that the ZyGEM/Acrosolv method was overall the best extraction method to use in a forensic DNA laboratory. This is due to the one-tube hands-off characteristic of the ZyGEM/Acrosolv method. Because of this feature, the ZyGEM/Acrosolv method is easier to use, faster and more reliable than the other extraction methods. It also has the least amount of analyst interaction so the samples should be more consistent. During this study, the ZyGEM/Acrosolv protocol had higher and more consistent percent recoveries with both sperm and e-cells but there was a downward trend in recovery as the amount of DNA increased. This downward trend was seen more prominently with ZyGEM/Acrosolv than with the EZ1 and manual Qiagen methods. Electropherograms were also produced with selected EZ1 and ZyGEM/Acrosolv samples and both methods produced accurate and reliable profiles but the EZ1 method had less variability in peak heights in comparison to ZyGEM/Acrosolv. Overall, both the ZyGEM/Acrosolv and the EZ1 extraction methods would be good procedures to use within a forensic DNA laboratory. The manual Qiagen extraction method generated very low percentage recoveries and had more variation when compared to the other two methods, therefore, it would not be recommended to use this method within a forensic laboratory today. The EZ1 BioRobot is currently used in many forensic laboratories and produces reliable results but this study proved that the ZyGEM/Acrosolv method is overall a better technique in all aspects tested in this study and should follow European laboratories and be implemented in laboratories within the United States.