@2024 Afarand., IRAN

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ISSN: 2383-3483    Journal of Police Medicine 2018;7(4):167-172

Background
DNA Extraction from forensic samples is the first and one of the most crucial steps of any genetic forensic tests.
Previous Studies
… [1-4]. Generally, DNA quality is measured by factors such as no contamination caused by RNA, lipid, and other structures that interact with restriction enzymes and polymerases, as well as time and cost [5, 6]. Due to the large size of the DNA molecule, extraction techniques should have the minimum level of mechanical stress through the extraction processes. Accordingly, the methods used to purify DNA, in addition to be time-consuming and fast, should be safe to remove inhibitors and purify all nucleic acids. White blood cells are used to purify DNA from blood samples. Several techniques are used to purify DNA, including phenol-chloroform extraction, proteinase K extraction, boiling, acid guanidinium thiocyanate-phenol-chloroform extraction (AGPC), and various types of kits [7-10].
Aim(s)
The present study aimed at achieving a suitable, faster, and low-cost method of DNA extraction from different samples for further molecular studies in criminal sciences and forensic application.
Research type
Blood samples were collected from different animals, including mice, dogs and cats by the vet from a pet hospital
Research society, place and time
None declared.
Sampling method and number
In this study, human and animal blood samples were used for DNA extraction. 1-2 cc of blood were collected from the animals, and were poured in special tubes with and without anticoagulants. The samples were divided into several parts after transferring to the lab. Some samples were stored at -20, -30 and -70°C for DNA extraction at 1, 2, and 3 weeks as well as one-month. Human blood samples were also collected from laboratory staff and were stored like animal samples, at different temperatures and at the considered times. In addition, 5 μl of the human and animal samples were poured on cotton cloths. The samples were dried and stored at room temperature. DNA was extracted at 1, 2, and 3 weeks as well as one-month from these samples, as well.
Used devices&materials
DNA extraction was performed using all blood samples using three different methods, as follows: 1) Non-enzymatic DNA extraction 2) Chelex DNA extraction 3) Modified rapid alkaline lysis Determination of the concentration and purity of DNA Following DNA extraction and drying, all samples were dissolved in a certain amount of Tris-EDTA (TE) buffer, and the appropriate dilutions were prepared and their concentration was measured using a spectrophotometer. TE was first used as a Blank solution for calibration. Nano Drop apparatus at the wavelengths of 260 and 280 nm was used to measure the concentration of extracted samples. Nano Drop was cleaned with alcohol, and according to the DNA extraction method, distilled water or TE buffer was used as a control buffer to reset the apparatus. 1 µL of the extracted sample was placed on Nano Drop and its concentration was evaluated at the wavelengths 260 and 280 nm. Determination of the quality of extracted DNA 16S rRNA gene polymerase chain reaction (PCR) was used for amplification of a 300-bp fragment on the samples in order to ensure that all extracted samples contained a suitable and high quality DNA for using in molecular reactions including PCR. The sequence of primers used in the PCR reaction was as follows (Table 1): Forw L16S 300: 5′-GCCTGTTTACCAAAAACATCAC-3′ Revs H16S 300: 5′-CTCCATAGGGTCTTCTCGTCTT-3′ When the reaction was completed, the tubes were immediately transferred to the place with 4°C temperature and prepared for electrophoresis. The resulted product of the PCR reaction is a 300-bp fragment, which can be seen in each sample, if DNA is extracted. After PCR, PCR products were observed on 1.5% agarose gel.
Findings by Text
The common 16S rRNA gene was replicated in all studied samples (Fig. 1). A low quantity of DNA was extracted from the samples that were kept at room temperature for a longer time compared with other samples. The highest optical density (OD) and DNA concentration were obtained using non-enzymatic method. The efficiency and quality of the PCR were approximately equal in all three methods. In terms of time, the modified Alkalin method was more time-consuming than other methods, so it can be performed in less than 20 min. Furthermore, it was more convenient and simple than the other two methods, so that it can be done with the simplest tools in each laboratory.
Main comparison to the similar studies
DNA extraction with an appropriate quality, purity, level of contamination, etc., for molecular objectives is the starting point for many molecular and diagnostic biological methods. On the other hand, DNA extraction methods should have the least mechanical stress for the sample through the extraction process and possibly have fewer stages, so that they are time-consuming and have a low level of contamination [20-22]. As it is known, there are very different methods and kits for DNA extraction from the samples for various objectives, such as criminal sciences [23-25], which can be used according to the available equipment and conditions. Identification of the different methods of DNA extraction from the samples for various reasons is crucial and necessary. Accordingly, the investigators, based on the equipment and conditions available in the forensic science laboratories, especially those which are located in deprived areas with no easy access to DNA extraction kits, by understanding these techniques and using the simplest equipment, can extract DNA from the samples discovered from the crime scene.
Suggestions
It can be said that DNA extraction using modified alkaline is one of the easiest, cheapest and fastest techniques to extract DNA. A DNA with a good quality can be extracted by this method which can be used for PCR tasks.
Limitations
Chelex or the modified Alkalin extraction methods do not have the stage for complete purification; however, DNA can be extracted from the samples in the shortest possible time and with the least materials.
Conclusions
Given the ease of access to materials used in each of these methods, as well as their cost-effectiveness in terms of time and cost, these methods can be an appropriate alternative to DNA extraction kits.
Acknowledgments
Authors are thankful to those who helped us to perform this study, especially Dr. Sadegh Hassannia, a faculty member at Tarbiat Modares University.
Conflict of interest
None declared.
Ethical Permissions
None declared.
Funding Sources
This research was supported by the Research Institute for Police Sciences.

TABLES and CHARTS

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