preparation of necrophilic fly samples for dna extraction

הערכת טוהר וריכוז DNA בדגימות זבובים נקרופיליים טריים וישנים

The inference of the time of death has always been one of the key and difficult issues to be resolved in judicial practice. In the practice of forensic science, for a criminal case, determining postmortem interval plays a crucial role in deducing the time of the crime, locking in the suspect, and narrowing the scope of the investigation. Traditional methods of inferring the time of death are based primarily on early postmortem phenomena, which can generally only be used to infer the time of death within 24 h. However, the long time of death cannot be determined by postmortem phenomena. It is now generally recognized in the forensic science community that forensic entomology has the potential to be an effective method of inferring a longer time of death.
Necrophagous insects are named for their larvae that feed on corpses. Among them, whenever a corpse is present, the adult necrophilous flies will be the first to reach the corpse and lay their eggs or larvae. The larvae feed on corpse tissue and mature into pupae, which fledge into adults. Necrophilic flies play a huge role in the practice of forensic science because of their regular life cycle and geographic distribution, for example, deducting the time of death and determining the place of death1,2. However, the barrier to the use of necrophilous flies in forensic practice is species identification. Morphological methods are still used as the authoritative method for species identification of necrophilous flies. However, morphological species identification requires a high degree of insect integrity. If the flies were in different developmental stages, or due to morphological changes caused by environmental choices, those all make morphological examination more difficult. In particular, the morphology of pupae and pupal shells, which are most often found at the crime scene, is barely recognizable. This, together with the scarcity of morphological experts, makes huge difficulty to the identification of species morphologically. Therefore, the application of molecular biology methods for species identification of flies has emerged, which reduces the difficulty of morphological identification and is independent of developmental stage3,4,5,6,7.
The first step in species identification of necrophilous flies based on molecular biology methods is the efficient extraction of DNA, while specialized kits for insect DNA extraction are not commonly available currently. And how to use common blood kits for DNA extraction of necrophilic flies becomes more forensically relevant. Necrophilic flies found at crime scenes are often mutilated or have undergone decay and DNA degradation. Fly samples are not immediately available for DNA extraction after acquisition, or if possible, complete samples need to be retained due to evidence preservation requirements. Based on the above requirements, in this study, we applied the blood DNA kit based on proteinase K digestion, and selected three fresh Chrysomya megacephala (Diptera, Lepidoptera) samples (Figure 1), one old C. megacephala sample placed in alcohol for two years, and one C. megacephala sample placed in light-protected storage for two years, weighed each of the five samples, elected the insect thorax tissue for DNA extraction. Comparing the quality and purity of DNA extracted from the old and new samples, PCR amplification and electrophoresis of the extracted DNA were performed with necrophilic fly-specific primers located in the mitochondrial CO <img alt="Equation 1" src="/files/ftp_upload/66737/66737eq01v2.jpg" style="margin: auto;" /> gene sequence.

מחבר זרקור: קידום מיצוי DNA מדגימות זבוב נקרופילי באמצעות טכניקה פשוטה

מיצוי DNA והשוואה בין דגימות זבוב נקרופילי ישן וטרי

Our particle aims to extract fly DNA simply and conveniently, and compare the DNA concentrations between new and old samples of necrophilic flies. We're trying investigating whether blood DNA extraction kits effectively extract DNA from fly, and is there a change between DNA concentrations from new and old sample of necrophilic flies? We provided a method for extracting fly DNA using ordinary blood test kits, which solved the problem of expensive and scarce insect extraction kits, and paved the way for molecular biology analysis of these sets.After solving the problem of extracting fly DNA, necrophilic flies to avert the time of death.

הכנת דגימות זבוב נקרופילי למיצוי DNA

preparation-of-necrophilic-fly-samples-for-dna-extraction

Research

JoVE Journal

Medicine

Preparation of Necrophilic Fly Samples for DNA Extraction

6 Views. כדי להתחיל, להקריב זבובים עם אתיל אצטט, ולשים את הדגימות בשלושה בקבוקוני גז. שקלו את הדגימות, ותייגו אותן כטריות אחת, שתיים ושלוש. לאחר מכן, שלפו דגימות בנות שנתיים המאוחסנות בצינורות צנטריפוגות מלאות אלכוהול, ספגו נוזל משטח עם נייר סינון ושקלו את הדגימות.

Video: הכנת דגימות זבוב נקרופילי למיצוי DNA

DNA Extraction and Comparison Between Old and Fresh Necrophilic Fly Samples

A total of five samples of Chrysomya megacephala samples - three fresh samples, one sample stored in alcohol for 2 years, and one sample stored in dry sealed storage for 2 years protected from light only - were selected to investigate whether a blood DNA extraction kit could extract DNA from necrophilous flies and to determine whether alcohol could prolong the preservation of necrophilous flies&#39; DNA. First, the blood DNA extraction kit was used to extract DNA from their thorax tissues. Then, the DNA purity and concentration were examined using a microplate reader and a fluorometer. Finally, PCR amplification and electrophoresis of the extracted DNA were done with necrophilic fly-specific primers located in the mitochondrial CO I gene sequence. The results showed that the DNA purity of all samples was greater than 2.0. The DNA concentration was observed to be of the following order: fresh samples &#62; alcohol-preserved old samples &#62; untreated, old samples. All samples had specific electrophoretic bands after PCR amplification. In conclusion, a blood DNA extraction kit can be used to extract DNA from necrophilic flies successfully, and the DNA concentration of fresh fly samples is greater than that of old fly samples. The flies can be stored in alcohol for a long time.

This article describes a protocol for fresh and old necrophilic fly DNA extraction using a modified common DNA extraction kit.

A total of five samples of Chrysomya megacephala samples - three fresh samples, one sample stored in alcohol for 2 years, and one sample stored in dry ...

To begin, sacrifice flies with ethyl acetate, and place the samples in three gas vials. Weigh the samples, and label them as fresh one, two, and three. Next, retrieve 2-year-old samples stored in alcohol filled centrifuge tubes, and absorb surface liquid with filter paper and weigh the samples.Also, retrieve 2-year-old samples stored in centrifuge tubes and weigh them. Then, add a small amount of liquid nitrogen to a stainless steel container, and wait until all liquid nitrogen has evaporated. Place a fly sample in the container, and slowly pour liquid nitrogen.Freeze the samples in liquid nitrogen, and remove them after the liquid nitrogen evaporates. Separate the thorax individually, followed by slicing each thorax. Place the slices in a two milliliter centrifuge tube.

DNA Extraction from Necrophilic Fly Samples

To begin place the clipped fly thorax samples in a two-milliliter centrifuge tube. Add 180 microliters of tissue lysis buffer to the tube, followed by 20 microliters of Proteinase K.Vortex thoroughly at 3, 000 RPM for 10 seconds and incubate at 56 degrees Celsius until tissues are lysed. After incubation, vortex the samples again for 15 seconds.Add 200 microliters of lysis buffer, and mix thoroughly by vortexing. Then add 200 microliters of ethanol and vortex again for 15 seconds. Next, place a DNA absorption column in a two-milliliter collection tube and pipette the mixture into the column.Centrifuge at 6, 000 G for one minute. Discard flow through and the collection tube. Place the column in a new tube and add 500 microliters of protein removal buffer.Centrifuge for one minute and discard the flow through. Transfer the column to a new tube. Add 500 microliters of desalination buffer and centrifuge at 20, 000 G for three minutes to dry the membrane.Then add 100 microliters of DNA elution buffer onto the membrane. Incubate a room temperature for one minute and then centrifuge at 6, 000 G for one minute to elute the DNA. Store the DNA samples as appropriate.

מיצוי DNA מדגימות זבוב נקרופילי

Qualitative and Quantitative Analysis of DNA Extracted from Fly Samples

To begin, extract DNA from fly samples using a DNA extraction kit. Then use a microplate reader to determine the values of OD260 and OD280 to compare DNA purity and nucleic acid concentration. Next, prepare fluorometer standard solutions by adding 190 microliters of working solution to two 0.5 milliliters centrifuge tubes.Then add 10 microliters each of standard one and standard two to the working solutions. Keep the tubes away from light for at least 15 minutes. Mix two microliters of test DNA with 198 microliters of working solution in a 0.5 milliliter centrifuge tube and keep the mixture in the dark for at least 15 minutes.After 15 minutes, turn on the fluorometer and select the dsDNA concentration measurement, long range setting. Test standard one and standard two to obtain the standard curve. Then place the samples to be tested in the assay wells.Adjust the DNA spiking volume to two microliters and perform the measurement. To visualize the DNA first set up 20 microliters of the PCR amplification reaction. Place the tubes in a thermal cycler and run the PCR cycles.After PCR, subject the products to 2%agarose gel electrophoresis. Then place the gel on a gel imaging system for photo analysis. Assessment of extracted DNA purity by Microplate reader showed that all fresh samples and old sample one had OD260 to OD280 ratios above two.In contrast, old sample two displayed a lower ratio. DNA concentration derived from OD260 readings was highest in fresh samples, followed by old sample one and lowest in old sample two. Electrophoretic analysis revealed bands in the range of 250 to 400 base pairs, which were more pronounced in fresh samples, compared to old ones.