Name: Diatom DNA Extraction from Water Sample and Human Lung Tissue Using a Modified DNA Extraction Kit
Uploaded: 2023-11-10T13:10:00
Description: Watch this Scientific Journal Video about Diatom DNA Extraction from Water Sample and Human Lung Tissue Using a Modified DNA Extraction Kit at JoVE.com

diatom dna extraction from water sample and human lung tissue using a modified dna extraction kit

Modifiye edilmiş bir kit kullanarak ekonomik diatom DNA ekstraksiyonu

In forensic practice, determining whether a corpse found in the water drowning or was thrown into the water after death is essential for the proper resolution of the case1. It is also one of the difficult issues that need to be solved urgently in forensic practice2. Diatoms are abundant in the natural environment (especially in water)3,4. In the process of drowning, due to hypoxia and stress response, people will have intense breathing movements and inhale a large amount of drowning liquid. Therefore, the diatoms in the water enter the lung with the drowning liquid, and some diatoms can enter the blood circulation through the alveolar-capillary barrier and spread to internal organs with the blood flow5,6. The detection of diatoms in internal tissues and organs such as lung, liver, and bone marrow is a strong evidence of drowning before death7,8. Currently, forensic diatom testing is mainly based on morphological testing methods. After a series of pre-digestion of the tissue, the morphological qualitative and quantitative estimations of undigested diatoms are carried out under the microscope. During this period, dangerous and environmentally unfriendly reagents such as nitric acid need to be used. This process is time-consuming and requires researchers to have solid taxonomic expertise and extensive experience. These all bring certain challenges to the forensic staff9. Diatom DNA testing technology is a new technology for diatom testing developed in recent years10,11,12. This technology realizes the species identification of diatoms by analyzing the specific DNA sequence composition of diatoms13,14. PCR technology and sequencing technology are commonly used technical methods, but their basis is the successful extraction of DNA from diatoms. However, diatoms have a special structure different from other organisms, making their DNA extraction techniques also different.
The cell wall of diatom has a high degree of silicification, and its main component is silicon dioxide15,16,17. The siliceous cell wall is very hard, and it must be destroyed before extract the DNA. Ordinary DNA extraction kits are often difficult to use directly for the extraction of diatom DNA because they cannot destroy the siliceous shell of diatoms18. Therefore, destroying the siliceous shell of diatoms is one of the key technical problems to be solved in extracting diatom DNA.
At the same time, since the number of diatoms contained in forensic research samples, whether water samples or organs and tissues of drowned bodies, is often limited, it is necessary to enrich diatoms. The essence of enrichment is the separation of substances. While trying to gather diatoms together, minimize the content of other material components (interfering components). In forensic work, laboratories often use centrifugation or membrane filtration enrichment methods to separate diatom cells19. However, since vacuum pumping equipment is not widely used, the membrane enrichment method is not often used in ordinary primary forensic laboratories. So, the centrifugation method is still commonly diatom enrichment in forensic laboratories20.
DNA extraction from diatoms is currently used primarily in forensic practice, and there are significant limitations to its application. At present, there are few diatom DNA extraction kits used in forensic science on the market and they are generally expensive21. This article provides an improved diatom DNA extraction method, making diatom DNA extraction simple, convenient and cost effective. This increases the application of subsequent molecular biology testing of diatoms and can better solve problems related to drowning in forensic medicine through diatom testing. This method breaks the siliceous cell walls of diatoms by adding glass beads and setting an appropriate time for vortex. In this way, the proteinase K and the binding solution rapidly lyse the cells and inactivate various enzymes in the cells. The genomic DNA is absorbed in the matrix membrane in the adsorption column and finally eluted by the elution buffer. Such an improved whole blood gene extraction kit improves the diatom DNA extraction effect of the blood kit in forensic examination materials, reduces the cost of diatom DNA extraction in forensic practice, and can be better applied to grassroot forensic research.

Yazar Spotlight: Adli Boğulma Muayenesi için Diatom Testi 

Su Örneklerinden ve Dokulardan Diatom DNA'nın Ekstraksiyonu

Our research mainly focus on the topic of drowning. We wanted to answer the question of whether the body found in the water died from drowning, as well to answer another question of where the person fell into the water. That means to know the location of the drowning.Compared with other technologies, we provide a simple, convenient, and economical diatom DNA extraction protocol that we can use better in forensic research, especially in basic forensic practice. Our laboratory will further focus on the drowning related problems based on the pattern in the water, such as reason of death, post mortem interval and so on. What's more, we also carry on the research through the forensic entomology and a some special phenomenons in the tropical forensic medicines.

Watch this Scientific Journal Video about Diatom DNA Extraction from Water Sample and Human Lung Tissue Using a Modified DNA Extraction Kit at JoVE.com

Diatom DNA Extraction from Water Sample and Human Lung Tissue Using a Modified DNA Extraction Kit  

Modifiye DNA Ekstraksiyon Kiti Kullanılarak Su Numunesi ve İnsan Akciğer Dokusundan Diatom DNA Ekstraksiyonu

Başlamak için, bir santrifüj tüpüne 10 mililitre diatom su numunesi alın. Tüpü beş dakika boyunca 13.400 G'de santrifüjleyin. Bir pipet tabancasıyla, süpernatanın 9.8 mililitresini dikkatlice atın.Daha sonra 200 mikrolitre zenginleştirilmiş diatomlu su örneğini iki mililitrelik bir santrifüj tüpüne aktarın. Daha sonra, boğulmuş bir insan vücudundan 0.5 gram akciğer marjı dokusu alın. Makasla akciğer dokusunu çamurlu hale gelene kadar kesin.Hem diatom hem de akciğer örneklerinden DNA ekstraksiyonu için, önce her iki örneğe de cam boncuklar ekleyin. Numuneleri iyice karıştırdıktan sonra, tüplere 40 mikrolitre proteinaz K ekleyin. Oda sıcaklığında 15 dakikalık bir inkübasyondan sonra, her iki tüpe 200 mikrolitre bağlayıcı tampon ekleyin.Numuneleri hemen dört dakika boyunca bir vorteks karıştırıcı ile karıştırın. Ardından tüpleri 10 dakika boyunca 70 santigrat derecede bir su banyosuna yerleştirin. çözüm netleşene kadar.Daha sonra, berrak çözeltiyi topaklaştırıcı çökelti ile aktarın. silikon matris ile paketlenmiş üç santimetre uzunluğunda bir adsorpsiyon kolonuna. Kolonu 30 saniye boyunca 8.000 G'de santrifüjleyin.Ardından atık sıvıyı toplama tüpüne atın ve kolonu tekrar toplama tüpüne koyun. Şimdi kolona 500 mikrolitre inhibitör çıkarma tamponu ekleyin ve 30 saniye boyunca 13.400 G'de santrifüjleyin. Atık sıvıyı attıktan sonra, kolona 700 mikrolitre yıkama tamponu ekleyin ve tekrar santrifüjleyin.Atık sıvıyı attıktan sonra, adsorpsiyon kolonunu tekrar boş toplama tüpüne yerleştirin. Mümkün olduğunca fazla yıkama tamponunu çıkarmak için sütunu iki dakika boyunca 14.500 G'de santrifüjleyin. Şimdi sütunu çıkarın ve temiz bir santrifüj tüpüne yerleştirin.Adsorpsiyon membranının orta kısmına 100 mikrolitre elüsyon tamponu ekleyin. Kolonu oda sıcaklığında beş dakikalık bir inkübasyondan sonra bir dakika boyunca 13.400 G'de santrifüjleyin. Toplama tüpünde elde edilen çözeltiyi tekrar santrifüjlemeden önce adsorpsiyon kolonuna geri aktarın.Ayrıştırılmış diatom DNA'sını ileride kullanmak üzere iki ila sekiz santigrat derecede saklayın. Agaroz jel elektroforezi, PCR amplifikasyonundan sonra hem su örneklerinde hem de dokularda 250 ila 500 BP arasında diatom DNA bantları gösterdi.

Research

JoVE Journal

Medicine

Diatom DNA Extraction from Water Sample and Human Lung Tissue Using a Modified DNA Extraction Kit

Extraction of Diatom DNA from Water Samples and Tissues

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. ...