Name: extraction of diatom dna from water samples and tissues
Uploaded: 2023-11-10T13:10:00.000+00:00
Duration: 4 min 20 s
Description: Watch this Scientific Journal Video about Diatom Testing for Forensic Drowning Examination at JoVE.com

This work is supported by the National Natural Science Foundation of China (82060341,81560304) and by the Academician Innovation Platform Scientific Research Project of Hainan Province (YSPTZX202134).

This study was approved by the Ethics Committee of Hainan Medical University. The tissue samples used in this study are not considered to be studies involving human subjects. These specimens were obtained for the purpose of forensic pathological diagnosis, and the rest were used for the extraction of diatom DNA in this experiment. Researchers cannot readily identify individuals to obtain informed consent from relevant stakeholders.
NOTE: To ensure the general applicability of the research method reported in this experiment, this experiment basically followed the operating instructions of the kit used, and only some steps were modified. The water samples used in this experiment were randomly taken from ponds near the laboratory (Supplementary Figure 1A). In this experiment, the lung tissue of the drowned body was confirmed as the research tissue to demonstrate the extraction protocol (Supplementary Figure 1B). In forensic practice, it is also sometimes necessary to use other organs and tissues of drowned bodies (such as liver, spleen, kidney, bone marrow, etc.) to extract diatoms DNA, which requires minor corresponding improvements to this experimental method, which will be explained in the corresponding section of the experiment. The lung tissue samples used in this experiment came from corpses that were clearly drowned in forensic cases. Morphological tests have been conducted to prove that the lung tissue contains diatoms (Supplementary&#160;Figure 2).
1. Pretreatment of samples
<ol>
	<li>Pretreatment of water samples
	<ol>
		<li>Take 10 mL of water sample into the centrifuge tube and centrifuge at 13,400 x g for 5 min. Carefully discard 9.8 mL of supernatant with a pipette gun, and transfer about 200 &#181;L of enriched diatom water sample remaining at the bottom into a 2 mL centrifuge tube. 
		NOTE: Pre-experiment can be carried out first, and the initial amount can be increased if a 10 mL water sample is not enough for enrichment.</li>
	</ol>
	</li>
	<li>Pretreatment of tissue samples
	<ol>
		<li>Take 0.5 g of the lung margin tissue from the drowned body, fully chop or grind the lung tissue until it becomes muddy. Preventing contamination of exogenous diatoms is the core of this step. Cut the tissue into pieces repeatedly using scissors.</li>
	</ol>
	</li>
</ol>
2. DNA extraction
NOTE: All centrifugation steps are completed at room temperature. Using a desktop centrifuge with a centrifugal force of 14,500 x g; a water bath (or metal bath) preheated to 70 &#176;C needs to be prepared before the experiment starts. All steps must strictly follow the principles of aseptic operation.
<ol>
	<li>Assembly of water samples and tissues 
	NOTE: Water sample and tissue diatom DNA extraction method are the same.
	<ol>
		<li>Add glass beads to a 2 mL centrifuge tube containing the pretreated water sample. Invert 10x-15x to mix well. The added glass beads are composed of large and small glass beads mixed in a mass ratio of 1:1. The diameter of large glass beads is 1.5-2.0 mm and of the small glass beads is 0.4-0.6 mm.</li>
		<li>Take 0.5 g of finely chopped tissue, add glass beads to a 2 mL centrifuge tube containing the tissue as described above. Invert 10x-15x to mix.</li>
	</ol>
	</li>
	<li>Add 40 &#181;L of proteinase K (20 mg/mL) to the tubes. Place at room temperature for 15 min, during this period, invert, and mix 10x every 3 min. 
	NOTE: If the tissue is not fully digested, the amount of proteinase K can be appropriately increased until the solution becomes clear.</li>
	<li>Add 200 &#181;L of binding buffer to the centrifuge tubes, immediately vortex, and mix for 4 min (oscillation mixing frequency: 3000 rpm). 
	NOTE: In this step, the shaking intensity and time should be strictly controlled to ensure sufficient mixing intensity and time.</li>
	<li>Put the centrifuge tubes in a 70 &#176;C water bath for 10 min, and the solution becomes clear.</li>
	<li>Add 100 &#181;L of isopropanol to the centrifuge tubes, vortex and mix for 15 s, flocculent precipitation may appear at this time. 
	NOTE: It is very important to mix well with appropriate strength in the above operation steps, but vigorous shaking should be avoided to prevent DNA shearing.</li>
	<li>Put the solution obtained in the previous step together with the flocculent precipitate into the adsorption column (the adsorption column is placed in the collection tube). The adsorption column length is 3.0 cm, the diameter is 1.0 cm, and the adsorption matrix is silicon matrix membrane.</li>
	<li>Centrifuge at 8,000 x g for 30 s, discard the waste liquid in the collection tube and put the adsorption column back into the collection tube.</li>
	<li>Add 500 &#181;L of inhibitor removal buffer to the adsorption column. Centrifuge at 13,400 x g for 30 s and discard the waste liquid in the collection tube.</li>
	<li>Add 700 &#181;L of washing buffer to the adsorption column. Centrifuge at 13,400 x g for 30 s and discard the waste liquid in the collection tube. 
	NOTE: Add the specified amount of absolute ethanol to the wash buffer bottle before first use.</li>
	<li>Add 500 &#181;L of wash buffer to the adsorption column. Centrifuge at 13,400 x g for 30 s and discard the waste liquid in the collection tube.</li>
	<li>Put the adsorption column back into the empty collection tube. Centrifuge at 14,500 x g for 2 min, and remove the washing buffer as much as possible, to avoid the residual ethanol in the washing buffer from inhibiting downstream reactions.</li>
	<li>Take out the adsorption column and put it into a clean centrifuge tube. Add 100 &#181;L of elution buffer to the middle part of the adsorption membrane.</li>
	<li>Place the adsorption column at room temperature for 3-5 min, and centrifuge at 13,400 x g for 1 min.</li>
	<li>Add the solution obtained in the previous step to the centrifugal adsorption column again. Place the centrifugal adsorption column at room temperature for 2 min, and centrifuge at 13,400 x g for 1 min. 
	NOTE: The elution buffer should be preheated in a 70 &#176;C water bath for about 10 min. The elution volume should not be less than 50 &#181;L; otherwise, the DNA yield will be reduced.</li>
	<li>Store the extracted diatom DNA at 2-8 &#176;C for future use. If the DNA solution is to be stored for a long time, store it at -20 &#176;C.</li>
</ol>
3. PCR test
NOTE: Since the content of diatoms in forensic samples is often low, the tissue sample extracts of the drowned bodies may also contain varying degrees of tissue and organs (such as the lungs in this experiment) with their own DNA. Therefore, direct detection of total DNA in DNA extracts does not reflect the situation of diatom DNA extraction. In this experiment, diatom-specific primers were selected, and PCR products were used to evaluate the diatom DNA extraction in the extract. The products can be observed and analyzed by agarose gel electrophoresis and can also be analyzed by real-time fluorescent quantitative PCR-melting curve, which has higher sensitivity.
<ol>
	<li>Add 2 &#181;L of the extracted DNA from water samples and tissues as the template. Choose one of the following two methods for inspection.</li>
	<li>Conventional PCR test
	<ol>
		<li>Use primers22 that can specifically amplify diatom 18S rDNA fragments. For details, see Table 1.</li>
		<li>Establish the PCR reaction system and amplification conditions according to the characteristics of the primers. For details, see Table 2.</li>
		<li>Run the products of PCR amplification on 2% agarose gel. Observe and analyze the imaging with a gel imager.</li>
	</ol>
	</li>
	<li>Fluorescent quantitative PCR test
	<ol>
		<li>Use the above primers that can specifically amplify diatom 18S rDNA fragments to prepare a real-time fluorescent quantitative PCR reaction system. For details, see Table 3.</li>
		<li>Perform PCR amplification and analyze the obtained amplification curve and Ct values. At the same time, set a program, melt the double-strands of the amplified products into single-strands gradually through fluorescent quantitative PCR-melting curve technology, and then analyze the obtained melting curves.</li>
	</ol>
	</li>
</ol>

Economical Diatom DNA Extraction Using a Modified Kit

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The authors declare that they have no competing financial interests.

Diatom cells are protected by hard siliceous cell walls17, and this structure must be destroyed to extract diatom DNA. Ordinary kits do not easily destroy the siliceous shell of diatoms; thus it is difficult to successfully extract diatom DNA21. Our laboratory improved the most commonly used blood DNA extraction kit, adding glass beads of different diameters and different mass ratio in the process of diatom extraction. Vortex oscillation is carried out at the same time, whi...

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.

<table><tbody><tr><td>Binding Buffer</td><td>BioTeke</td><td>B010006022</td><td>rapidly lysing cells</td></tr><tr><td>ChemoHS qPCR Mix</td><td>Monad</td><td>00007547-120506</td><td>qPCR Mix</td></tr><tr><td>D2000 DNA ladder</td><td>Real-Times(Beijing) Biotechnology</td><td>RTM415</td><td>Measure the position of electrophoretic bands</td></tr><tr><td>D512</td><td>Taihe Biotechnology</td><td>TW21109196</td><td>forword primer</td></tr><tr><td>D978</td><td>Taihe Biotechnology</td><td>TW21109197</td><td>reverse primer</td></tr><tr><td>Elution buffer</td><td>BioTeke</td><td>B010006022</td><td>A low-salt elution buffer washes off the DNA</td></tr><tr><td>Glass bead</td><td>Yingxu Chemical Machinery(Shanghai)&amp;nbsp;</td><td>70181000</td><td>Special glass beads for dispersing and grinding</td></tr><tr><td>Import adsorption column</td><td>BioTeke</td><td>B2008006022</td><td>Adsorption column with silica matrix membrane</td></tr><tr><td>Inhibitor Removal Buffer</td><td>BioTeke</td><td>B010006022</td><td>Removal of Inhibitors in DNA Extraction</td></tr><tr><td>Isopropanol</td><td>BioTeke</td><td>B010006022</td><td>Precipitate or isolate DNA</td></tr><tr><td>MIX-30S Mini Mixer</td><td>Miulab</td><td>MUC881206</td><td>oscillatory action</td></tr><tr><td>Proteinase K</td><td>BioTeke</td><td>B010006022</td><td>Inactivation of intracellular nucleases and other proteins</td></tr><tr><td>Rotor-Gene Q 5plex HRM</td><td>Qiagen</td><td>R1116175</td><td>real-time fluorescence quantification PCR</td></tr><tr><td>Speed Micro-Centrifuge</td><td>Scilogex</td><td>9013001121</td><td>centrifuge</td></tr><tr><td>Tanon 3500R Gel Imager</td><td>Tanon</td><td>16T5553R-455</td><td>gel imaging</td></tr><tr><td>Taq Mix Pro</td><td>Monad</td><td>00007808-140534</td><td>PCR Mix</td></tr><tr><td>Thermo Cycler</td><td>Zhuhai Hema</td><td>VRB020A</td><td>ordinary PCR</td></tr><tr><td>Wash Buffer</td><td>BioTeke</td><td>B010006022</td><td>Remove impurities such as cell metabolites</td></tr></tbody></table>

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. Diatom testing is also an important research content in the field of the environment and plankton. The diatom molecular biology testing technology, which focuses on diatom DNA as the primary research object, is a new method of diatom testing. Diatom DNA extraction is the basis of diatom molecular testing. At present, the kits commonly used for diatom DNA extraction are expensive, which increases the cost of carrying out related research. Our laboratory improved the general whole blood genomic DNA rapid extraction kit and obtained a satisfactory diatom DNA extraction effect, thus providing an alternative economical and affordable DNA extraction solution based on glass beads for related research. The diatom DNA extracted using this protocol could satisfy many downstream applications, such as PCR and sequencing.

Since the DNA solution extracted by the currently used DNA extraction method contains all DNA components from different sources in the sample, the DNA obtained by this protocol was no exception. So, the DNA solution was not just a solution of diatom genomic DNA. The primers that can specifically amplify diatom 18S rDNA fragments were selected by consulting the literature22,23,24. The primers were verified by NCBI-Blast Primer, a...

Watch this Scientific Journal Video about Diatom Testing for Forensic Drowning Examination at JoVE.com

Author Spotlight: Diatom Testing for Forensic Drowning Examination 

This article describes a protocol for diatom DNA extraction using a modified common 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. ...

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Research

JoVE Journal

Medicine

1.8K Views.  Hainan Medical University, Hainan Provincial Academician Workstation (tropical forensic medicine). This article describes a protocol for diatom DNA extraction using a modified common DNA extraction kit.

Video: Author Spotlight: Diatom Testing for Forensic Drowning Examination 

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

Environment

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Capture of Environmental DNA (eDNA) from Water Samples by Flocculation

The analysis of environmental DNA (eDNA) has become a widely used approach to problem solving in species management. The detection of cryptic species including invasive and (or) species at risk is the goal, typically accomplished by testing water and sediment for the presence of characteristic DNA signatures. Reliable and efficient procedures for the capture of eDNA are required, especially those that can be performed easily in the field by personnel with limited training and citizen scientists. The capture of eDNA using membrane filtration is widely used currently. This approach has inherent issues that include the choice of filter material and porosity, filter fouling, and time required on site for the process to be performed. Flocculation offers an alternative that can be easily implemented and applied to sampling regimes that strive to cover broad territories in limited time.

Capture of Environmental DNA eDNA from Water Samples by Flocculation

Environmental DNA (eDNA) is typically captured from water samples using alcohol precipitation, filtration, or flocculation. Presented here is an alternative protocol that uses lanthanum chloride to enable collection of dissolved and particulate-bound nucleic acids from water samples containing eukaryotic cells, prokaryotic cells, and virus.

The analysis of environmental DNA (eDNA) has become a widely used approach to problem solving in species management. The detection of cryptic species ...

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...

Biology

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Visualizing Methane-Cycling Microbial Dynamics in Coastal Wetlands

Coastal wetlands are the largest biotic source of methane, where methanogens convert organic matter into methane and methanotrophs oxidize methane, thus playing a critical role in regulating the methane cycle. The wetlands in South Texas, which are subject to frequent weather events, fluctuating salinity levels, and anthropogenic activities due to climate change, influence methane cycling. Despite the ecological importance of these processes, methane cycling in South Texas coastal wetlands remains insufficiently explored. To address this gap, we developed and optimized a method for detecting genes related to methanogens and methanotrophs, including mcrA as a biomarker for methanogens and pmoA1, pmoA2, and mmoX as biomarkers for methanotrophs. Additionally, this study aimed to visualize the spatial and temporal distribution patterns of methanogen and methanotroph abundance utilizing the geographic information system (GIS) software ArcGIS Pro. The integration of these molecular techniques with advanced geospatial visualization provided critical insights into the spatial and temporal distribution of methanogen and methanotroph communities across South Texas wetlands. Thus, the methodology established in this study offers a robust framework for mapping microbial dynamics in wetlands, enhancing our understanding of methane cycling under varying environmental conditions, and supporting broader ecological and environmental change studies.

The protocol detects key methane-cycling genes in South Texas coastal wetlands and visualizes their spatial distribution to enhance understanding of methane regulation and its environmental impacts in these dynamic ecosystems.

Coastal wetlands are the largest biotic source of methane, where methanogens convert organic matter into methane and methanotrophs oxidize methane, thus ...

DNA-based Fish Species Identification Protocol

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. This versatile method employs PCR amplification of genomic DNA extracted from fish samples, followed by restriction fragment length polymorphism (RFLP) analysis to generate fragment patterns that can be resolved on the Agilent 2100 Bioanalyzer and matched to the correct species using RFLP pattern matching software.
The fish identification method uses a simple, reliable, spin column- based protocol to isolate DNA from fish samples. The samples are treated with proteinase K to release the nucleic acids into solution. DNA is then isolated by suspending the sample in binding buffer and loading onto a micro- spin cup containing a silica- based fiber matrix. The nucleic acids in the sample bind to the fiber matrix. The immobilized nucleic acids are washed to remove contaminants, and total DNA is recovered in a final volume of 100 &mu;l. The isolated DNA is ready for PCR amplification with the provided primers that bind to sequences found in all fish genomes. The PCR products are then digested with three different restriction enzymes and resolved on the Agilent 2100 Bioanalyzer. The fragment lengths produced in the digestion reactions can be used to determine the species of fish from which the DNA sample was prepared, using the RFLP pattern matching software containing a database of experimentally- derived RFLP patterns from commercially relevant fish species.

This publication describes how to use the Agilent Fish Species Identification System to identify the species of a fish by extracting DNA and performing PCR and RFLP analysis.

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...