Extraction of Diatom DNA from Water Samples and Tissues

Summary

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

Abstract

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.

Introduction

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 flow^5,6. The detection of diatoms in internal tissues and organs such as lung, liver, and bone marrow is a strong evidence of drowning before death^7,8. 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 years^10,11,12. This technology realizes the species identification of diatoms by analyzing the specific DNA sequence composition of diatoms^13,14. 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 dioxide^15,16,17. 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.

Protocol

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 Figure 2).

1. Pretreatment of samples

1. Pretreatment of water samples
   1. 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 µL 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.
2. Pretreatment of tissue samples
   1. 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.

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 °C needs to be prepared before the experiment starts. All steps must strictly follow the principles of aseptic operation.

1. Assembly of water samples and tissues
   NOTE: Water sample and tissue diatom DNA extraction method are the same.
   1. 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.
   2. 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.
2. Add 40 µL 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.
3. Add 200 µL 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.
4. Put the centrifuge tubes in a 70 °C water bath for 10 min, and the solution becomes clear.
5. Add 100 µL 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.
6. 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.
7. 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.
8. Add 500 µL 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.
9. Add 700 µL 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.
10. Add 500 µL 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.
11. 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.
12. Take out the adsorption column and put it into a clean centrifuge tube. Add 100 µL of elution buffer to the middle part of the adsorption membrane.
13. Place the adsorption column at room temperature for 3-5 min, and centrifuge at 13,400 x g for 1 min.
14. 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 °C water bath for about 10 min. The elution volume should not be less than 50 µL; otherwise, the DNA yield will be reduced.
15. Store the extracted diatom DNA at 2-8 °C for future use. If the DNA solution is to be stored for a long time, store it at -20 °C.

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.

1. Add 2 µL of the extracted DNA from water samples and tissues as the template. Choose one of the following two methods for inspection.
2. Conventional PCR test
   1. Use primers²² that can specifically amplify diatom 18S rDNA fragments. For details, see Table 1.
   2. Establish the PCR reaction system and amplification conditions according to the characteristics of the primers. For details, see Table 2.
   3. Run the products of PCR amplification on 2% agarose gel. Observe and analyze the imaging with a gel imager.
3. Fluorescent quantitative PCR test
   1. 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.
   2. 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.

Results

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 literature^22,23,24. The primers were verified by NCBI-Blast Primer, a...

Discussion

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...

Materials

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