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 liqu....

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 ....

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

List of materials used in this article

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