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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 meth.......

Representative 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 literature22,23,24. 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.......

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
Binding BufferBioTekeB010006022rapidly lysing cells
ChemoHS qPCR MixMonad00007547-120506qPCR Mix
D2000 DNA ladderReal-Times(Beijing) BiotechnologyRTM415Measure the position of electrophoretic bands
D512Taihe BiotechnologyTW21109196forword primer
D978Taihe BiotechnologyTW21109197reverse primer
Elution bufferBioTekeB010006022A low-salt elution buffer washes off the DNA
Glass beadYingxu Chemical Machinery(Shanghai) 70181000Special glass beads for dispersing and grinding
Import adsorption columnBioTekeB2008006022Adsorption column with silica matrix membrane
Inhibitor Removal BufferBioTekeB010006022Removal of Inhibitors in DNA Extraction
IsopropanolBioTekeB010006022Precipitate or isolate DNA
MIX-30S Mini MixerMiulabMUC881206oscillatory action
Proteinase KBioTekeB010006022Inactivation of intracellular nucleases and other proteins
Rotor-Gene Q 5plex HRMQiagenR1116175real-time fluorescence quantification PCR
Speed Micro-CentrifugeScilogex9013001121centrifuge
Tanon 3500R Gel ImagerTanon16T5553R-455gel imaging
Taq Mix ProMonad00007808-140534PCR Mix
Thermo CyclerZhuhai HemaVRB020Aordinary PCR
Wash BufferBioTekeB010006022Remove impurities such as cell metabolites

References

  1. Liu, C., Cong, B. Review and prospect of diagnosis of drowning deaths in water. Fa Yi Xue Za Zhi. 38 (1), 3-13 (2022).
  2. Frisoni, P., et al. Forensic diagnosis of freshwater or sal....

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Diatom DNAWater SamplesTissuesDrowningForensic ResearchDNA Extraction ProtocolForensic PracticeForensic EntomologyTropical Forensic MedicineDiatom TestingDiatom Molecular BiologyPlanktonGlass Beads

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