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Related Concept Videos

DNA Isolation01:24

DNA Isolation

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DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
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Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications
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State-Specific Extraction of Environmental DNA: Spike-and-Recovery Controls to Validate and Optimise Extraction

Julia Zöhrer1, Judith Ascher-Jenull2, Eva Maria Prem1

  • 1Department of Microbiology, Universität Innsbruck, Innsbruck, Austria.

Environmental Microbiology
|December 2, 2025
PubMed
Summary

Separating extracellular DNA (exDNA) and intracellular DNA (iDNA) is crucial for understanding environmental DNA (eDNA) in microbial ecology. This study validates extraction protocols, revealing that buffer composition and lysis methods significantly impact DNA recovery.

Keywords:
Bacillus subtilisEscherichia coliextracellular DNAintracellular DNAtotal environmental DNA

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Area of Science:

  • Environmental microbiology
  • Molecular ecology
  • Biogeochemistry

Background:

  • Environmental DNA (eDNA) analysis is vital for biodiversity assessment.
  • Distinguishing between extracellular DNA (exDNA) and intracellular DNA (iDNA) is critical for accurate eDNA interpretation.
  • Existing eDNA extraction protocols often lack thorough validation for state-specific recovery.

Purpose of the Study:

  • To evaluate and compare the efficacy of different eDNA extraction protocols in separating extracellular DNA (exDNA) and intracellular DNA (iDNA).
  • To identify key factors influencing the recovery rates of exDNA and iDNA from environmental samples.
  • To optimize a protocol for reliable, state-specific eDNA extraction.

Main Methods:

  • Selection and testing of four established eDNA extraction protocols.
  • Utilized spike-and-recovery controls representing different bacterial origins (Gram-positive, Gram-negative) and DNA states (exDNA, iDNA).
  • Quantification of spike-in recovery using digital PCR across various environmental matrices.

Main Results:

  • Significant differences in exDNA and iDNA recovery were observed among protocols and environmental matrices, with protocols being more influential.
  • exDNA recovery was primarily influenced by washing buffer composition and duration.
  • iDNA recovery was predominantly determined by the cell lysis method.

Conclusions:

  • The choice of extraction protocol and specific steps (washing, lysis) critically affect the accurate recovery of extracellular and intracellular DNA.
  • Optimizing these parameters is essential for advancing the field of molecular microbial ecology and understanding eDNA dynamics.
  • This research provides a foundation for developing more precise eDNA extraction methods.