What Is The Optimal Protocol For Preserving And Extracting Ancient DNA From Frozen Permafrost-preserved Plant Tissues That Have Undergone Repeated Freeze-thaw Cycles, And How Can I Minimize The Risk Of DNA Degradation And Contamination From Modern Microbial Communities During The Extraction Process?

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To address the preservation and extraction of ancient DNA from permafrost-preserved plant tissues, especially those subjected to freeze-thaw cycles, the following protocol is recommended:

Sampling and Storage

  1. Sampling: Collect samples when permafrost is frozen to minimize thawing. Use sterile tools (e.g., scoops or knives) and wear gloves to prevent contamination. Store samples in airtight containers or bags to maintain dryness and prevent contamination.

  2. Transportation: Transport samples in a cold state using ice packs or portable freezers. Avoid repeated freeze-thaw cycles during transport.

  3. Storage: Store samples at -80°C to slow DNA degradation. Use airtight containers to prevent moisture exposure.

Laboratory Setup

  1. Dedicated Lab Space: Use a dedicated ancient DNA lab equipped with UV decontamination, HEPA filters, and laminar flow hoods to minimize contamination.

  2. Personal Protective Equipment (PPE): Wear lab coats, gloves, face masks, and hairnets to prevent transferring modern DNA.

Extraction Protocol

  1. Pre-Treatment: Grind frozen samples using liquid nitrogen to prevent thawing and DNA degradation.

  2. Extraction Method: Use a silica-based method, which is effective for fragmented DNA typical in ancient samples. Consider using commercial kits designed for ancient DNA, which may include inhibitor removal and RNA degradation steps.

  3. Enzymatic Treatment: Use proteinase K to digest proteins and lysozyme to lyse microbial cells, reducing contamination risk.

  4. Negative Controls: Include negative controls in extraction and PCR steps to monitor contamination.

DNA Assessment and Sequencing

  1. DNA Integrity: Use qPCR with primers of varying lengths and a Bioanalyzer to assess DNA fragmentation.

  2. Sequencing: Employ shotgun metagenomics to capture all DNA, followed by bioinformatic filtering to identify plant DNA. Consider enrichment methods if targeting specific DNA.

Authentication and Minimizing Contamination

  1. Authentication: Confirm ancient origin by checking degradation patterns and comparing with modern species. Consider radiocarbon dating if feasible.

  2. Contamination Control: Use UV-treated surfaces and equipment, sterile reagents, and dedicated lab areas. Include replication steps to ensure consistency.

Documentation

Maintain detailed records of sample handling, extraction, and analysis to track each sample's journey and ensure reproducibility.

This protocol balances the need to preserve DNA integrity while minimizing contamination, leveraging current methods in ancient DNA research.