What Is The Optimal Primer Design And PCR Protocol For Detecting The Presence Of Bowhead Whale (Balaena Mysticetus) EDNA In Arctic Seawater Samples With High Levels Of PCR Inhibitors, Such As Humic Acids And Phenolic Compounds, While Minimizing The Risk Of False Positives From Closely Related Species Like The Gray Whale (Eschrichtius Robustus)?

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To detect bowhead whale eDNA in Arctic seawater efficiently, follow this structured approach:

Primer Design

  1. Target Selection: Choose mitochondrial genes like COI, 16S rRNA, or the control region for their species-specific sequences.
  2. Specificity: Use in silico tools (Primer-BLAST, PrimerExplorer) to design primers with high specificity for bowhead whale, ensuring minimal binding to gray whale DNA.
  3. Primer Characteristics:
    • Length: 18-25 bp.
    • Tm: 50-65°C.
    • Amplicon Size: 100-250 bp for degraded eDNA.
    • Avoid Self/Primer Dimer Formation.

Sample Preparation

  1. DNA Extraction: Use silica columns or magnetic beads to remove humic acids and phenolics.
  2. PCR Additives: Incorporate BSA (0.1-1 μg/μL), DMSO (1-5%), or betaine (1M) to mitigate inhibitors.

PCR Protocol

  1. Reaction Setup: Use high-fidelity Taq polymerase. Optimize primer concentrations (0.1-1 μM) and MgCl2 (1.5-2.5 mM).
  2. Thermocycling:
    • Initial Denaturation: 95°C for 5-10 minutes.
    • Touchdown PCR: Start at 65°C, decrease by 0.5°C each cycle for 20 cycles, then 20 cycles at 55°C.
    • Extension: 72°C for 30-60 seconds.
  3. Detection: Use qPCR with SYBR Green or probes for specificity. Include a melt curve analysis for SYBR Green.

Controls and Validation

  1. Negative Controls: Include no-template and no-DNA controls.
  2. Technical Replicates: Run multiple replicates for reliability.
  3. Confirmation: Use a second PCR or sequencing for positive samples.

Optimization

  1. Gradient PCR: Determine optimal annealing temperature.
  2. Test Additives: Evaluate different combinations to enhance efficiency.

By following this approach, you can enhance the specificity and reliability of detecting bowhead whale eDNA while minimizing false positives from related species.