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Keywords:

  • alkaline lysis;
  • DNA barcoding;
  • fast sequencing;
  • FTA Elute;
  • next-generation PCR enzymes

Abstract

Although devices combining microfluidic and advanced sequencing technologies promise a future where one can generate a DNA barcode in minutes, current analytical regimes typically involve workflows that extend over 2 days. Here we describe simple protocols enabling the advance from a specimen to barcode-based identification in less than 2 h. The protocols use frozen or lyophilized reagents that can be prepackaged into ‘kits’ and support barcode analysis across the animal kingdom. The analytical procedure allows 5 min for DNA extraction, 25 min for polymerase chain reaction amplification of the barcode region, 25 min for cycle-sequencing, 10 min for cleanup, 45 min for capillary sequencing and 5 min for trace file analysis to complete DNA-based identification. This study involved the comparison of varied DNA preservation and extraction methods, and evaluated Taq polymerases with high processivity and resistance to inhibitors.