Canadian Centre for DNA Barcoding, Biodiversity Institute of Ontario, University of Guelph, 579 Gordon Street, Guelph, ON, Canada N1G 2W1.
Mol Ecol Resour. 2009 May;9 Suppl s1:35-41. doi: 10.1111/j.1755-0998.2009.02630.x.
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.
尽管结合微流控和先进测序技术的设备有望实现人们在数分钟内生成 DNA 条码的未来，但当前的分析方案通常涉及耗时超过 2 天的工作流程。在这里，我们描述了简单的方案，可将样本转化为基于条码的鉴定，耗时不到 2 小时。这些方案使用冷冻或冻干试剂，可以预包装成“试剂盒”，并支持动物界的条码分析。分析程序允许 5 分钟用于 DNA 提取，25 分钟用于条码区域的聚合酶链反应扩增，25 分钟用于循环测序，10 分钟用于清洗，45 分钟用于毛细管测序，5 分钟用于痕量文件分析，以完成基于 DNA 的鉴定。本研究涉及比较不同的 DNA 保存和提取方法，并评估了具有高进程性和抗抑制剂性的 Taq 聚合酶。
