Data from: Collecting in collections: a PCR strategy and primer set for DNA barcoding of decades-old dried museum specimens

Natural history museums are vastly underutilized as a source of material for DNA analysis because of perceptions about the limitations of DNA degradation in older specimens. Despite very few exceptions, most DNA barcoding projects, which aim to obtain sequence data from all species, generally use specimens collected specifically for that purpose, instead of the wealth of identified material in museums, constrained by the lack of suitable PCR methods. Any techniques that extend the utility of museum specimens for DNA analysis therefore are highly valuable. This study first tested the effects of specimen age and PCR amplicon size on PCR success rates in pinned insect specimens, then developed a PCR primer set and amplification strategy allowing greatly increased utilization of older museum specimens for DNA barcoding. PCR success rates compare favourably with the few published studies utilizing similar aged specimens, and this new strategy has the advantage of being easily automated for high-throughput laboratory workflows. The strategy uses hemi-nested, degenerate, M13-tailed PCR primers to amplify two overlapping amplicons, using two PCRs per amplicon (i.e. four PCRs per DNA sample). Initial PCR products are reamplified using an internal primer and a M13 primer. Together the two PCR amplicons yield 559 bp of the COI gene from Coleoptera, Lepidoptera, Diptera, Hemiptera, Odonata and presumably also other insects. BARCODE standard-compliant data were recovered from 67% (56 of 84) of specimens up to 25 years old, and 51% (102 of 197) of specimens up to 55 years old. Given the time, cost and specialist expertise required for fieldwork and identification, ‘collecting in collections’ is a viable alternative allowing researchers to capitalize on the knowledge captured by curation work in decades past.由于学界普遍认为老旧标本的DNA存在降解限制，自然历史博物馆作为DNA分析材料来源的潜力远未得到充分挖掘。尽管仅有极少数例外，大多数旨在获取所有物种序列数据的DNA条形码（DNA barcoding）项目，通常仅使用专门为该项目采集的标本，而非馆藏中大量已完成鉴定的标本——这一现状正是由于缺乏适配的聚合酶链式反应（PCR）方法所导致的。因此，任何能够拓展博物馆标本在DNA分析中应用价值的技术都具有极高的科研价值。本研究首先探究了标本保存时长与PCR扩增子（amplicon）长度对针插昆虫标本PCR成功率的影响，随后开发了一套PCR引物（primer）组与扩增策略，可大幅提升老旧博物馆标本在DNA条形码分析中的利用率。该方法的PCR成功率与少数已发表的、针对相近保存时长标本的研究结果相当，且这一新策略易于实现自动化，适配高通量实验室工作流程。该策略采用半巢式、简并且带有M13尾序列的（M13-tailed）PCR引物，扩增两段重叠的扩增子，每个扩增子需进行两轮PCR反应（即每份DNA样本共需四轮PCR反应）。首轮PCR产物将通过内部引物与M13引物进行二次扩增。两段PCR扩增子可共同覆盖鞘翅目、鳞翅目、双翅目、半翅目、蜻蜓目及其他多数昆虫的细胞色素C氧化酶亚基I基因（COI gene）的559 bp片段。在保存时长不超过25年的标本中，有67%（84份中的56份）可获得符合DNA条形码标准的序列数据；保存时长不超过55年的标本中，该比例为51%（197份中的102份）。考虑到野外采集与标本鉴定需要投入大量时间、成本及专业技术，“利用馆藏标本开展研究”是一种可行的替代方案，可让研究者充分利用数十年来博物馆藏品管理工作所积累的物种鉴定知识。