King R A, Read D S, Traugott M, Symondson W O C
Cardiff School of Biosciences, Biomedical Sciences Building, Cardiff University, Museum Avenue, Cardiff CF10 3US, UK.
Mol Ecol. 2008 Feb;17(4):947-63. doi: 10.1111/j.1365-294X.2007.03613.x. Epub 2008 Jan 14.
Molecular analysis of predation, through polymerase chain reaction amplification of prey remains within the faeces or digestive systems of predators, is a rapidly growing field, impeded by a lack of readily accessible advice on best practice. Here, we review the techniques used to date and provide guidelines accessible to those new to this field or from a different molecular biology background. Optimization begins with field collection, sample preservation, predator dissection and DNA extraction techniques, all designed to ensure good quality, uncontaminated DNA from semidigested samples. The advantages of nuclear vs. mitochondrial DNA as primer targets are reviewed, along with choice of genes and advice on primer design to maximize specificity and detection periods following ingestion of the prey by the predators. Primer and assay optimization are discussed, including cross-amplification tests and calibratory feeding experiments. Once primers have been made, the screening of field samples must guard against (through appropriate controls) cross contamination. Multiplex polymerase chain reactions provide a means of screening for many different species simultaneously. We discuss visualization of amplicons on gels, with and without incorporation of fluorescent primers. In more specialized areas, we examine the utility of temperature and denaturing gradient gel electrophoresis to examine responses of predators to prey diversity, and review the potential of quantitative polymerase chain reaction systems to quantify predation. Alternative routes by which prey DNA might get into the guts of a predator (scavenging, secondary predation) are highlighted. We look ahead to new technologies, including microarrays and pyrosequencing, which might one day be applied to this field.
通过聚合酶链反应扩增捕食者粪便或消化系统中残留的猎物来进行捕食的分子分析，是一个快速发展的领域，但由于缺乏关于最佳实践的易于获取的建议而受到阻碍。在这里，我们回顾了迄今为止使用的技术，并为该领域的新手或具有不同分子生物学背景的人提供指导方针。优化从野外采集、样本保存、捕食者解剖和DNA提取技术开始，所有这些都旨在确保从半消化样本中获得高质量、未受污染的DNA。我们回顾了以核DNA与线粒体DNA作为引物靶标的优势，以及基因选择和引物设计建议，以最大限度地提高特异性和捕食者摄取猎物后的检测期。讨论了引物和检测方法的优化，包括交叉扩增试验和校准喂养实验。一旦制备好引物，对野外样本的筛选必须通过适当的对照防止交叉污染。多重聚合酶链反应提供了一种同时筛选许多不同物种的方法。我们讨论了在凝胶上对扩增子的可视化，包括是否掺入荧光引物。在更专业的领域，我们研究了温度和变性梯度凝胶电泳在研究捕食者对猎物多样性反应方面的效用，并回顾了定量聚合酶链反应系统在量化捕食方面的潜力。强调了猎物DNA进入捕食者肠道的其他途径（ scavenging、二次捕食）。我们展望了新技术，包括微阵列和焦磷酸测序，这些技术有朝一日可能应用于该领域。
