Universal primers for fluorescent labelling of PCR fragments-an efficient and cost-effective approach to genotyping by fluorescence
Universal primers for fluorescent labelling of PCR fragments-an efficient and cost-effective approach to genotyping by fluorescence
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DOI：
10.1111/j.1755-0998.2011.03104.x
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发表时间：
2012-05-01
影响因子：
7.7
通讯作者：
Miller, A. D.
中科院分区：
文献类型：
--
作者：
Blacket, M. J.;Robin, C.;Miller, A. D.
直接标记基因座特异性引物用于微卫星分析是昂贵的，并且是小预算分子生态学项目的常见限制。通过三引物PCR方法可以实现PCR产物的更具成本效益的末端标记，该方法涉及荧光标记的通用引物与具有5'通用引物序列尾的经修饰的基因座特异性引物的组合。该技术已被广泛使用，但主要由于缺乏适用于共扩增大量大小重叠基因座且不需要修改基因座特异性PCR条件的可用通用引物而受到限制。在这项研究中，我们报告了一套四个高性能的通用引物，可以采用三引物PCR的方法，高效和具有成本效益的荧光末端标记的PCR片段。由于高通用引物Tm值（约60+ ℃），扩增效率最大化，与常用的通用引物如M13相比，高通用引物Tm值增强了引物通用性并能够采用更高的退火温度。我们证明，这些通用引物可以与多种荧光团组合，通过多重PCR有效地共扩增多个位点。该方法提供了类似于使用直接标记引物的微卫星荧光检测测定的多重和PCR效率水平，同时显著降低了项目成本。引物的性能进行了测试，使用几种替代的PCR策略，涉及单一和多个荧光团在单一和多重PCR在广泛的分类群。
Directly labelling locus-specific primers for microsatellite analysis is expensive and a common limitation to small-budget molecular ecology projects. More cost-effective end-labelling of PCR products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus-specific primers with 5' universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co-amplifying large numbers of size overlapping loci and without requiring locus-specific PCR conditions to be modified. In this study, we report a suite of four high-performance universal primers that can be employed in a three primer PCR approach for efficient and cost-effective fluorescent end-labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ degrees C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co-amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa.