A novel strategy to design highly specific PCR primers based on the stability and uniqueness of 3′-end subsequences
A novel strategy to design highly specific PCR primers based on the stability and uniqueness of 3′-end subsequences
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DOI：
10.1093/bioinformatics/bti716
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发表时间：
2005-12-15
期刊：
影响因子：
5.8
通讯作者：
Ito, T
中科院分区：
文献类型：
--
作者：
Miura, F;Uematsu, C;Ito, T
动机：与使用一对特异性引物的常规PCR相比，一些应用利用单个独特引物与共同引物的组合，从而仅依赖于前者的特异性。这些应用包括cDNA末端快速扩增（RACE）、衔接子标记的竞争性PCR（ATAC-PCR）、PCR介导的基因组步移等。由于常规方法设计的引物在这些应用中往往不起作用，因此需要改进策略，特别是对于大规模分析。结果：基于ATAC-PCR中“脱靶”产物的结构，我们推断，引物特异性的实际决定因素可能不是整个序列的唯一性，而是最短的3 ′-结束子序列超过双工稳定性阈值。我们将这样的子序列称为“特异性决定子序列”（SDSS），并开发了一种简单的算法来预测引物的性能：该算法识别每个引物的SDSS并检查其在靶基因组中的独特性。在ATAC-PCR和5 '-RACE实验中，使用该算法设计的引物比使用常规方法设计的引物工作得更好。因此，该算法通常可用于改进各种基于PCR的应用。
Motivation: In contrast with conventional PCR using a pair of specific primers, some applications utilize a single unique primer in combination with a common primer, thereby relying solely on the former for specificity. These applications include rapid amplification of cDNA ends (RACE), adaptor-tagged competitive PCR (ATAC-PCR), PCR-mediated genome walking and so forth. Since the primers designed by conventional methods often fail to work in these applications, an improved strategy is required, particularly, for a large-scale analysis.Results: Based on the structure of 'off-target' products in the ATAC-PCR, we reasoned that the practical determinant of the specificity of primers may not be the uniqueness of entire sequence but that of the shortest 3'-end subsequence that exceeds a threshold of duplex stability. We termed such a subsequence as a 'specificity-determining subsequence' (SDSS) and developed a simple algorithm to predict the performance of the primer: the algorithm identifies the SDSS of each primer and examines its uniqueness in the target genome. The primers designed using this algorithm worked much better than those designed using a conventional method in both ATAC-PCR and 5'-RACE experiments. Thus, the algorithm will be generally useful for improving various PCR-based applications.