Kushwaha Garima, Srivastava Gyan Prakash, Xu Dong
Informatics Institute and Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, USA.
Methods Mol Biol. 2015;1275:181-99. doi: 10.1007/978-1-4939-2365-6_14.
Highly specific and efficient primer and probe design has been a major hurdle in many high-throughput techniques. Successful implementation of any PCR or probe hybridization technique depends on the quality of primers and probes used in terms of their specificity and cross-hybridization. Here we describe PRIMEGENSw3, a set of web-based utilities for high-throughput primer and probe design. These utilities allow users to select genomic regions and to design primer/probe for selected regions in an interactive, user-friendly, and automatic fashion. The system runs the PRIMEGENS algorithm in the back-end on the high-performance server with the stored genomic database or user-provided custom database for cross-hybridization check. Cross-hybridization is checked not only using BLAST but also by checking mismatch positions and energy calculation of potential hybridization hits. The results can be visualized online and also can be downloaded. The average success rate of primer design using PRIMEGENSw3 is ~90 %. The web server also supports primer design for methylated sequences, which is used in epigenetic studies. Stand-alone version of the software is also available for download at the website.
高度特异且高效的引物和探针设计一直是许多高通量技术中的主要障碍。任何聚合酶链反应（PCR）或探针杂交技术的成功实施都取决于所用引物和探针的特异性及交叉杂交方面的质量。在此，我们描述PRIMEGENSw3，这是一套基于网络的用于高通量引物和探针设计的实用工具。这些实用工具允许用户以交互式、用户友好且自动的方式选择基因组区域并为选定区域设计引物/探针。该系统在后端的高性能服务器上运行PRIMEGENS算法，使用存储的基因组数据库或用户提供的自定义数据库进行交叉杂交检查。交叉杂交不仅通过BLAST进行检查，还通过检查潜在杂交命中的错配位置和能量计算来进行。结果可以在线可视化，也可以下载。使用PRIMEGENSw3进行引物设计的平均成功率约为90%。该网络服务器还支持用于表观遗传学研究的甲基化序列引物设计。该软件的独立版本也可在网站上下载。
