Arányi Tamás, Tusnády Gábor E
Institute of Enzymology, BRC, Hungarian Academy of Sciences, Karolina, Hungary.
Methods Mol Biol. 2007;402:385-402. doi: 10.1007/978-1-59745-528-2_20.
The design of adequate primers for polymerase chain reaction (PCR) is sometimes a difficult task. This is the case when either the target sequence harbors unusual nucleotide motifs or the template is complex. Unusual nucleotide motifs can be repeat elements, whereas complex templates are targets for mispriming and alternative amplification products. Such examples are GC-rich native or bisulfite-treated genomic DNA sequences. Bisulfite treatment leads to the specific conversion of non-methylated cytosines to uracyls. This is the key step of bisulfite genomic sequencing, widely used to determine DNA methylation of a sequence. Here, we describe BiSearch Web server (http://bisearch.enzim.hu), a primer design software created for designing primers to amplify such target sequences. Furthermore, we developed a unique post-design primer analysis module, to carry out genome wide searches to identify genomic mispriming sites and to test by electronic (in silico) PCR (ePCR) for alternative PCR products. This option is currently available on four native or bisulfite-treated mammalian genomes.
为聚合酶链反应（PCR）设计合适的引物有时是一项艰巨的任务。当靶序列含有异常核苷酸基序或模板复杂时，情况就是如此。异常核苷酸基序可以是重复元件，而复杂模板则是错误引物结合和产生替代扩增产物的靶点。富含GC的天然或亚硫酸氢盐处理的基因组DNA序列就是这样的例子。亚硫酸氢盐处理导致未甲基化的胞嘧啶特异性转化为尿嘧啶。这是亚硫酸氢盐基因组测序的关键步骤，广泛用于确定序列的DNA甲基化。在这里，我们描述了BiSearch网络服务器（http://bisearch.enzim.hu），这是一个用于设计引物以扩增此类靶序列的引物设计软件。此外，我们开发了一个独特的设计后引物分析模块，用于进行全基因组搜索以识别基因组错误引物结合位点，并通过电子（虚拟）PCR（ePCR）测试替代PCR产物。此选项目前可用于四个天然或亚硫酸氢盐处理的哺乳动物基因组。
