Pattyn Filip, Hoebeeck Jasmien, Robbrecht Piet, Michels Evi, De Paepe Anne, Bottu Guy, Coornaert David, Herzog Robert, Speleman Frank, Vandesompele Jo
Center for Medical Genetics, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium.
BMC Bioinformatics. 2006 Nov 9;7:496. doi: 10.1186/1471-2105-7-496.
DNA methylation plays an important role in development and tumorigenesis by epigenetic modification and silencing of critical genes. The development of PCR-based methylation assays on bisulphite modified DNA heralded a breakthrough in speed and sensitivity for gene methylation analysis. Despite this technological advancement, these approaches require a cumbersome gene by gene primer design and experimental validation. Bisulphite DNA modification results in sequence alterations (all unmethylated cytosines are converted into uracils) and a general sequence complexity reduction as cytosines become underrepresented. Consequently, standard BLAST sequence homology searches cannot be applied to search for specific methylation primers.
To address this problem we developed methBLAST, a sequence similarity search program, based on the original BLAST algorithm but querying in silico bisulphite modified genome sequences to evaluate oligonucleotide sequence similarities. Apart from the primer specificity analysis tool, we have also developed a public database termed methPrimerDB for the storage and retrieval of validated PCR based methylation assays. The web interface allows free public access to perform methBLAST searches or database queries and to submit user based information. Database records can be searched by gene symbol, nucleotide sequence, analytical method used, Entrez Gene or methPrimerDB identifier, and submitter's name. Each record contains a link to Entrez Gene and PubMed to retrieve additional information on the gene, its genomic context and the article in which the methylation assay was described. To assure and maintain data integrity and accuracy, the database is linked to other reference databases. Currently, the database contains primer records for the most popular PCR-based methylation analysis methods to study human, mouse and rat epigenetic modifications. methPrimerDB and methBLAST are available at http://medgen.ugent.be/methprimerdb and http://medgen.ugent.be/methblast.
We have developed two integrated and freely available web-tools for PCR based methylation analysis. methBLAST allows in silico assessment of primer specificity in PCR based methylation assays that can be stored in the methPrimerDB database, which provides a search portal for validated methylation assays.
DNA甲基化通过对关键基因的表观遗传修饰和沉默在发育和肿瘤发生中发挥重要作用。基于聚合酶链反应（PCR）的亚硫酸氢盐修饰DNA甲基化检测方法的发展，在基因甲基化分析的速度和灵敏度方面带来了突破。尽管有这项技术进步，但这些方法需要逐个基因进行繁琐的引物设计和实验验证。亚硫酸氢盐DNA修饰会导致序列改变（所有未甲基化的胞嘧啶都转化为尿嘧啶），并且由于胞嘧啶的比例降低，序列复杂性总体下降。因此，标准的BLAST序列同源性搜索不能用于搜索特定的甲基化引物。
为了解决这个问题，我们开发了methBLAST，这是一个序列相似性搜索程序，基于原始的BLAST算法，但在计算机模拟的亚硫酸氢盐修饰基因组序列中进行查询，以评估寡核苷酸序列的相似性。除了引物特异性分析工具外，我们还开发了一个名为methPrimerDB的公共数据库，用于存储和检索经过验证的基于PCR的甲基化检测方法。该网络界面允许公众免费访问以进行methBLAST搜索或数据库查询，并提交基于用户的信息。数据库记录可以通过基因符号、核苷酸序列、使用的分析方法、Entrez基因或methPrimerDB标识符以及提交者姓名进行搜索。每个记录都包含一个指向Entrez基因和PubMed的链接，以检索有关该基因、其基因组背景以及描述甲基化检测方法的文章的更多信息。为了确保和维护数据的完整性和准确性，该数据库与其他参考数据库相链接。目前，该数据库包含用于研究人类、小鼠和大鼠表观遗传修饰的最流行的基于PCR的甲基化分析方法的引物记录。methPrimerDB和methBLAST可在http://medgen.ugent.be/methprimerdb和http://medgen.ugent.be/methblast上获取。
我们开发了两个用于基于PCR的甲基化分析的集成且免费的网络工具。methBLAST允许在计算机模拟中评估基于PCR的甲基化检测中引物的特异性，这些检测可以存储在methPrimerDB数据库中，该数据库为经过验证的甲基化检测提供了一个搜索门户。
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