FluoMEP: A new genotyping method combining the advantages of randomly amplified polymorphic DNA and amplified fragment length polymorphism
FluoMEP: A new genotyping method combining the advantages of randomly amplified polymorphic DNA and amplified fragment length polymorphism
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DOI：
10.1002/elps.200600715
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发表时间：
2007-02-01
期刊：
影响因子：
2.9
通讯作者：
Orban, Laszlo
中科院分区：
文献类型：
--
作者：
Chang, Alex;Liew, Woei Chang;Orban, Laszlo
基于PCR的两个未知基因组之间差异的鉴定通常需要在用手动方法扩增和/或凝胶电泳分离大量样品之前对模板进行复杂的操作。在这里，我们描述了一种新的基因分型方法，称为荧光基序增强多态性（fluoremotion enhanced polymorphism，fluoMEP）。fluoMEP方法基于随机扩增多态性DNA（RAPD）测定，但结合了大量未标记的10聚体引物（ca. 5000）和用于检测扩增片段长度多态性（AFLP）模式的自动CE装置的能力。这两种成分之间的联系是由荧光标记的“共同引物”提供的，该引物与未标记的RAPD引物一起用于双引物PCR。利用同一“通用引物”和一系列RAPD引物，可以快速有效地筛选DNA模板进行多态性检测。我们的手稿描述了该方法的优化及其在不同模板上的表征。我们证明，通过使用几种不同的方法，除了“共同引物”的PCR改变了扩增片段的配置文件，允许筛选基因组的各个部分与相同的一组未标记的引物。我们还提出了一个在计算机上分析的基因组定位的RAPD引物扩增的片段与两个不同的“共同引物”和单独的。
PCR-based identification of differences between two unknown genomes often requires complex manipulation of the templates prior to amplification and/or gel electrophoretic separation of a large number of samples with manual methods. Here, we describe a new genotyping method, called fluorescent motif enhanced polymorphism (fluoMEP). The fluoMEP method is based on random amplified polymorphic DNA (RAPD) assay, but combines the advantages of the large collection of unlabelled 10mer primers (ca. 5000) from commercial sources and the power of the automated CE devices used for the detection of amplified fragment length polymorphism (AFLP) patterns. The link between these two components is provided by a fluorescently labeled "common primer" that is used in a two-primer PCR together with an unlabeled RAPD primer. By using the same "common primer" and a series of RAPD primers, DNA templates can be screened quickly and effectively for polymorphisms. Our manuscript describes the optimization of the method and its characterization on different templates. We demonstrate by using several different approaches that the addition of the "common primer" to the PCR changes the profile of amplified fragments, allowing for screening various parts of the genome with the same set of unlabeled primers. We also present an in silico analysis of the genomic localization of fragments amplified by a RAPD primer with two different "common primers" and alone.