FluoMEP: A new genotyping method combining the advantages of randomly amplified polymorphic DNA and amplified fragment length polymorphism

Abstract

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.

Polygenic Sex Determination System in Zebrafish

“…The first experiments we performed were a series of comparative FluoMEP assays [39]. After screening through 258 FluoMEP primer combinations, no confirmed sex marker was obtained from zebrafish.…”

“…Pooled male and female samples were generated by combining equal quantity of individual gDNA samples (nine individuals of rosy barb, eight individuals of guppy and four individuals of zebrafish for each sex). FluoMEP screening was carried out as described previously [39]. Bulk segregant analysis [52] was performed using the male and female pooled gDNA samples to screen for potentially sex-linked markers.…”

“…[36], [37], [38]). Earlier, we have developed a new PCR-based mass genotyping technique called fluorescent motif enhanced polymorphism (FluoMEP; [39]) that combines the advantages of RAPD and AFLP. In this study, we have utilized this technology to search for sex-linked DNA markers in the genome of three fish species, including zebrafish.…”

Sex-associated DNA markers from turbot

“…The new procedure is based on RAPD, but the inclusion of a fluorescently labelled 'common primer' makes it compatible with high-throughput detection, thereby reducing the number of reactions needed to search for sex specificity in a given number of PCR products. Moreover, the 'common primer' can be designed to bind to frequently occurring motifs in the genome, opening the possibility for targeting the products onto particular regions of the genome (Chang et al 2007). Preliminary data from the Singaporean lab indicate that fluoMEP is indeed an efficient tool for the isolation of sex markers (Liew, Lim & Orban, unpublished data).…”

“…Recently, a new genotyping method called fluo-MEP has been described and recommended for such screens (Chang et al 2007). The new procedure is based on RAPD, but the inclusion of a fluorescently labelled 'common primer' makes it compatible with high-throughput detection, thereby reducing the number of reactions needed to search for sex specificity in a given number of PCR products.…”