High-resolution melting curve analysis for rapid detection of mutations in a Medaka TILLING library

Abstract

BackgroundDuring the last two decades, DNA sequencing has led to the identification of numerous genes in key species; however, in most cases, their functions are still unknown. In this situation, reverse genetics is the most suitable method to assign function to a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse-genetic strategy that combines random chemical mutagenesis with high-throughput discovery of the induced mutations in target genes. The method has been applied to a variety of plant and animal species. Screening of the induced mutations is the most important step in TILLING. Currently, direct sequencing or nuclease-mediated screening of heteroduplexes is widely used for detection of mutations in TILLING. Both methods are useful, but the costs are substantial and turnaround times are relatively long. Thus, there is a need for an alternative method that is of higher throughput and more cost effective.ResultsIn this study, we developed a high resolution melting (HRM) assay and evaluated its effectiveness for screening ENU-induced mutations in a medaka TILLING library. We had previously screened mutations in the p53 gene by direct sequencing. Therefore, we first tested the efficiency of the HRM assay by screening mutations in p53, which indicated that the HRM assay is as useful as direct sequencing. Next, we screened mutations in the atr and atm genes with the HRM assay. Nonsense mutations were identified in each gene, and the phenotypes of these nonsense mutants confirmed their loss-of-function nature.ConclusionsThese results demonstrate that the HRM assay is useful for screening mutations in TILLING. Furthermore, the phenotype of the obtained mutants indicates that medaka is an excellent animal model for investigating genome stability and gene function, especially when combined with TILLING.

Dmrt1 mutation causes a male-to-female sex reversal after the sex determination by Dmy in the medaka

“…1). In a gene-driven mutagenesis library, it has been calculated that each sperm has approximately 2,300 ENUinduced mutations per genome (Taniguchi et al 2006;Ishikawa et al 2010). Furthermore, the mutagenesis library has been made using the Cab strain.…”

“…The Cab strain is a sub-strain of the Carbio strain, which was established from fish obtained from Carolina Biological Supplies. Founder fish were repeatedly mutagenized with ENU and crossed with wild-type females, and the progeny were used to establish a permanently cryopreserved resource of 5,771 F 1 males (Taniguchi et al 2006;Ishikawa et al 2010). The Hd-rR strain was used for back-crossing and as a control in the gene expression analyses.…”

“…The final step was included to redistribute DNA strands derived from mutant and wild-type alleles to maximize heteroduplex formation. After PCR, the plates were imaged in a 96-well LightScanner (Idaho Technology Inc., Salt Lake City, UT, USA) for HRM analyses as previously described (Taniguchi et al 2006;Ishikawa et al 2010).…”

Mutation Detection by Analysis of DNA Heteroduplexes in TILLING Populations of Diploid Species

“…a SNP, is easily detected because of the formation of the heteroduplexes for which an altered melting profile is observed. Because heteroduplexes are less stable than homoduplexes, a loss of fluorescence is observed at lower temperatures than for homoduplexes (Ishikawa et al 2010;Hondow et al 2011;Martino et al 2010). Differences between homozygous samples can be observed as a shift in melting temperature, whereas changes in the shape of the melting curves (which are composites of the homo-and heteroduplex) demonstrate different heterozygous samples (Gundry et al 2003).…”

“…BRCA1 and BRCA2 genes, Hondow et al 2011). This method has also been used to detect mutations (single-nucleotide polymorphisms, SNPs and small insertions/deletions, InDels) in plant genetic studies, including TILLING in Lycopersicon esculentum (Gady et al 2009), Oryzias latipes (Ishikawa et al 2010), Triticum aestivum (Botticella et al 2011), Brassica rapa (Lochlainn et al 2011) and genetic mapping in Hordeum vulgare (Lehmensiek et al 2008), Zea mays (Li et al 2010) and Oryza sativa (Li et al 2011). HRM has also been used for the identification of transgenic plants (Milner et al 2014) and the detection of DNA methylation (Wojdacz and Dobrovic 2007).…”