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.
Highlights
During the last two decades, DNA sequencing has led to the identification of numerous genes in key species; in most cases, their functions are still unknown
We developed high resolution melting (HRM) assays to evaluate the effectiveness of this methodology for screening ENU-induced mutations in the TILLING library
Screening of mutations in p53 gene To evaluate whether the HRM assay is applicable to the screening of a TILLING library, we first used it to screen in exons 5 and 6 of the p53 gene (Fig. 1) and compared the results with those obtained previously by screening the same library with direct sequencing [9]
Summary
During the last two decades, DNA sequencing has led to the identification of numerous genes in key species; in most cases, their functions are still unknown In this situation, reverse genetics is the most suitable method to assign function to a gene. Genome sequencing projects over the past few decades have identified numerous genes in key species, and the completion of these sequences produced a situation in which most of the. One of these approaches is TILLING (Targeting Induced Local Lesions IN Genomes). TILLING is a reverse-genetic strategy that combines random chemical mutagenesis with high-throughput discovery of the induced mutations in target genes. Once a mutation is identified, homozygous mutant animals can be obtained by crossing progeny from heterozygous F1 matings
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