Department of Radiation Biology and Medical Genetics, Graduate School of Medicine, Osaka University, B4, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
BMC Mol Biol. 2010 Sep 15;11:70. doi: 10.1186/1471-2199-11-70.
During 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.
In 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.
These 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.
在过去的二十年中，DNA 测序技术已经鉴定了许多关键物种中的基因；然而，在大多数情况下，它们的功能仍然未知。在这种情况下，反向遗传学是赋予基因功能的最合适方法。TILLING（靶向诱导基因组局部突变）是一种反向遗传学策略，它将随机化学诱变与目标基因中诱导突变的高通量发现相结合。该方法已应用于多种植物和动物物种。诱变的筛选是 TILLING 中最重要的步骤。目前，直接测序或核酸酶介导的异源双链体筛选广泛用于检测 TILLING 中的突变。这两种方法都很有用，但成本很高，周转时间相对较长。因此，需要一种具有更高通量和更具成本效益的替代方法。
在这项研究中，我们开发了一种高分辨率熔解（HRM）分析，并评估了其在筛选斑马鱼 TILLING 文库中 ENU 诱导突变中的有效性。我们之前通过直接测序筛选了 p53 基因的突变。因此，我们首先通过筛选 p53 中的突变来测试 HRM 分析的效率，这表明 HRM 分析与直接测序一样有用。接下来，我们用 HRM 分析筛选了 atr 和 atm 基因的突变。在每个基因中都发现了无义突变，这些无义突变体的表型证实了它们的失活性质。
这些结果表明 HRM 分析可用于筛选 TILLING 中的突变。此外，获得的突变体的表型表明，斑马鱼是研究基因组稳定性和基因功能的优秀动物模型，尤其是与 TILLING 结合使用时。
