The hypoxanthine-phosphoribosyltransferase (HPRT) mutation assay has been widely used to investigate gene mutations induced by radiation. Here, we developed a novel method detecting deletions of multiple exons of the HPRT gene based on real-time quantitative PCR (qPCR). Immortalized normal human fibroblasts (BJ1-hTERT) were irradiated at various doses with γ rays, subjected to the 6-thioguanine (6-TG) selection, and more than one hundred 6-TG-resistant (6-TGR) clones were isolated. High-molecular-weight genomic DNA was extracted, and real-time qPCR was performed with the nine exon-specific primers. Optimization of the primer concentration, appropriate selection of PCR enzyme and refinement of the reaction profiles enabled simultaneous quantitative amplification of each exon. We were able to identify 6-TGR clones with total deletions, which did not show any amplification of the nine exons, and partial deletion mutants, in which one or some of the nine exons were missing, within a few days. This novel technique allows systematic determination of multiple deletions of the HPRT exons induced by ionizing radiation, enabling high-throughput and robust analysis of multiple HPRT mutants.

中文翻译：

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技术报告：一种用于检测辐射诱导的人类 HPRT 基因多外显子缺失的简单而可靠的实时定量 PCR 方法。

次黄嘌呤-磷酸核糖基转移酶 (HPRT) 突变测定已广泛用于研究辐射诱导的基因突变。在这里，我们开发了一种基于实时定量 PCR (qPCR) 检测 HPRT 基因多个外显子缺失的新方法。永生化的正常人成纤维细胞 (BJ1-hTERT) 以不同剂量的 γ 射线照射，进行 6-硫鸟嘌呤 (6-TG) 选择，并分离出一百多个 6-TG 抗性 (6-TGR) 克隆。提取高分子量基因组 DNA，并使用 9 个外显子特异性引物进行实时 qPCR。Optimization of the primer concentration, appropriate selection of PCR enzyme and refinement of the reaction profiles enabled simultaneous quantitative amplification of each exon. 我们能够识别完全缺失的 6-TGR 克隆，几天之内，它没有显示九个外显子的任何扩增，以及部分缺失突变体，其中九个外显子中的一个或一些缺失。这种新技术允许系统地确定由电离辐射引起的 HPRT 外显子的多个缺失，从而实现对多个 HPRT 突变体的高通量和稳健分析。