Technical Report: A Simple and Robust Real-Time Quantitative PCR Method for the Detection of Radiation-Induced Multiple Exon Deletions of the Human HPRT Gene
Technical Report: A Simple and Robust Real-Time Quantitative PCR Method for the Detection of Radiation-Induced Multiple Exon Deletions of the Human HPRT Gene
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DOI：
10.1667/rade-21-00047.1
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发表时间：
2021-06
影响因子：
3.4
通讯作者：
Kasumi Kawamura;Keiji Suzuki;N. Mitsutake
中科院分区：
文献类型：
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作者：
Kasumi Kawamura;Keiji Suzuki;N. Mitsutake
次黄嘌呤-磷酸核糖转移酶(HPRT)突变试验已被广泛用于研究辐射诱发的基因突变。在此，我们发展了一种基于实时定量聚合酶链式反应(QPCR)的检测HPRT基因多个外显子缺失的新方法。用不同剂量的γ照射永生化的正常人成纤维细胞(BJ1hTERT)，经6-Tg筛选，获得100多个6-TGR抗性克隆。提取大分子量基因组DNA，用9条外显子特异的引物进行实时定量聚合酶链式反应。通过优化引物浓度、选择合适的聚合酶链式反应和优化反应图谱，可以同时定量扩增每个外显子。我们能够在几天内鉴定出9个外显子没有任何扩增的6-TGR克隆，以及9个外显子中的一个或部分缺失的部分缺失突变体。这项新技术允许系统地检测电离辐射诱导的HPRT外显子的多个缺失，从而能够高通量和稳健地分析多个HPRT突变体。
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.