Display options
Cite
Kawamura K, Suzuki K, Mitsutake N. 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. Radiat Res. 2021;doi: 10.1667/RADE-21-00047.1.
Kawamura, K., Suzuki, K., & Mitsutake, N. (2021). 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. Radiation research, . https://doi.org/10.1667/RADE-21-00047.1
Kawamura, Kasumi, et al. "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." Radiation research vol. (2021). doi: https://doi.org/10.1667/RADE-21-00047.1
Kawamura K, Suzuki K, Mitsutake N. 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. Radiat Res. 2021 Jun 18; doi: 10.1667/RADE-21-00047.1. Epub 2021 Jun 18. PMID: 34143221.
Copy Download .nbib Format: NLM
Share it on

Radiat Res. 2021 Jun 18; doi: 10.1667/RADE-21-00047.1. Epub 2021 Jun 18.

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.

Radiation research

Kasumi Kawamura, Keiji Suzuki, Norisato Mitsutake

Affiliations

  1. Department of Radiation Medical Sciences, Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.

PMID: 34143221 DOI: 10.1667/RADE-21-00047.1

Abstract

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.

©2021 by Radiation Research Society.

Publication Types