Quantification Of The MtDNA 4977 Bp Deletion Induced By Ionizing Radiation In Human Peripheral Blood Using Real-time PCR

Background: Nowadays, along with the wide and friendly use of radiation sources and nuclear energy, tremdendous benefits have been brought to human being. Exposure dose was estimated by usually using the methods of physical and biological dosage. At present, most common and general accepted approach applying to biological dosage estimation is chromosomal aberration. Nevertheless this approach also has evident defect. Therefore, Establishment of the methods on quick and accurate biological dose evaluation using modern biological technology has become the focus of current research.Human mitochondrial DNA(mtDNA) lacks the protection of histones and other DNA-binding proteins, and effective gene repair system, so mtDNA easy suffer damage from external factor, and generate mutation. Point mutation,deletion and insert deletion are the frequent mutations. Among these, the deletion of mtDNA 4977bp is the most common. Some researches indicated that the deletion could be induced by ionizing radiation.In order to explore to establish the molecular biodosimetry, cloning and sequence analyses of mtDNA 4977bp deletion induced ionizing radiation were performed, and the deletion was quantified by real-time PCR in the study. Meanwhile, the mtDNA 4977bp deletion of peripheral blood in China healthy adult were detected and quantified by real-time PCR.Methods: Human peripheral blood samples were collected from two healthy youngster donors and irradiated by ⁶⁰Co gamma ray. After 2 hours in 37°C, mtDNA was extracted from WBC. The amplification of the fragment of mtDNA4977bp deletion was performed by using PCR with a pair of specific primers designed according to the gene sequence issued by Genbank. The PCR products were cloned into pMD18-T-vector after recovering of PCR products made on the gel. The DNA sequence was determined by dideoxy-mediated chain termination. Analyses of the DNA sequences obtained were processed by using Biosoft. The quantity of mtDNA4977bp deletion induced by ionizing radiation was detected by using Real-Time PCR and the curve of dose-effect was primarily ploted. Meantime, the peripheral blood of twenty-seven healthy adult, aged from 17 to 44, was collected. The deleted and undeleted mitochondrial DNA in each sample were detected and quantified by real-time PCR.Results: (1) The fragment of mtDNA4977bp deletion obtained by PCR amplification was consistent with the expected fragment (129bp); The result of Blast sequence analysis indicated that the fragment was the sequence of cross mtDNA4977bp deletion; The primary Real-Time PCR analyses showed that the mtDNA4977bp deletion increased along with irradiation dose in the range of 0 to 4 Gy. (2) The nineteen samples contained mitochondrial DNA 4977 bp deletion in all 27 samples, and the positive rate of the DNA 4977bp deletion was 70.37%(19/27); Quantity of the deleted mitochondrial DNA were not statistically significant in different age group, as well as that of undeleted mitochondrial DNA.Conclusion: (1) The 129bp DNA fragment of cross mtDNA4977bp deletion by ionizing radiation was obtained by the PCR; The primary results showed that the mtDNA4977bp deletion by using Real-Time quantitative analyses may be a hopeful molecular biodosimetry. (2) The peripheral blood of about 70% healthy adult contained mitochondrial DNA 4977 bp deletion in the research; Approximately 1 in 10⁶ to 107 copies carried the mitochondrial DNA 4977 bp deletion in the samples containing the deletion; The data did not show any accumulation of the DNA 4977 bp deletion with increasing age.