Quantification of total mitochondrial DNA and the 4977-bp common deletion in Pearson's syndrome lymphoblasts using a fluorogenic 5'-nuclease (TaqMan) real-time polymerase chain reaction assay and plasmid external calibration standards.

This study describes a multiplex real-time polymerase chain reaction (PCR) assay that quantifies total mitochondrial DNA (mtDNA(total)) and mtDNA bearing the 4977-base pair 'common deletion' (deltamtDNA4977) in lymphoblasts derived from an individual diagnosed with Pearson's syndrome. The method is unique in its use of plasmids as external quantification standards and its use of multiplex conditions. Standards are validated by comparison with purified mtDNA amplification curves and by the fact that curves are largely unaffected by nuclear DNA (nucDNA). Finally, slopes of standard curves and unknowns are shown to be similar to each other and to theoretical predictions. From these data, mtDNA(total) in these cells is calculated to be 3258 (+723/-592) copies per cell while deltamtDNA4977 averages 232 (+136/-86) copies per cell or 7% (+4.65/-2.81).

Biological dosimetry using human interphase peripheral blood lymphocytes.

Conventional metaphase-spread chromosome-aberration-based biodosimetry techniques for radiation dose assessment, although robust, are laborious and time consuming. The molecular cytogenetic laboratory of the Armed Forces Radiobiology Research Institute is developing simple and rapid interphase-based cytological assays that will be applicable to a broad range of radiation exposure scenarios. These assays include analysis of chromosome aberrations (premature chromosome condensation-fluorescence in situ hybridization assay) and mitochondrial DNA mutations (mtDNA4977 deletion assay) using resting human peripheral blood lymphocytes. The dose-effect relationship for radiation-induced aberrations involving chromosome 1 after 24 hours of repair at 37 degrees C in resting human peripheral blood lymphocytes was studied using fluorescence in situ hybridization after chemical induction of premature chromosome condensation as previously explained. In the present study, we examined whether gamma irradiation in the range of 0 to 7.5 Gy induces a dose-dependent increase in aberrations manifested as "excess spots." The number of excess spots per cell, reflecting aberrations involving chromosome 1, increased from 0.035 at 0.5 Gy to 0.236 at 7.5 Gy. This observed dose-effect relationship was fit with a nonlinear power model. This technique may be extended to the study of radiation-induced translocations in interphase cells for retrospective dose reconstruction. With a recently developed in situ polymerase chain reaction method to detect and quantify mtDNA deletion in interphase cells after radiation exposure in cultured human peripheral blood lymphocytes, 90% to 95% of cells are analyzable. We discuss the potential use of the mtDNA deletion assay in biological dosimetry applications. Interphase-based cytological assays may eliminate some inherent problems associated with metaphase-spread-based assays. These problems involve (1) the limited number of analyzable cells containing chromosome aberrations, which is due to various factors including radiation-induced cell death and delay in cell cycle progression into mitosis, and (2) the requirements for radiation cytogenetics expertise and tedious labor to manually score chromosome aberrations.

Nucleic acid molecular biomarkers for diagnostic biodosimetry applications: use of the fluorogenic 5'-nuclease polymerase chain reaction assay.

A reliable, relatively easy method for diagnostic assessment of radiation exposure is needed to support the triage of radiation casualties and medical treatment decisions in military defense operations. Our strategy is to identify radiation-responsive DNA mutations and gene expression targets that can be analyzed using polymerase chain reaction (PCR) assays and an existing fluorescence-based nucleic acid analysis system designed for forward-deployable laboratory applications. Using an in vitro model system of human peripheral blood lymphocytes, we identified a candidate nucleic acid biomarker (i.e., gene expression target) that is responsive to ionizing radiation. In this report, we describe our preliminary Haras gene expression findings. A dose-dependent elevation in Haras gene expression levels was demonstrated using Northern-blot analysis 17 hours after exposure to a 250-kVp dose of X-rays (25-100 cGy, 1 Gy/minute); c-Haras expression levels at 100 cGy were ninefold higher than those of controls. An alternative protocol to quantify the Haras cDNA target, using the rapid, real-time reverse transcriptase fluorogenic 5'-nuclease PCR assay, is described, along with a preliminary characterization of the dynamic range for detection. Our research shows that the analysis of multitarget nucleic acid biomarkers, using the multiplex fluorogenic 5'-nuclease PCR assay, has beneficial applications in radiation epidemiology, radiation therapy, and biodosimetry.