Nucleic acid molecular biomarkers for diagnostic biodosimetry applications: Use of the fluorogenic 5′-nuclease polymerase chain reaction assay

Abstract

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.