REAL TIME QUANTITATIVE PCR THERMOCYCLER

Tufts Medical Center, Boston MA

Investigators

Abstract

This proposal is a request for the ABI Prism 7700, a thermocycler with the capability to perform real-time quantitation of the amplified products. Real-time quantitative PCR is the state-of-the-art method for the quantitation of specific nucleic acid sequences. The high sensitivity and the large linear response range of the fluorescence-based detection facilitate the quantitation of sequences that might otherwise be difficult or impossible to detect, as well as the comparison of multiple nucleic acid target sequences within the same reaction. The diversity of projects for which this instrument will be used demonstrates the versatility of the system. Real-time quantitative PCR will allow members of this user group to quantitate pathogenic bacteria (Borrelia burgdorferi, Vibrio cholerae) in tissues from infected animals and humans, and to quantitate Bacillus subtilis, Clostridium difficile, B. burgdorferi, E. coli, and V. cholerae gene expression under various in vitro and in vivo conditions. Other members of this user group will employ the ABI Prism 7700 to quantitate retroviral mutations arising in a population and retroviral recombination events. Cytokine and chemokine expression in response to bacterial infection, HIV infection, and physical training will be monitored by additional members of the user group. This thermocycler system will be incorporated into an existing core facility, and will be open to all laboratories on this campus, as time permits, after appropriate training of the personnel. Access to the ABI Prism 7700 will be essential for the progression of numerous NIH- funded basic science and clinical studies already underway at New England Medical Center and Tufts University School of Medicine.