SGER: Pressure Dependence of DNA Synthesis

Massachusetts Institute Of Technology, Cambridge MA

Investigators

Abstract

0085355 Lerman This project is based on the assumption that the polymerase chain reaction can be driven by changes in hydrostatic pressure at constant temperature. The PI and co-workers will examine each step of the cycle to determine its compatibility with the hydrostatic pressure and temperature necessary to maintain helicity and enzyme activity at an elevated temperature and strand separation at low pressure. The pressure-driven procedure would provide an alternative to conventional PCR with some advantages - more rapid transition between stages of the cycle, unlimited processing volume, simplified assurance of processing uniformity, and perhaps different sequence restrictions. The project will proceed through measuring the dependence of DNA helicity on temperature, pressure, and composition of the medium. Initially the PI and co-PI will follow in detail the progress of a single cycle using DNA templates of well characterized thermal stability and domain structure and determine the relation of the parameters of each domain structure to pressure. They will use a simple temperature- and pressure-controlled cell, to be constructed at MIT, equipped for spectrophotometric measurement of UV absorbance and fluorescent emission of specific labels. The function of appropriate DNA polymerases will be explored with respect to physical parameters and some aspects of medium composition that affect protein stability and enzyme activity.