Development of a New Technique for DNA Single Base Pair Mismatch Analysis

摘要

Research on the development and progression of diseases has shown that single base pair mismatches in key genes can be associated with increased risk of particular cancers. Analytical techniques for determining these mismatches currently exist, but faster, simpler, and cheaper techniques are desirable. We present a new technique based upon DNA melting curve analysis using a temperature gradient established using a silicon wafer. Along one edge of a square silicon substrate, two electrical connectors are mounted to deliver electrical current from a power supply. Since both edge connectors are on the same side of the square, the current flow in the silicon substrate is highest near the edge with the connectors, and decreases as the distance from that edge increases. This non-uniform current distribution generates resistive heating in a manner which, when combined with thermal conductivity effects, results in a nearly linear gradient of measured temperature along the axis normal to the electrode mounting edge. Finite element modeling analysis confirms the results measured with infrared thermal imaging. Using a control loop consisting of a PID controller and an RTD to monitor temperature, gradients of varying temperature ranges can be established. For example, we have routinely created gradients of 0.3°C per millimeter for the temperature range of 50 to 70°C, with which we have experimentally shown that single base pair mismatches in a section of a commonly studied gene (N-ras gene) can be distinguished from sections which do not contain the mismatch.