Rapid and Accurate Mitochondrial DNA Analysis in Amino Glycoside Sensitive Patients

Abstract

The use of Restriction Fragment Length Polymorphism to assess the position of mutation in nuclear or mitochondrial DNA is very common but the effectiveness of this methodology is limited and expensive since it utilizes many reagents that are not easy to acquire. We propose the use of a Denaturing Gradients Gel Electrophoresis to analyze the presence of mutation T1189C in the 12S rRNA region of human mitochondrial DNA, formerly detected by nucleotide sequence analysis, in order to develop an optimized method that would allow the simultaneous detection of this mutation in several patient mitochondrial DNA and further finding the relationship of its presence with sudden deafness produced by hypersensitivity to aminoglycoside antibiotic treatment. The technique was improved optimizing the denaturing gradient gel electrophoresis parameters, such as optimum temperature, voltage, concentration of denaturing agents and time for carrying out the method, which allowed us to precisely distinguish whether there has been a change in the sequence of a given sample, analyzing a wild type mitochondrial 12S rRNA against the patient sample simultaneously, by simply observing the differences in running time of a band in which mutation is present, and corroborated by sequence analysis. The application of the technique to samples of various patients at the same time would be very valuable to assess the presence of the mutation prior to treatment of a given infection is started and to recommend the use of an alternative therapeutic agent innocuous for the inner ear.