Observable real-time pulsed-field gel electrophoresis

Abstract

Pulsed-Field Gel Electrophoresis (PFGE) is a very important molecular research and diagnostic technique, used for the separation of very large DNA molecules. It enables DNA ngerprint analysis which can di erentiate small number of sequence differences in genomes belonging to the same species. In this method the genome digested rst by a restriction enzyme into large DNA fragments analyzed by PFGE. While DNA ngerprinting is used in forensic medicine for identifying the suspects or paternity, it enabled also the development of molecular epidemiology of infectious diseases. It is possible to identify the source and transmission routes of an epidemic by DNA ngerprinting of the strains of infectious agent isolated from a series of samples. It is very important to follow the transmission routes of the infectious agent in a hospital, country or even around the world, in order to take the precautions to stop the epidemic. Although DNA ngerprinting by PFGE is a very important and powerful technique, its application is very cumbersome, requires long time, experienced personnel and expensive equipment. These hinder the method from being widely used. In a project that was completed four years ago, a method and an instrument called "Observable Real Time Electrophoresis (ORTE)" was developed and it won the Ak n C akmak c Award, of Turkish Technology Development Foundation. In this thesis work the aim is to develope an Observable Real Time PFGE system. Its e ciency is evaluated by comparing the genomic ngerprints of bacterial and fungal strains that were previously isolated from hospital infections by Observable and classical PFGE. Observable PFGE has the potential to eliminate the need for hazardous ultraviolet light and carcinogen ethidium bromide. It provided pictures at every stage of electrophoresis enabling to choose the best picture for analysis of the results. Since Observable PFGE enables to see the results early during electrophoresis we believe that compared to classical PFGE, it could save a lot of time and e ort, in many diverse applications, once the system that we present the technological feasibility in this thesis work, is developed and tested further.