A stain-free detection system for electrophoresis

Abstract

In this thesis, a novel stain free detection system for slab gel electrophoresis is examined. Currently, stained techniques are used to identify electrophoretic bands in gels. The stains utilized in these methods involve health risks since they are mutagenic. Also stains like EtBr are intercalating agents meaning they wedge themselves into the grooves of DNA and stay there. Since this includes a physical contact the stains remain in the DNA at the end of the experiment. This makes further use DNA very difficult. The stains need to be removed by chemical techniques which are timewise very costly. Also these operations are very inefficient, retrieve rates are very low which leads to waste of most of the analyte. The specific method we addressed aims to eliminate the use of any kind of stains and therefore inherently increase the end product efficiency. The method introduces the absorption method as the means of detection. The physical law governing the absorption technique is the Beer-Lambert Law. The Beer-Lambert Law defines the linear relation, which correlates absorption value to the analyte concentration, path length of the light and wavelength-dependent absorptivity coefficient. Although the proposed method is intended to apply to all kind of different analytes, to achieve primary goals and prove the feasibility of the method, as the first step detection of DNA molecules are targeted. Hence absorption pattern at a wavelength of 254 nm (which is characteristic absorption peak for DNA) is examined. After the method is proven to work robustly, it will be extended to all kind of different analytes. The unique approach used in the proposed detection system is the use of a scanning technique incorporated with absorption technique utilizing a high QE (Quantum efficiency) CCD camera as the detector. Experiments have been performed to determine the only unknown parameter -wavelength-dependent absorptivity coefficient a([gamma])- in the Beer-Lambert Law. The value of a([gamma]) is dependent on the wavelength and also on the transmission media. In our case wavelength of interest is 254 nm and the specific transmission media is agarose gel with 0.8% concentration. Each lane in the agarose gel is scanned under UV light and transmittance values at 254 nm are recorded as a function of position. The recorded data are processed to see the absorption pattern along the lane. The drop in the signal indicates the existence of a DNA band. Experiments have been performed on three different agarose gels, which are 4 mm thick, and with 0.8% concentration. The value of wavelength-dependent absorptivity coefficient a([gamma]) was determined within an error margin. The resolution of the method was found to be 4 ng/[mu]l.