Electrophoresis is a separation technique in which molecules or other units are separated, eg for analysis or purification purposes, by application of an electric field. This causes differential migration of the units, the rate of migration of each unit depending on its charge and frictional resistance (which is related to its size and shape). The technique is used to separate mixtures e.g. of proteins or DNA fragments, with the mixture being located on a suitable porous gel, typically of starch, agarose or polyacrylamide, referred to herein as an electrophoretic gel. Differential migration on application of the electric field results in either a one dimensional array of bands or a two dimensional array of spots. The resulting bands or spots can be detected by a number of techniques, including the following:
1. By staining with a suitable dye, rendering the bands or spots opaque and visible. PA1 2. By staining with fluorescent markers or labels which emit light when stimulated by an U/V radiation source. PA1 3. By use of radioactive markers or labels, e.g. using the technique known as autoradiography. PA1 treating the samples with fluorescent marking material so that components of the sample are fluorescently marked; PA1 applying the samples to an electrophretic gel; PA1 running the gel to effect electrophoresis causing differential migration of different components; PA1 irradiating the gel with a U/V source to render the marked components visible; PA1 detecting the pattern of light emanating from the marked components by means of a light sensitive charge coupled device (CCD), said CCD being a silicon CCD having a two-dimensional detector array; and PA1 cooling the CCD to a temperature less than -25 degrees C. during detection. PA1 a source of U/V radiation for illuminating an electrophoretic gel to which the fluorescently marked samples have been applied; PA1 means to control running of gel to effect electrophoresis, causing differential migration of different components of the samples giving rise to a pattern of light due to illumination of the gel by the U/V source; and
Analysis of electrophoresis results by these techniques has serious limitations. With stained dyes viewed in transmission, the faintest spots or bands have to be detected against a bright background, while the darkest spots or bands may transmit so little light that they are impossible accurately to measure with conventional detectors such as T.V. cameras or photographic films. Likewise, fluorescent marker dyes produce high level of fluorescent background since the dyes also bind to the gel as well as to the spots or bands. This makes it hard to see weak spots or bands as they are easily lost in the fluorescent background.
Autoradiography has all the problems associated with the handling of radioactive materials, plus poor resolution and extremely long visualisation times, of the order of days to weeks. However, autoradiography is able to detect the presence of smaller quantities of the components, e.g. protein or DNA fragments, than hitherto known techniques using dyes or markers. In particular, the use of fluorescent markers has been considered impractical for detection of components present in very small amounts, due to problems of sensitivity. However, protein and DNA fragments are frequently present only in these small amounts.
It is a primary object of this invention to provide a highly sensitive method for effecting analysis of the results of electrophoresis, without incurring the disadvantages associated with autoradiography techniques.