It is a common practice in biological experimentation to separate molecules according to their relative mobilities through a polymer network under the influence of an electric potential. As commonly practiced, this technique of electrophoresis is done by applying several samples to one end of a thin slab of the polymer network or gel and applying an electric potential to the gel for a certain period of time, so that charged components of the samples move in parallel directions for various distances depending on their relative mobilities which depend on their chemical natures. After the period of electrophoresis, the location of the various components in the gel are determined by various means.
In some experiments, components of the samples are rendered radioactive and their locations are determined by detecting ionizations produced by the radioactive disintegrations. The most common means of identifying the position of the radioactive components in the gel is by the process of autoradiography, whereby a photographic emulsion is apposed to the gel for a period of time which may range from several hours to several days. Ionizing radioactivity causes silver grains in the emulsion to develop at positions correponding to the positions of the various radioactive components of the samples. The amount of radioactivity in the various components can be approximated by the optical density of the developed photographic emulsion.
This technique is used in procedures for the determination of the sequences of nucleotides in nucleic acid chains as described by Maxam et al., Proc. Natl. Acad. Sci., 74,560 (1977), by Sanger et al., Proc. Natl. Acad. Sci., 74,5463 (1977), and by Simoncsits et al., Nature 269,833 (1977). In these procedures, 4 or 5 samples containing nucleic acid chains labeled with phosphorus-32 migrate simultaneously in adjacent channels of a polyacrylamide electrophoresis gel for a predetermined period of time. The nucleotide sequence is determined by noting the relative positions on the autoradiograph of radioactively labeled components in the 4 or 5 adjacent channels. The radioactive components are well separated only toward the anodic end of the electrophoresis gel, and the same samples are usually electrophoresed two or three times for different time periods, or on different electrophoresis gels, so that all the radioactive components of interest are well separated at the anodic end of a gel at least once to acquire all the required sequence information.
An alternative to autoradiography for determining the positions of ionizing radioactivity in electrophoresis gels is by detecting ionizations produced in a gas by the use of a position sensitive radiation detector. Such devices have been reported by Gabriel et al., FEBS Letters 39,307 (1974), Markham et al., Nuclear Inst. and Methods 160,49 (1974), Goulianos et al., Anal. Biochem. 103,64 (1980), and Petersen et al., Nuclear Inst. and Methods 176,239 (1980). A device which uses a position sensitive radiation detector for detecting the position of radioactivity in electrophoresis or chromatography supports was disclosed in U.S. Pat. No. 4,019,057 to Bram.
The resolution with which these position sensitive detectors localize the radioactively labeled material depends on several factors, including the energy of the ionizing particle. Resolution is generally poorer for highly penetrating particles, such as the beta particles from phorphorus-32 (0.70 MeV average energy). Markham et al., Nuclear Inst. and Methods, 160,49 (1979), reported resolution of 2.2 to 3.8 mm full width at half maximum (FWHM) for localizing phosphorus-32 with their device. For experiments such as nucleic acid sequencing, this degree of resolution would not be adequate to resolve components in the direction of electrophoretic migration because the radioactive components are not separated by greater than 2 mm except at the anodic end of the electrophoresis gel. The degree of resolution could be adequate to resolve components in different channels corresponding to the various samples.
In many laboratories, DNA sequencing, has become a routine, but somewhat tedious procedure. The protocol for carrying out DNA sequencing using electrophoresis is a manual-intensive (rather than instrument-intensive) procedure which requires attention over an extended period of time to complete the analysis of a single sequencing experiment. Even after the electrophoresis experiment, which may require 24 hours, has been run, the exposure of the x-ray film necessary to get a read-out may require an additional 16-48 hours. Thus, it is not uncommon to have a two day hiatus between the completion of the DNA synthesis or modification and the analytical results from the electrophoresis.
USPTO Backgrounds
USPTO Backgrounds