Electrophoresis is a fundamental laboratory technique used to separate and analyze biological molecules based on their movement through a medium under the influence of an electric field. This process allows scientists to resolve complex mixtures of substances like DNA, RNA, and proteins into distinct components for study. The invention of gel electrophoresis was a progressive series of innovations that spanned several decades, transforming a basic physical phenomenon into a precise tool for molecular biology.
The Fundamental Principle of Electrophoresis
The underlying mechanism of electrophoresis is rooted in the physical properties of charged molecules. When biological molecules such as nucleic acids or proteins are dissolved in a conductive buffer solution and subjected to an electric field, they begin to migrate. Negatively charged molecules move toward the positive electrode (anode), while positively charged molecules move toward the negative electrode (cathode).
The rate at which a molecule travels is determined by its electrophoretic mobility, influenced primarily by its charge-to-mass ratio and the resistance encountered as it moves through the medium. A higher charge relative to size generally results in faster movement. By controlling the voltage and the properties of the separation medium, this differential movement can be harnessed to separate molecules effectively.
The Pioneering Era and Solution-Based Separation
The initial application of the electrophoretic principle to separate biological material began in the 1930s. Swedish chemist Arne Tiselius developed the first highly effective apparatus for this purpose, earning him the Nobel Prize in Chemistry in 1948. Tiselius’s technique, known as moving-boundary electrophoresis, performed the separation in a free, liquid buffer solution within a U-shaped glass tube.
This liquid-based method allowed for the separation of proteins, such as those found in blood serum, which were previously thought to be homogenous. However, the technique had limitations, primarily due to convection currents and the difficulty in isolating the separated components. Without a solid support structure, molecules tended to diffuse back together, resulting in low resolution and making it challenging to analyze complex mixtures.
The Transition to Gel Media and Modern Techniques
The transition from liquid to a solid support matrix was the defining step in the creation of true gel electrophoresis. This innovation introduced the concept of “molecular sieving,” dramatically improving resolution and stability. The earliest successful use of a gel medium was Oliver Smithies’ introduction of starch gel electrophoresis in 1955.
Starch forms a porous gel that acts like a physical sieve, separating molecules not just by charge but also by size and shape. Larger molecules are physically impeded by the gel’s matrix and move slower than smaller molecules. This capability immediately doubled the number of components that could be resolved in a sample, such as proteins in human blood serum.
Researchers quickly explored other synthetic and natural polymers. Polyacrylamide Gel Electrophoresis (PAGE) was developed in the early 1960s by Leonard Ornstein and Baruch Davis, becoming the standard for protein analysis. Polyacrylamide offers a highly controllable pore size and chemical inertness, providing superior resolution for proteins and small nucleic acid fragments.
For larger molecules, specifically DNA, the use of agarose gel became widespread in the late 1960s and early 1970s. Agarose forms a robust gel with larger pores ideal for separating DNA fragments up to tens of thousands of base pairs in length. The development of starch, polyacrylamide, and agarose gels established gel electrophoresis as a versatile and standard laboratory practice.
Modern Significance and Key Applications
The adoption of gel media revolutionized molecular biology by providing a reliable, high-resolution method for analyzing biological samples. The separation capabilities of gel electrophoresis provided the foundation for the rapid advancement of genetic research.
One of its earliest applications was in DNA sequencing, where polyacrylamide gels resolved DNA fragments differing by only a single nucleotide, enabling the reading of the genetic code. Gel electrophoresis also became a cornerstone of forensic science, facilitating DNA fingerprinting and profiling. In clinical diagnostics, the technique remains a common method for analyzing specific proteins or detecting genetic mutations.