Pipetting sample into a gel well

Most life science researchers will run a gel at some point in their careers. Running a gel, formally called gel electrophoresis, is an effective method to separate biomolecules, such as nucleic acids and proteins, based on size. How does nucleic acid gel electrophoresis work, and how was the technique conceived?

What is gel electrophoresis?

Gel electrophoresis is a common laboratory technique in molecular biology to identify, quantify, and purify nucleic acids. Because of its speed, simplicity, and versatility, the method is widely employed for separation and analysis of nucleic acids. Using gel electrophoresis, nucleic acids in the range of approximately 100 bp–25 kb can be separated for analysis in a matter of minutes to hours, and separated nucleic acids can be recovered from the gels with relatively high purity and efficiency [1,2].

How does gel electrophoresis work?

Gel electrophoresis involves applying an electrical field across a gel matrix through which a mixture of nucleic acid fragments or protein fragments migrate. The rate of migration of molecules is based on size, charge, and structure of the molecule. The phosphate groups of the ribose-phosphate backbones of nucleic acids are negatively charged at neutral to basic pH (Figure 1A). As such, each nucleotide carries a net negative charge, and the overall charge of a nucleic acid molecule is proportional to the total number of nucleotides or its mass.

Therefore, DNA and RNA molecules carry a constant charge-to-mass ratio. When subjected to an electrical field, nucleic acids migrate from the negative electrode (cathode) toward the positive electrode (anode); the shorter fragments, which have a lower mass, move more rapidly than longer ones, with a higher mass, resulting in size-based separation (Figure 1B). The distances that nucleic acids fragments migrate in a gel matrix correlate with their size.

Figure 1. Mechanism of gel electrophoresis(A) Net negative charges carried by a nucleic acid fragment due to the phosphate backbone. (B) Negatively charged DNA samples are pipetted on the cathode side of the gel rig and will move towards the anode side. Separation of nucleic acid fragments in gel electrophoresis are based on fragment lengths.

The size-based separation is reliable when the fragments have a comparable structure. For linear double-stranded DNA fragments, migration distance is inversely proportional to the log of the molecular weight, within a certain range (Figure 2A) [3]. For approximate sizing, migration distances of samples are compared to migration distances of molecules of known sizes (molecular weight standards), sometimes referred to as ladders, which are often included in the gel run.

“Biased reptation” is a widely accepted model of nucleic acid mobility through a gel. According to the biased reptation model, the mechanism of migration is biased towards the applied electrical force and involves a snaking movement where the leading edge pulls the rest of the fragment (Figure 2B) [4,5]. This model has been visualized by fluorescence microscopy [6].

A brief history of gel electrophoresis

The use of electrophoresis to separate nucleic acids began in the early 1960s. At the time, nucleic acids were commonly fractionated by density gradient centrifugation based on sedimentation velocities, which are determined by size and conformation of the nucleic acids. Density gradient centrifugation was time intensive, required heavy equipment, and needed high inputs of samples. Researchers then explored DNA mobility in ionic, or electrolytic, solutions when an electrical field was applied; the process was termed electrophoresis [7,8].

1960s: Emergence of gel electrophoresis

In the 60s, scientists employed a gel matrix as a separation medium for nucleic acid electrophoresis, borrowing the technique from protein electrophoresis. In the mid- to late 1960s, the following gels were found to be successful matrices for DNA and RNA electrophoresis [9–11]:

  • Agar, a naturally derived carbohydrate 
  • Agarose, a component of agar
  • Polyacrylamide, a synthetic gel, and
  • Agarose-acrylamide composite 

With better understanding and advances in the manufacturing of agarose in the late 1960s, agarose gradually replaced agar as the preferred gel electrophoresis medium [12].

Figure 3. Timeline of early development of nucleic acid gel electrophoresis to present day. Notable current advancements include the introduction of microfluidic electrophoresis systems in 2010 and minimizing run times down to 18 minutes in 2021.

1970s: Expansion of gel electrophoresis applications

In the 1970s, the use of gel electrophoresis for separation and analysis of nucleic acids became more prevalent with the discovery of restriction enzymes and their application in recombinant DNA technology. Sucrose density gradient centrifugation, a common separation method at the time, involved cumbersome processes and did not adequately distinguish similarly sized DNA fragments from restriction digestion. 

In 1971, The viscosity of a solution was used empirically as an indicator of the success of restriction digests, since the digestion of DNA from larger to smaller fragments results in less viscous solutions [13]. Cloning of DNA fragments was revolutionized in 1971 when Danna and Nathans first reported sizing of restriction-digested fragments of SV40 DNA by polyacrylamide gel electrophoresis [14]. Although agarose and agarose-polyacrylamide gels were used for separation of RNA and single-stranded DNA in the late 1960s [15,16], the works on analysis of restriction-digested fragments by agarose gel electrophoresis were not published until 1973 [17,18].

1970s: Advancements in gel electrophoresis

In the early stages, electrophoresis methods relied on radioactive labeling for visualizing the separated nucleic acid fragments. Although highly sensitive, radiolabeling protocols are lengthy and necessitate training in radiation safety.

In 1972, two laboratories independently described gel staining with the fluorescent molecule ethidium bromide (EtBr), providing a simpler method for detecting nucleic acids. Ethidium bromide is highly sensitive and detects as low as a few nanograms of double-stranded DNA [19–21].

As of 2023, fluorescent stains that are safer and more sensitive and specific than EtBr are available, improving the detection of nucleic acids after gel electrophoresis.

The advent of “slab” gel formats around 1970, led to major developments in gel technology. Prior to slab gels, studies with gel electrophoresis were performed using tube gels cast into glass tubes of 1–3 mm diameter (Figure 4A). It was a very low-throughput method since each tube could accommodate only one sample. Tube gels were succeeded by vertical slab gels (Figure 4B), which were simpler to prepare and allowed running multiple samples simultaneously [22–24]. The horizontal slab gel for agarose (Figure 4C), similar to the present-day format, was first described by McDonell et al. in 1977 [24].

Figure 4. Common gel formats for electrophoresis. (A) Before “slab” gel formats, researchers employed the use of tube gels. (B) The vertical slab gel runs from top (wells) to bottom. (C) The horizontal slab gel runs from side to side, as it lays down horizontally.

1980s and beyond: Developments and electrophoresis applications

The 1980s saw the introduction of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE), which use a gradient of denaturants or temperature to separate DNA fragments based on sequence differences. Pulsed field electrophoresis made its arrival in 1983 [25]. The same year, agarose gels were first used to separate PCR products.

The first commercial vertical gel electrophoresis systems were introduced in the 1990s, which allowed for better resolution of nucleic acids and efficient use of space in the laboratory. The E-Gel system, the first pre-cast buffer-free system, launched in 1998. The first-generation E-Gel system consisted of a 12-well minigel contained in a clear, UV-transparent cassette. Ethidium bromide was incorporated into the gel for consistent DNA staining results. The minigel is placed within the specially designed E-Gel Base, which hooks up to any standard power supply. The system used a TAE-based dry buffer technology that eliminated the need for buffer preparation. This system decreased the gel run time to 25 minutes [26]. The 1990s also saw the advent of a new generation of stains, SYBR Green, for safer gel staining [27].

The dawn of the 21st century was marked by the development of new gel formulations, such as high-resolution agarose and acrylamide gels, for better separation of nucleic acid fragments of different sizes and lengths. Microfluidic electrophoresis systems, which use microscale channels and electrodes to separate and analyze nucleic acids with high sensitivity and throughput, were introduced in 2021 [28].

As gel electrophoresis matured, state-of-the-art electrophoresis systems, such as the E-Gel Power Snap Plus System, are integrated with digital imaging and analysis software. These devices have a sleek look, a user-friendly touch interface, and the run time is as few as ten minutes, which vastly improves the efficiency and convenience of the electrophoresis workflow. Development of new electrophoresis systems and technologies, such as droplet-based microfluidics and digital PCR, seek to continuously improve the resolution and sensitivity in nucleic acid analysis.

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