Agarose gel electrophoresis

Agarose gel electrophoresis is a widely used analytical technique in biochemistry, molecular biology, genetics, and clinical chemistry for separating mixtures of macromolecules, primarily nucleic acids and certain proteins. The method exploits the migration of charged biomolecules through a semi-solid agarose matrix under the influence of an electric field, allowing fragments to be distinguished largely on the basis of size. Its simplicity, versatility, and ability to handle DNA within the size range commonly encountered in laboratories contribute to its status as one of the most frequently applied molecular separation methods.

Principles and Context

Agarose is one of the two major polysaccharide components of agar. When dissolved and cooled, it forms a three-dimensional gel matrix composed of helical agarose molecules aggregated into supercoiled bundles. These aggregates create a network of aqueous channels and pores through which biomolecules can migrate. As nucleic acids possess a negatively charged phosphate backbone, they move towards the anode during electrophoresis, and their movement is impeded by the gel matrix in a size-dependent manner. Smaller fragments pass more easily through the pores and therefore migrate more rapidly, whereas larger fragments experience greater hindrance.
The method is routinely used to analyse DNA resulting from restriction digests, PCR amplification, plasmid preparations, and genomic DNA fragmentation. It is also applicable to RNA and, in some cases, to large proteins or biological particles with effective radii exceeding approximately 5–10 nm. The separated fragments are typically visualised using intercalating dyes such as ethidium bromide or modern fluorescent alternatives, followed by illumination under ultraviolet or blue light.

Structure and Properties of Agarose Gel

Agarose gel exhibits physical and chemical properties that make it particularly suitable for electrophoresis. The matrix structure is stabilised by hydrogen bonds, which can be disrupted by heating. The gelling temperature differs from the melting temperature, allowing manipulation of the gel over a useful thermal range. Chemically modified variants—such as low-melting point and low-gelling agaroses—are employed when downstream recovery of DNA is required.
Key properties include:

• Large pore size, which allows free movement of DNA ranging from approximately 50 base pairs up to many kilobases. A typical 1% agarose gel has pore sizes estimated between 100 nm and 200–500 nm.
• High gel strength, enabling even very dilute gels (around 0.15%) to form usable slabs, although gels below about 0.2% become fragile.
• Relatively low density of charged groups, thereby reducing electroendosmosis compared to agar.

Agarose does contain negatively charged substituents, notably pyruvate and sulphate groups. These create electroendosmosis (EEO), a phenomenon in which water flows in the opposite direction to the movement of DNA. Excessive EEO can retard DNA migration and cause diffuse bands. Low-EEO agarose is therefore preferred for nucleic acid analysis, particularly when recovery of DNA for cloning, ligation, or polymerase chain reaction is required. High-EEO gels, however, may be chosen for specific applications such as serum protein electrophoresis.

Range and Resolution of Separation

Agarose gels permit separation over a wide fragment size range—from approximately 50 base pairs to nearly 20,000 base pairs in standard conditions. They possess lower resolving power than polyacrylamide gels for small DNA fragments but offer a greater workable size range. Standard agarose electrophoresis loses resolving efficiency at sizes above roughly 750 kilobases; however, by employing pulsed-field gel electrophoresis (PFGE), fragments exceeding 6 megabases can be separated by repeatedly alternating the direction of the electric field.
The concentration of agarose strongly influences resolution:

• Low-concentration gels (0.2–0.7%): permit the separation of very large DNA fragments.
• Medium-concentration gels (0.8–2%): suitable for most routine DNA analyses.
• High-concentration gels (>2%): required for small fragments but necessitate prolonged run times.

Migration of Nucleic Acids: Influencing Factors

Several variables govern the rate and pattern of DNA migration in agarose:

• Gel concentration: Higher concentrations reduce pore size and slow migration.
• Fragment size: Migration speed is inversely proportional to the logarithm of fragment length for most DNA sizes.
• Voltage: Higher voltage increases migration rate, but excessive voltage reduces resolution, particularly for large fragments.
• Buffer ionic strength: Affects heat generation, conductivity, and DNA mobility.
• DNA conformation: Supercoiled DNA migrates faster than linear or relaxed circular forms because of its compact shape.
• Presence of intercalating dyes: Dyes such as ethidium bromide alter charge and flexibility of DNA; they may reduce mobility by up to approximately 15% under some conditions.
• Contaminants: Salts or proteins can retard migration or distort bands.

For large DNA fragments exceeding 1 kilobase, the simple sieving model becomes insufficient. Instead, a reptation model—representing DNA as an elongated, snakelike molecule navigating through the pore network—more accurately depicts behaviour. Under strong electric fields, biased reptation occurs, with the leading end becoming oriented and pulling the remainder of the molecule forward.

Mechanisms of Separation

Multiple theoretical models have been developed to describe migration through polymer matrices:

• Ogston Model: Appropriate for small globular proteins or random coil DNA. Molecules diffuse through pores, and their probability of encountering obstructions increases with size.
• Reptation and Biased Reptation Models: Applied to long DNA that cannot freely pass through pores. DNA adopts an extended conformation and moves by snake-like deformation.
• Dynamic observations: Fluorescence microscopy has demonstrated that DNA stretches and contracts during movement, occasionally forming hooked or U-shaped configurations when caught on polymer fibres.

These models collectively emphasise that separation arises from the differential hindrance experienced by molecules of varying size.

Migration Anomalies

Certain irregularities may occur during electrophoresis:

• Smiley gels: Distorted band shapes caused by excessively high voltage in relation to gel concentration.
• Overloading: Excess sample slows migration and results in broad or smeared bands.
• Band inversion: Observed in field inversion gel electrophoresis when particular switching frequencies cause larger fragments to migrate more rapidly than smaller ones.
• Impurities: Residual salts, solvents, or proteins can modify mobility patterns.

Such anomalies highlight the importance of optimising gel composition and operating conditions.

General Procedure

Although specific protocols vary, agarose gel electrophoresis generally follows a consistent workflow:

1. Casting the Gel: Agarose is dissolved in electrophoresis buffer by heating. Once cooled to an appropriate temperature, it is poured into a casting tray containing a comb that forms sample wells. The gel solidifies into a uniform slab.
2. Preparing Samples: DNA samples are mixed with loading dye, which adds density and enables visual tracking during loading and electrophoresis.
3. Running the Gel: The solidified gel is placed in a tank filled with buffer. Samples and molecular weight markers are loaded into the wells. An electric field is applied, and DNA fragments migrate through the matrix.
4. Visualisation: After electrophoresis, the gel is stained if necessary and examined under ultraviolet or blue light to reveal separated bands.
5. Documentation and Recovery: Images may be captured for records, and particular DNA fragments can be excised for purification and further analysis.

Applications and Significance

Agarose gel electrophoresis is indispensable in modern molecular analysis. It supports the verification of DNA constructs in cloning workflows, assessment of PCR amplification success, estimation of fragment sizes, detection of plasmid topologies, and preparation of DNA for downstream sequencing or enzymatic manipulation. Its ease of use, low cost, and adaptability ensure its continuing relevance across research, forensic, medical, and educational laboratories.