Gel Electrophoresis
Gel electrophoresis is a laboratory technique used to separate macromolecules — primarily DNA, RNA, or proteins — by size and charge as they migrate through a porous gel matrix under the influence of an electric field. Negatively charged nucleic acids migrate toward the positive electrode (anode), with smaller fragments travelling faster and further than larger ones, producing a pattern of bands that can be visualised by staining with ethidium bromide or SYBR Green and exposing to UV light. Gel electrophoresis is one of the most widely used techniques in molecular biology, underpinning applications from forensic DNA profiling and paternity testing to restriction mapping, PCR product verification, and Southern blotting.
Worked Example
Problem
A researcher runs a DNA ladder alongside three unknown PCR products on a 1% agarose gel at 100 V for 45 minutes. The ladder has bands at 100, 200, 500, 1000, and 2000 bp. The three unknowns migrate to the same position as the 500 bp and 1000 bp ladder bands respectively (band 1 co-migrates with 500 bp, band 2 with 1000 bp, band 3 is halfway between 500 and 1000 bp on a log scale). Estimate the size of band 3.
Solution
Step 1: On an agarose gel, migration distance is proportional to the log₁₀ of DNA fragment size. Step 2: Band 3 is at the midpoint between 500 bp and 1000 bp on a log scale. Step 3: Midpoint on log scale = (log₁₀(500) + log₁₀(1000)) / 2 = (2.699 + 3.000) / 2 = 2.850 Step 4: Antilog: 10^2.850 = 707 bp (approximately).
Answer
Band 3 is approximately 707 bp in size.
Comparison of Gel Electrophoresis Types
| Type | Matrix | Separates | Size Range | Common Application |
|---|---|---|---|---|
| Agarose gel (DNA) | 0.5–3% agarose | DNA/RNA by size | 100 bp – 50 kb | PCR analysis, restriction digest |
| Polyacrylamide gel (DNA) | 4–20% PAGE | DNA by size (high resolution) | 1 bp – 500 bp | DNA sequencing, SSCP |
| SDS-PAGE (protein) | Polyacrylamide + SDS | Proteins by molecular weight | 1 – 300 kDa | Western blot, protein analysis |
| Native PAGE (protein) | Polyacrylamide, no SDS | Proteins by size + charge | Variable | Enzyme activity assays |
| Pulsed-field gel (PFGE) | Agarose + alternating field | Very large DNA | 50 kb – 10 Mb | Bacterial typing, genome mapping |
Interactive Tools
Agarose Gel Electrophoresis Simulator — Bio-Rad
Educational resources and virtual electrophoresis demonstrations from Bio-Rad
Open ToolKhan Academy — Gel Electrophoresis
Illustrated tutorial on agarose gel electrophoresis principle and procedure
Open ToolWolframAlpha — DNA Size Calculator
Use to calculate log-linear interpolations for DNA band sizing from gel data
Open Tool
Wikimedia Commons, CC BY-SA
Related Terms
Restriction Enzyme
A restriction enzyme (restriction endonuclease) is a bacterial enzyme that cuts double-stranded DNA at or near a specific short nucleotide sequence called a recognition site, typically 4–8 base pairs in length. Bacteria produce these enzymes as part of a restriction-modification defence system to degrade foreign viral DNA that lacks the bacterium's own methylation marks. In molecular biology, type II restriction enzymes such as EcoRI and HindIII are indispensable tools for cutting DNA predictably to produce defined fragments with either blunt ends or "sticky" (cohesive) overhangs, enabling gene cloning, restriction mapping, and DNA fingerprinting.
Gene Cloning
Gene cloning is the process of making multiple identical copies of a specific gene or DNA fragment by inserting it into a vector (such as a plasmid or bacteriophage) and replicating it inside a host organism, typically Escherichia coli. The technique involves cutting both the target DNA and the vector with the same restriction enzyme to generate compatible sticky ends, ligating them with DNA ligase, transforming the recombinant construct into host cells, and selecting colonies that contain the insert. Gene cloning is foundational to modern biotechnology and medicine, enabling the production of recombinant proteins (such as insulin), gene therapy constructs, diagnostic probes, and transgenic organisms.
Post-translational Modification
Post-translational modification (PTM) refers to the covalent chemical changes made to a protein after its synthesis by the ribosome, altering its activity, localisation, stability, or interactions with other molecules. Common modifications include phosphorylation, glycosylation, ubiquitination, acetylation, and methylation, each carried out by specific enzymes at defined amino acid residues. PTMs vastly expand the functional diversity of the proteome beyond what is encoded in the genome, and their dysregulation underpins diseases ranging from diabetes to neurodegeneration.
From Greek "elektron" (amber, for electrical properties) + Greek "phoresis" (carrying, from "pherein" to carry) + "gel" (from Latin "gelu," frost, referring to the gel-like matrix). The technique was developed in the 1950s–1960s, with agarose gel electrophoresis of DNA becoming standard after the work of Sharp, Sugden, and Sambrook in the early 1970s.