Lesson 20 of 34 · Separation and Detection
Gel Electrophoresis
Overview
Gel electrophoresis is the workhorse method for separating nucleic acids by size. Once DNA or RNA has been isolated and manipulated, a laboratory almost always needs to ask a basic question of the product: how big are the fragments, how many distinct sizes are present, and is the material intact? Electrophoresis answers all three. An electric field pulls the nucleic acid through a porous gel, and because larger molecules are held back more than smaller ones, the sample sorts itself into size-ordered bands that can be visualized and measured 1.
This lesson explains why nucleic acids separate by size at all, the two gel materials used to do it, the buffers and size markers involved, how to read the result, and what the method is used for at the bench.
Why nucleic acids migrate by size
Two facts about nucleic-acid structure make the separation work.
First, every nucleic acid carries a uniform negative charge. The sugar-phosphate backbone has one phosphate group per nucleotide, and each phosphate is negatively charged at the pH of laboratory buffers (this backbone-charge property is established in the nucleic-acid structure lesson). A DNA or RNA molecule is therefore a polyanion whose total charge is roughly proportional to its length. When placed in an electric field, it moves toward the positive electrode, the anode 2. Because charge scales with size, the charge-to-mass ratio is nearly the same for fragments of every length, so charge alone does not separate them.
Second, the gel is a molecular sieve. It is a meshwork of pores, and a nucleic acid must thread its way through that mesh to move toward the anode. Small fragments slip through easily and travel far; large fragments are retarded by the matrix and travel less 3. The combination — a uniform pull that does not discriminate by size, acting through a mesh that does — means the distance a fragment migrates depends mainly on its size, not its sequence or charge.
cathode (-) anode (+)
| wells |
| [====] large fragments (move slowly) ---> |
| [====] |
| [====] small fragments (move quickly) ----> |
| |
+----------- direction of migration ----------->
negatively charged DNA/RNA --> toward (+)
Agarose versus polyacrylamide
The gel material sets the size range and the resolution.
Agarose gels are cast from a seaweed-derived polysaccharide simply by melting agarose in buffer and letting it set. They are easy to prepare, non-toxic, and have large pores, which suits them to larger fragments and lower resolution. The agarose concentration tunes the useful range: a low percentage (around 0.5–0.7%) resolves large fragments of many kilobases, while a higher percentage (around 2%) has smaller pores and resolves shorter fragments of a few hundred base pairs 1. Agarose is the everyday choice for checking PCR products and restriction digests.
Polyacrylamide gels are formed by chemically polymerizing acrylamide into a much finer, more uniform mesh. The smaller pore size gives higher resolution for small fragments — short enough to separate molecules differing by a single base in length, which agarose cannot do. Polyacrylamide is therefore used where fine sizing matters, such as small oligonucleotides and the readout of sequencing reactions 1. The trade-off is a more involved preparation and a narrower size range than agarose.
Property Agarose Polyacrylamide
-------- ------- --------------
Pore size large small, fine mesh
Resolution lower higher (single-base)
Fragment range ~100 bp to ~25 kb a few bp to ~1 kb
Typical use PCR products, digests oligos, sequencing reads
Buffers
Electrophoresis is run in a conductive buffer that both carries the current and holds the pH steady so the backbone stays negatively charged. Two buffers are standard for nucleic acids: TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). Both work well; TAE gives slightly better separation of large fragments, while TBE has greater buffering capacity for long runs 1. The EDTA in each chelates metal ions that nucleases need, helping protect the sample during the run.
Estimating size: the log-linear relationship and the ladder
Over the useful range of a gel, the distance a fragment migrates is approximately linear in the logarithm of its size — that is, migration distance falls off as fragment length rises, and plotting distance against the log of size gives a nearly straight line 1. This relationship is what lets a gel report sizes rather than just rank them.
To convert distance into an actual size, a size ladder (also called a marker or standard) is run in its own lane alongside the samples. A ladder is a mixture of fragments of known lengths, so it produces a column of reference bands. The size of an unknown band is read off by comparing how far it migrated to the ladder bands that bracket it 2. Without a ladder a gel shows relative sizes only; with one it gives estimates in base pairs.
Visualization
Nucleic acids are colorless, so the gel must be stained to be seen. The common approach uses an intercalating or fluorescent dye that inserts between the stacked bases of the nucleic acid and fluoresces brightly when bound. Under ultraviolet (UV) or blue-light illumination, the stained bands glow while the empty gel stays dark, producing the familiar image of bright bands on a dark background 1. Stains may be cast into the gel, added to the buffer, or applied after the run. This whole-sample staining is distinct from attaching a label to a specific target, the subject of the nucleic-acid labeling lesson.
Reading a gel
A gel image is read by lane and by band:
- Wells are the slots at the loading end (near the cathode) where each sample was pipetted in; migration proceeds away from them toward the anode.
- A lane is the vertical track a single sample travels down. One lane is reserved for the size ladder.
- A band is a sharp zone where many fragments of the same size have comigrated and piled up. Its position indicates size; a fainter or thicker band carries less or more material.
Lane 1 Lane 2 Lane 3 Lane 4
Ladder Sample A Sample B Sample C
+-------+ +-------+ +-------+ +-------+
wells | ===== | | ===== | | ===== | | ===== | <- loading end (-)
| | | | | | | |
large | -10kb | | | | ----- | | |
| --5kb | | ----- | | | | |
| --2kb | | | | | | ----- |
| --1kb | | ----- | | | | |
small | -0.5kb| | | | ----- | | ----- |
+-------+ +-------+ +-------+ +-------+
|
v toward anode (+)
Read each sample band against the ladder to estimate its size.
Applications
Several routine laboratory tasks rest on gel electrophoresis:
- Checking PCR products. A single band of the expected size confirms the reaction made the intended amplicon; extra bands or a smear signal nonspecific products or contamination 2.
- Restriction digests. Cutting DNA with restriction enzymes yields fragments whose sizes form a predictable pattern; resolving them on a gel is the basis of restriction fragment length polymorphism (RFLP) analysis, taken up with restriction enzymes and RFLP 1.
- Assessing nucleic-acid integrity. Intact high-molecular-weight DNA runs as a tight high band, whereas degraded material appears as a low smear; this electrophoretic check complements the spectrophotometric and fluorometric measures discussed in the nucleic-acid quality and quantity lesson 2.
Where this goes next
Slab-gel electrophoresis is robust and inexpensive, but it is manual, modest in resolution, and read by eye. The next lesson takes up capillary electrophoresis, which performs the same size separation inside a thin fluid-filled capillary with automated detection. It delivers higher resolution and quantitative, single-base sizing, and is the platform behind automated fragment analysis and Sanger sequencing — the higher-resolution successor to the slab gel.
References
- Michael R. Green, Joseph Sambrook. Molecular Cloning: A Laboratory Manual. 4th ed. Cold Spring Harbor Laboratory Press. 2012. verified
- Lela Buckingham. Molecular Diagnostics: Fundamentals, Methods, and Clinical Applications. 3rd ed. F.A. Davis Company. 2019. verified
- Bruce Alberts, Rebecca Heald, Alexander Johnson, David Morgan, Martin Raff, Keith Roberts, Peter Walter. Molecular Biology of the Cell. 7th ed. W. W. Norton & Company. 2022. verified