Table of Contents
Principle of Gel Electrophoresis
Gel electrophoresis is a method used to separate charged biological molecules—most commonly DNA, RNA, or proteins—according to their size (and sometimes shape or charge) as they move through a gel in an electric field.
In genetic engineering, gel electrophoresis is especially important for:
- Checking the size of DNA fragments (e.g., after restriction enzyme digestion or PCR).
- Isolating specific fragments from a mixture.
- Analyzing the results of cloning or sequencing preparations.
Why Molecules Move in a Gel
Charged molecules in solution move in an electric field: negatively charged molecules migrate towards the positive pole (anode), positively charged ones towards the negative pole (cathode).
- DNA and RNA are strongly negatively charged because of their phosphate groups, so under typical conditions they move from the negative to the positive electrode.
- Proteins can carry different net charges depending on pH, so specialized conditions are needed for protein electrophoresis (covered elsewhere).
The gel itself acts as a molecular “sieve”:
- It contains pores.
- Smaller molecules move more easily through these pores and migrate faster.
- Larger molecules are slowed down and remain closer to the starting point.
Types of Gels
Two main gel materials are used in genetic engineering:
Agarose Gels
Agarose is a polysaccharide extracted from certain types of algae.
Characteristics:
- Forms a relatively loose network with large pores.
- Commonly used for separation of larger DNA and RNA fragments (from about a few hundred base pairs up to tens of thousands of base pairs).
- Gel concentration (e.g., 0.7%, 1%, 2% agarose) controls pore size:
- Lower percentage → larger pores → better separation of large fragments.
- Higher percentage → smaller pores → better separation of small fragments.
Advantages:
- Easy and safe to prepare: agarose powder is dissolved in buffer by heating and then poured into a mold to cool and solidify.
- Gels are relatively robust and easy to handle and cut.
Polyacrylamide Gels
Polyacrylamide is a synthetic polymer formed by chemical cross‑linking.
Characteristics:
- Forms a much finer network with smaller, more uniform pores than agarose.
- Used for:
- Very small DNA or RNA fragments (e.g., less than ~500 bases).
- High‑resolution separation (single‑base differences).
- Protein electrophoresis (with additional specific conditions).
Compared to agarose:
- Provides higher resolution for small fragments.
- Preparation and handling are more technically demanding.
- Acrylamide monomer is toxic before polymerization, requiring more safety precautions.
Components of a Gel Electrophoresis Setup
A basic DNA gel electrophoresis system consists of:
Gel Casting Tray and Comb
- A flat tray in which the liquid gel solution is poured.
- A comb is placed before the gel sets. The teeth of the comb form wells (small slots) where the samples will later be loaded.
- After the gel solidifies, the comb is removed, leaving wells at one end of the gel.
Buffer Solution
The gel is placed in a tank filled with a conductive buffer solution. The same or similar buffer is mixed into the gel before it solidifies.
Functions of the buffer:
- Conducts electricity (ions carry the current).
- Maintains a stable pH to protect DNA and ensure stable charge.
- Minimizes heating during electrophoresis.
Common DNA electrophoresis buffers include:
- TAE (Tris–acetate–EDTA).
- TBE (Tris–borate–EDTA).
Power Supply and Electrode Chamber
- Two electrodes: a negative electrode (cathode) and a positive electrode (anode) are placed at opposite ends of the gel chamber.
- The gel is oriented so that:
- Wells are closer to the negative electrode.
- DNA migrates through the gel toward the positive electrode.
- A power supply applies a constant voltage (e.g., 50–150 V for typical agarose gels), creating the electric field.
Preparing Samples for DNA Gel Electrophoresis
DNA Samples
Typical DNA samples in genetic engineering include:
- Restriction enzyme digestion products.
- PCR products.
- Plasmid DNA preparations.
- DNA markers or ladders (standard fragments of known sizes, used as references).
Samples are often purified or at least cleaned up enough so that:
- Salt concentration is not too high (which can disturb migration).
- Proteins and other contaminants are reduced.
Loading Buffer (Sample Buffer)
Before loading samples into the wells, they are mixed with a loading buffer. This buffer usually contains:
- Tracking dyes
- Colored molecules (e.g., bromophenol blue, xylene cyanol) that migrate through the gel.
- Allow the experimenter to monitor how far the electrophoresis has progressed.
- Do not bind to DNA and are visible during the run.
- Density agents
- Substances like glycerol or sucrose make the sample denser than the surrounding buffer.
- Ensures that the sample sinks into the well instead of diffusing away into the buffer.
- Sometimes EDTA or other stabilizing agents
- To protect DNA from degradation by chelating metal ions needed by nucleases.
Running the Gel
Loading the Gel
- The solidified gel is placed in the electrophoresis chamber and covered with buffer.
- Wells are checked to ensure they are fully submerged and intact.
- Using a micropipette, the mixture of DNA sample and loading buffer is carefully pipetted into each well.
- At least one lane is reserved for a DNA size standard (DNA ladder).
Applying the Electric Field
- The lid with electrodes is placed on the chamber, ensuring correct orientation:
- Wells must be near the negative electrode.
- The power supply is turned on and set to the desired voltage.
- During the run:
- Negatively charged DNA migrates through the gel toward the positive electrode.
- Smaller fragments move faster and farther than larger fragments.
- The tracking dyes show the progress of the front of migrating molecules.
Factors Influencing Migration
Key factors that affect how DNA fragments move:
- Size (length) of DNA fragments
- Main determinant: smaller fragments move faster.
- Agarose (or polyacrylamide) concentration
- Higher concentration → smaller pores → slower migration and better resolution of small fragments.
- Lower concentration → larger pores → faster migration of large fragments.
- Voltage
- Higher voltage → faster movement but more heat and potential distortion (band “smiling” or poor resolution).
- Lower voltage → better separation but longer run time.
- Gel and buffer composition
- Different buffers or additives can change migration behavior.
- Gel thickness and length also influence resolution and run time.
Visualizing DNA in the Gel
After electrophoresis, DNA bands in the gel are not visible by eye without a stain. Visualization uses dyes that bind to DNA and fluoresce or absorb light.
Intercalating Dyes
Traditional and widely used dyes include:
- Ethidium bromide (EtBr) – intercalates between DNA bases and fluoresces under UV light.
- Safer alternatives (e.g., SYBR Safe, GelRed, GelGreen) – designed to reduce toxicity and mutagenicity.
Two main staining approaches:
- Pre‑staining (in‑gel staining)
- Dye is added to the gel solution before it sets and/or to the running buffer.
- DNA picks up the dye during migration.
- Bands can sometimes be watched as they form.
- Post‑staining
- The gel is soaked in a solution of dye after the run.
- Dye binds to the DNA already separated in the gel.
Detection
- For fluorescent dyes (EtBr, SYBR, etc.):
- Gel is placed on a UV or blue‑light transilluminator.
- Bands appear as bright lines at different positions in each lane.
- A camera system can record images for documentation.
- For some dyes that absorb visible light:
- Gels can be viewed directly under normal light after staining and destaining.
Interpreting Results
Interpretation of a DNA agarose gel usually involves:
Comparing to a DNA Ladder
A DNA ladder contains many fragments of known lengths. By comparing:
- The migration distance of unknown fragments to the bands of the ladder.
- You can estimate the size (in base pairs) of your fragments.
Patterns can show:
- Whether digestion with restriction enzymes was complete (appearance of expected bands).
- Whether PCR amplified a fragment of the expected size.
- Whether a plasmid is present and in which form (supercoiled, linear, nicked often migrate differently).
Qualitative and Semi‑Quantitative Analysis
From the bands’ appearance:
- Presence/absence: Does a DNA fragment exist in the sample?
- Relative quantity: Band brightness roughly reflects the amount of DNA (brighter band → more DNA).
- Purity: Smears or multiple unexpected bands can indicate degradation or contamination.
Isolating DNA from Gels (Gel Extraction)
Gel electrophoresis is not only an analytical method; it is also preparative:
- A DNA mixture is separated on a gel.
- The band corresponding to the desired fragment size is visualized under safe light (dim UV or blue light).
- Using a clean scalpel or blade, the gel piece containing that band is cut out.
- The DNA is then recovered from the gel piece using a gel extraction protocol:
- The gel slice is dissolved with a specialized buffer.
- DNA is bound to a purification matrix (often a silica column).
- Impurities and gel components are washed away.
- Purified DNA is eluted in a small volume.
This purified fragment can then be used for:
- Cloning into a vector.
- Sequencing.
- Further manipulations in genetic engineering.
Specialized Forms of Gel Electrophoresis
For specific analytical tasks, variations of the basic method are used (details covered in related chapters, but important to recognize):
Polyacrylamide Gel Electrophoresis (PAGE)
- Provides very high resolution.
- Used for:
- Short DNA or RNA fragments.
- Many protein studies (including SDS‑PAGE).
Denaturing Gels
- Contain agents (e.g., urea, formamide) or conditions (high temperature) that keep DNA or RNA single‑stranded and denature secondary structures.
- Used to separate single‑stranded nucleic acids by length.
Pulsed‑Field Gel Electrophoresis (PFGE)
- The direction of the electric field is periodically changed (“pulsed”).
- Allows separation of very large DNA molecules (up to millions of base pairs), which would otherwise move together in a standard agarose gel.
- Useful for analyzing whole chromosomes in microbes, or large genomic fragments.
Role of Gel Electrophoresis in Genetic Engineering Workflows
In the context of genetic engineering, gel electrophoresis is used at many stages, for example:
- After restriction digestion: to check if DNA has been cut into fragments of expected sizes.
- After PCR: to verify the presence and size of amplified products.
- During cloning:
- To confirm insertion of the correct fragment into a vector.
- To screen colonies by analyzing their plasmid DNA.
- Before sequencing: to assess DNA quality and fragment length.
- In combination with other methods:
- After PCR (from “Methods of Investigation: PCR”) to visualize amplified DNA.
- After digestion with restriction enzymes (from the corresponding section) to analyze restriction patterns.
- Prior to transfer to membranes in hybridization techniques (e.g., Southern blotting) for further analysis.
Thus, gel electrophoresis serves as a central “check‑point” technique, allowing researchers to see the size and approximate amount of DNA fragments they are working with, and to physically separate and purify specific pieces of DNA needed for downstream genetic engineering procedures.