Gel

Gel Electrophoresis, using either agarose or polyacrylamide, is a cornerstone technique in the clinical molecular lab. It allows us to separate DNA (and RNA) fragments based on size, providing critical information for verifying PCR results, analyzing genetic variations (like RFLPs), assessing nucleic acid quality, and serving as a component of many other molecular workflows

Gel electrophoresis technique is fundamental for visualizing and analyzing nucleic acids (DNA and RNA). Think of it as a molecular race track or sieve where we separate molecules based primarily on their size

Gel Electrophoresis: Separating Molecules by Size

• The Core Principle: It’s beautifully simple!
  1. Charge Nucleic acids (DNA/RNA) have a negatively charged phosphate backbone
  2. Electric Field When placed in an electric field (generated by a power supply connected to an electrophoresis chamber), these negatively charged molecules will migrate towards the positive electrode (anode, usually red)
  3. The Matrix (The Gel) This is where the separation happens! The gel acts as a porous matrix, a bit like a microscopic sieve or obstacle course
  4. Size Matters Smaller molecules navigate this matrix more easily and quickly, traveling further down the gel in a given amount of time. Larger molecules get tangled up more, move slower, and therefore travel shorter distances. So, smaller fragments migrate further, larger fragments stay closer to the starting wells.

We primarily use two types of gels in the clinical molecular lab for nucleic acid separation: Agarose and Polyacrylamide

Agarose Gel Electrophoresis

This is probably the most common type you’ll encounter for routine DNA analysis

• What it is: Agarose is a natural polysaccharide extracted from seaweed. It’s purchased as a powder
• How it’s made
  1. Mix agarose powder with a buffer solution (like TAE - Tris-acetate-EDTA, or TBE - Tris-borate-EDTA). The buffer provides ions for conductivity and maintains pH
  2. Heat the mixture (e.g., in a microwave) until the agarose dissolves completely (solution becomes clear)
  3. Cool it slightly (so you don’t melt the plastic tray!)
  4. Pour the molten agarose into a casting tray containing a “comb”. The comb creates the wells where you’ll load your samples
  5. Let it solidify (it forms a semi-solid, Jell-O like matrix with pores)
  6. Place the solidified gel (often in its tray) into the electrophoresis chamber (“gel box”), cover it with buffer, and carefully remove the comb
• Pore Size & Resolution
  • Forms relatively large pores compared to polyacrylamide
  • The pore size can be adjusted by changing the concentration of agarose (e.g., 0.8% to 2.5%)
    • Lower % Agarose: Larger pores, better for separating very large DNA fragments (e.g., > 5-10 kilobases, kb). Gel is more fragile
    • Higher % Agarose: Smaller pores, better for separating smaller DNA fragments (e.g., 50 base pairs, bp, to 2 kb). Gel is firmer
  • Generally used to resolve DNA fragments that differ in size by at least 50-100 bp, depending on the fragment size and gel percentage
• Typical Format: Horizontal “submarine” gels, where the gel lies flat in the buffer tank
• Clinical Applications
  • Confirming PCR products: Checking if your PCR worked and if the amplified product is the expected size. This is super common!
  • Restriction Fragment Length Polymorphism (RFLP) analysis: Analyzing fragment sizes after cutting DNA with restriction enzymes (a more traditional technique)
  • Checking plasmid integrity.
  • Quick check of genomic DNA quality/presence (though high MW DNA doesn’t resolve well into distinct bands)
• Pros: Easy and quick to prepare, relatively non-toxic (the agarose itself), good for a wide range of larger DNA sizes
• Cons: Lower resolving power than polyacrylamide (can’t easily separate very small fragments or those differing by only a few base pairs), can be fragile at low concentrations

Polyacrylamide Gel Electrophoresis (PAGE)

When you need higher resolution, especially for smaller fragments or even single-base differences, PAGE is the way to go

• What it is: A synthetic gel matrix created by polymerizing acrylamide monomers with a cross-linking agent (usually N,N’-methylenebisacrylamide or “bis-acrylamide”)
• How it’s made
  1. Mix acrylamide and bis-acrylamide monomers, buffer, and water
  2. Initiate polymerization chemically using catalysts like APS (ammonium persulfate) and TEMED (tetramethylethylenediamine). Caution: Unpolymerized acrylamide is a neurotoxin! Handle with care, gloves mandatory
  3. The solution is quickly poured between two glass plates separated by spacers, with a comb inserted at the top to form wells
  4. Polymerization occurs, forming a thin, firm gel slab
• Pore Size & Resolution
  • Forms much smaller, more uniform pores than agarose
  • Pore size is precisely controlled by:
    • Total acrylamide concentration (%T): Higher concentration = smaller pores
    • Amount of cross-linker (%C): Affects pore structure
  • Provides very high resolution, capable of separating molecules that differ by only a single base pair (essential for older Sanger sequencing methods) or distinguishing small DNA/RNA fragments very clearly
  • Excellent for fragments from ~5 bp up to ~1000 bp (1 kb)
• Typical Format: Vertical gels, where the thin gel slab stands upright between the buffer chambers
• Clinical Applications
  • Analysis of small PCR products: requiring high resolution
  • Mutation detection techniques: like Single-Strand Conformation Polymorphism (SSCP) or heteroduplex analysis (less common now but rely on PAGE)
  • Historically crucial for Sanger sequencing fragment separation
  • Analysis of short tandem repeats (STRs) - though capillary electrophoresis is now standard
  • Also the standard for protein separation (SDS-PAGE), but our focus here is nucleic acids
• Pros: Superior resolution for small fragments, gels are mechanically stable
• Cons: Acrylamide monomer is toxic before polymerization, gels are more complex and time-consuming to prepare than agarose

Comparison: Agarose vs. Polyacrylamide

Feature	Agarose Gel	Polyacrylamide Gel (PAGE)
Material	Natural Polysaccharide (Seaweed)	Synthetic Polymer (Acrylamide)
Pore Size	Larger, Less Uniform	Smaller, More Uniform & Controllable
Resolution	Lower (Good for >50 bp difference)	High (Can resolve 1 bp difference)
Typical Range	~50 bp to >20 kb	~5 bp to ~1 kb
Typical Format	Horizontal (“Submarine”)	Vertical
Primary NA Use	PCR products, RFLP, Plasmids	Sequencing (classic), Small fragments, Mutation analysis
Toxicity	Gel itself is non-toxic	Monomer is a neurotoxin
Preparation	Simple (dissolve, pour, cool)	More complex (polymerization rxn)

Essential Components & The Process

• Gel: Agarose or Polyacrylamide matrix

• Electrophoresis Buffer (TAE or TBE): Fills the tank, covers the gel, provides ions for current, maintains pH

• Power Supply: Provides the electric current (controlled voltage)

• Gel Box/Apparatus: Holds the gel and buffer, has electrodes (+ and -)

• Samples: Your DNA/RNA! Usually mixed with Loading Dye

  • Loading Dye: Contains glycerol or sucrose (makes sample dense so it sinks into the well) and one or more tracking dyes (e.g., bromophenol blue, xylene cyanol) that migrate at predictable rates, allowing you to monitor the run’s progress. Loading dye does NOT stain the DNA itself

• DNA Size Standard (“Ladder”): A mixture of DNA fragments of known sizes, run in one lane for comparison to estimate the size of your sample fragments

• Detection/Visualization System: How you see the results after the run

• Basic Steps

  1. Prepare the gel
  2. Place gel in the buffer-filled tank
  3. Mix samples with loading dye
  4. Carefully load samples and DNA ladder into the wells
  5. Connect the power supply (ensure correct orientation: wells near negative electrode!)
  6. Run the gel at a set voltage/current for a specific time
  7. Stop the run before the tracking dye runs off the end
  8. Stain the gel (if stain wasn’t included during casting)
  9. Visualize the separated fragments (bands) using an appropriate imaging system (e.g., UV light for EtBr/SYBR)

Detection: Seeing the Bands

Since nucleic acids are invisible to the naked eye, we need to stain them. Common methods:

• Intercalating Dyes: These molecules slip in between the stacked base pairs of the DNA double helix. When exposed to specific wavelengths of light (usually UV or blue light), they fluoresce, revealing the location of the DNA bands
  • Ethidium Bromide (EtBr): The classic. Very sensitive, fluoresces orange under UV. BUT it’s a potent mutagen - handle with extreme caution and dispose of properly!
  • SYBR Safe, SYBR Gold, GelRed, GelGreen: Newer, much safer alternatives designed to be less mutagenic. Often fluoresce green or orange under blue or UV light. They are often preferred in clinical labs now
• Autoradiography: Used if the nucleic acids were labeled with radioactivity (e.g., ³²P). The gel is exposed to X-ray film, and the radioactive bands create dark spots on the developed film. (Less common now due to safety and disposal issues)

Key Factors Influencing Migration Rate

• Molecular Size: The primary determinant. Smaller = faster
• Agarose/Acrylamide Concentration: Higher concentration = smaller pores = slower migration, especially for larger fragments
• Voltage: Higher voltage = faster migration. But too high generates heat, which can melt agarose gels or cause band distortion (“smiling”)
• Conformation of DNA: Supercoiled plasmids, nicked circles, and linear DNA of the same mass migrate differently (supercoiled is fastest in agarose). Less critical for PCR products which are linear
• Buffer Composition & Ionic Strength: Affects conductivity and pH. Buffer exhaustion during long runs can affect migration

Key Terms

• Electrophoresis: A technique used to separate charged molecules, such as DNA, RNA, or proteins, based on their differential migration rate through a matrix (the gel) in response to an applied electric field. Nucleic acids, being negatively charged, migrate towards the positive electrode (anode)
• Gel Matrix: The porous medium (either agarose or polyacrylamide) through which molecules are separated during electrophoresis. It acts as a sieve, impeding the movement of larger molecules more than smaller ones
• Agarose Gel: A type of electrophoresis gel matrix made from a natural polysaccharide (agarose) extracted from seaweed. It forms relatively large pores and is typically used in a horizontal format (“submarine gel”) to separate larger DNA fragments (approx. 50 bp to >20 kb) with moderate resolution
• Polyacrylamide Gel Electrophoresis (PAGE): A type of electrophoresis using a synthetic gel matrix formed by polymerizing acrylamide and a cross-linker (bis-acrylamide). PAGE forms smaller, more uniform pores than agarose, providing higher resolution, especially for separating smaller nucleic acid fragments (approx. 5 bp to 1 kb) or molecules differing by only a single base pair. It’s typically run in a vertical format
• Well: An indentation or small reservoir created in the gel (using a comb during casting) into which the nucleic acid sample, mixed with loading dye, is loaded before starting electrophoresis
• Loading Dye (or Loading Buffer): A solution mixed with the nucleic acid sample before loading it onto the gel. It contains a density agent (like glycerol or sucrose) to help the sample sink into the well and one or more visible tracking dyes (like bromophenol blue or xylene cyanol) to monitor the progress of the electrophoresis run. It does not stain the nucleic acid itself
• DNA Ladder (or Size Standard/Marker): A commercially prepared mixture of DNA fragments of known, predefined sizes. It is run in a lane alongside experimental samples during gel electrophoresis to allow for the estimation of the sizes of the sample fragments by comparison
• Intercalating Dye: A type of fluorescent molecule (e.g., Ethidium Bromide, SYBR Safe, GelRed) that inserts itself between the stacked base pairs of a DNA double helix (or sometimes binds to RNA). When exposed to specific wavelengths of light (often UV or blue light), these dyes fluoresce, allowing visualization of the nucleic acid bands in the gel
• Resolution (in Electrophoresis): The ability of a gel electrophoresis system to distinguish between two molecules of similar size. Higher resolution means the system can separate bands that are closer together in size (e.g., PAGE has higher resolution than agarose for small fragments)
• Migration Rate: The speed and distance traveled by a molecule through the gel matrix during electrophoresis. It is primarily determined by the molecule’s size (smaller moves faster/further), the gel concentration (higher % slows migration), and the applied voltage (higher voltage increases speed)