Electrophoresis Technique Used for DNA Analysis

Why Is Electrophoresis Important in DNA Studies?
Electrophoresis is an electrokinetic technique used to separate charged particles in a fluid by applying an electric field. In life sciences, it is widely used to separate biomolecules such as DNA and proteins.

Although different types of electrophoresis exist, they all require certain basic components—an electrical power source, a buffer solution, and a supporting medium like a gel. By applying an electric field, molecules can be separated based on their size, charge, purity, and density.

Because of its ability to precisely distinguish and isolate DNA fragments, electrophoresis has become an essential tool in molecular biology and genetic research.

1.0Electrophoresis

Electrophoresis is a laboratory method used to separate DNA, RNA, or protein molecules based on their size, type, density, and electrical charge. An electric current is passed through a gel, causing the molecules to move and separate.

The gel contains tiny pores that act like a sieve—smaller molecules travel more quickly, while larger ones move more slowly. By adjusting the experimental conditions, scientists can separate molecules within a specific size range.

2.0Principle of Electrophoresis

Molecules naturally carry electrical charges. When an electric field is applied, these charged molecules experience a force that causes them to move. The strength of this force depends on the magnitude of the molecule’s charge—the greater the charge, the stronger the force acting on it.

As a result, each molecule migrates through the supporting medium (such as a gel) at a rate determined by its size and mass. Smaller or more highly charged molecules move faster, while larger ones move more slowly.

Electrophoresis is widely used in the analysis of DNA, RNA, and proteins. It is an important medical and laboratory technique for separating and examining molecules present in fluid samples.

3.0Types of Electrophoresis

Gel Electrophoresis

• Gel electrophoresis is the most widely used technique for DNA separation.
• The gel acts as a molecular sieve, allowing smaller fragments to move more easily.

Capillary Electrophoresis

• Utilizes thin capillary tubes filled with a gel or polymer.
• Separation is faster and allows for high-resolution analysis.
• Common in automated DNA sequencing.

Other Variants

• Paper electrophoresis (less common for DNA).
• Isoelectric focusing (used for proteins).
• Pulsed-field gel electrophoresis (PFGE) for very large DNA fragments.

4.0Different Types of Electrophoresis

1. Routine Electrophoresis: Routine electrophoresis is the traditional form used in clinical laboratories. It is performed on a rectangular slab gel and is commonly used to separate proteins, DNA, and sometimes nucleic acids.
2. High-Resolution Electrophoresis: High-resolution electrophoresis is similar to routine electrophoresis but uses a higher voltage to achieve better separation. It is used when finer resolution is required, such as separating CSF proteins for diagnosing multiple sclerosis or detecting urinary light chains for early diagnosis of lymphoproliferative disorders like multiple myeloma.
3. Polyacrylamide (PAM) Electrophoresis: Polyacrylamide is an organic polymer formed from acrylamide subunits. Initially produced as a simple linear polymer, it can be modified into highly structured, cross-linked, or branched forms. While widely used in laboratories for protein separation (PAGE), polyacrylamide also has applications in municipal and industrial wastewater treatment for separating solids from liquids.
4. Capillary Electrophoresis: Capillary electrophoresis works on the principle of attraction and repulsion of charged particles, much like magnetic poles. It uses a capillary tube with two charged ends— the positively charged cathode and the negatively charged anode. This method is widely used in forensic DNA laboratories to separate and detect short tandem repeat (STR) alleles.
5. Isoelectric Focusing (IEF): In isoelectric focusing, chemicals create a pH gradient across the gel. Proteins migrate through the gel until they reach a point where they carry no net charge—their isoelectric point.
   IEF is used in prenatal screening to separate various haemoglobins and to detect oligoclonal bands in the gamma-globulin region.
6. Immunofixation Electrophoresis: In this method, proteins from a serum sample are separated using agarose gel electrophoresis. An antiserum specific to the protein of interest is applied directly onto the gel, forming a precipitate at that location. After washing and staining, the precipitated protein can be clearly visualized. This technique is essential for identifying abnormal proteins, such as those found in multiple myeloma, and analyzing protein breakdown products.
7. Pulsed-Field Electrophoresis (PFGE): Pulsed-field electrophoresis alternates electric fields across different pairs of electrodes, allowing the separation of very large DNA fragments that cannot be resolved by traditional methods.
   By switching between positive and negative electrodes, PFGE provides an effective way to analyze large genomic DNA pieces.

5.0Standard DNA Analysis Technique

Agarose Gel Electrophoresis

• Agarose gels are ideal for separating DNA fragments ranging from ~100 base pairs to several kilobases.
• Agarose concentration determines the pore size; higher concentrations are used for smaller DNA fragments.

Polyacrylamide Gel Electrophoresis (PAGE)

• PAGE is used for separating very small DNA or RNA fragments with high resolution.
• Commonly used in DNA sequencing and protein analysis.

6.0Interpretation of Results and Applications

• DNA Fingerprinting: Unique band patterns are generated for individual identification in forensic science and paternity testing.
• Genetic Engineering: Confirms successful gene insertion or removal during recombinant DNA technology.
• Forensic Science: Used for crime scene investigations by comparing DNA from suspects and evidence.
• Medical Diagnostics: Detects mutations, genetic disorders, and pathogens by analyzing DNA or RNA samples.
• Research and Biotechnology: Essential for gene mapping, cloning, sequencing, and molecular marker analysis.

7.0Factors Affecting Electrophoresis

Several factors influence the efficiency and resolution of DNA electrophoresis:

Gel Concentration

• Higher agarose concentration = smaller pores = better separation of small fragments.
• Lower concentration = larger pores = better for large fragments.

Voltage Applied

• Higher voltage increases migration speed but may reduce resolution and cause band smearing.

Buffer Composition

• Maintains pH and ionic strength; common buffers are TAE and TBE.
• Incorrect buffer leads to poor resolution or DNA degradation.

Sample Quality and Quantity

• Degraded or overloaded samples can result in unclear bands.

Temperature

• Excessive heat can melt the gel or distort results.

Dye and Visualization Methods

• Choice of stain (e.g., ethidium bromide, SYBR Green) affects sensitivity and safety.