Electrophoresis: The Technique for Separating Charged Molecules

The separation technique of charged molecules under the influence of electric current is called
(a) Colony hybridization
(b) Electrophoresis
(c) Dot blot technique
(d) Western blotting

 

The correct answer is (b) Electrophoresis.

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What is Electrophoresis?

Electrophoresis is a widely used technique in molecular biology, biochemistry, and biotechnology for the separation and analysis of charged molecules such as DNA, RNA, and proteins under the influence of an electric current.

When charged molecules are placed in an electric field, they migrate towards the electrode with an opposite charge. The rate of migration depends on the molecule’s size, shape, and charge. Electrophoresis allows researchers to separate complex biological mixtures and analyze the composition, purity, and molecular weight of biomolecules.

Electrophoresis is a fundamental technique in genomics, proteomics, and clinical diagnostics for the separation and identification of biomolecules.

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✅ Correct Answer: (b) Electrophoresis

Electrophoresis is the most effective technique for separating charged molecules, as it relies on the differential migration of molecules based on their charge and size under an electric field.

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How Electrophoresis Works

1. Sample Preparation

• Biological samples (DNA, RNA, or proteins) are prepared and mixed with a suitable buffer solution.
• The buffer maintains a stable pH and ionic strength during electrophoresis.

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2. Gel Preparation

• The separation matrix is typically made of:
  • Agarose gel – for DNA and RNA separation
  • Polyacrylamide gel – for protein separation
• The gel is poured into a mold and allowed to solidify.

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3. Loading the Sample

• Samples are mixed with a loading dye and carefully introduced into wells within the gel.
• Loading dye increases sample density and ensures that the sample sinks into the well.

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4. Application of Electric Current

• The gel is placed in an electrophoresis chamber filled with buffer.
• An electric field is applied across the gel.
• Negatively charged molecules migrate toward the positive electrode (anode).

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5. Separation of Molecules

• Molecules migrate at different rates depending on:
  • Charge: Strongly charged molecules migrate faster.
  • Size: Smaller molecules migrate faster through the gel matrix.
  • Shape: Linear molecules migrate differently than globular or circular molecules.

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6. Staining and Detection

• After separation, the gel is stained using:
  • Ethidium bromide (EtBr) – for DNA and RNA (fluorescent under UV light)
  • Coomassie blue – for protein detection
  • Silver staining – for high-sensitivity protein detection
• Bands are visualized using UV light or other imaging systems.

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Types of Electrophoresis

1. Agarose Gel Electrophoresis

• Commonly used for DNA and RNA separation.
• Agarose provides a porous matrix, allowing separation based on size.
• High-resolution separation of nucleic acids.

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2. Polyacrylamide Gel Electrophoresis (PAGE)

• Used for protein and small nucleic acid separation.
• Provides higher resolution than agarose gels.
• SDS-PAGE (Sodium Dodecyl Sulfate PAGE) separates proteins based on molecular weight.

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3. Capillary Electrophoresis

• Molecules are separated within a narrow capillary tube.
• High-speed and high-resolution separation.
• Used for DNA sequencing and protein profiling.

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4. Isoelectric Focusing (IEF)

• Separates proteins based on their isoelectric point (pI).
• Proteins migrate until they reach a pH where their net charge is zero.

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Applications of Electrophoresis

1. DNA and RNA Analysis

• Used to analyze the size and integrity of DNA and RNA.
• Essential for genotyping, gene expression analysis, and mutation detection.

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2. Protein Purification and Analysis

• SDS-PAGE and Western blotting are used to purify and identify proteins.
• Measures protein molecular weight and post-translational modifications.

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3. Clinical Diagnostics

• Used for diagnosing genetic diseases and protein disorders.
• Hemoglobin electrophoresis detects sickle cell anemia.

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4. Forensic Science

• DNA fingerprinting uses electrophoresis to identify individuals.
• Agarose gel electrophoresis separates DNA fragments for crime investigation.

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5. Viral and Bacterial Research

• Separates viral and bacterial proteins or nucleic acids.
• Used in vaccine development and pathogen identification.

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Advantages of Electrophoresis

✔ High resolution – Separates molecules with high precision.
✔ Versatile – Suitable for nucleic acids and proteins.
✔ Quantitative – Provides an estimate of molecular concentration and size.
✔ Scalable – Works for both small- and large-scale analysis.

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Why Other Techniques Are Incorrect

(a) Colony Hybridization

• Detects specific DNA or RNA sequences within bacterial colonies but does not separate molecules.

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(c) Dot Blot Technique

• Detects the presence of specific biomolecules without separating them.
• Lacks size-based resolution.

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(d) Western Blotting

• Western blotting involves protein separation by electrophoresis but includes additional steps for transfer and detection.
• Electrophoresis is a part of the Western blot technique but not synonymous with it.

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Importance of Electrophoresis in Molecular Biology

Electrophoresis is essential for modern molecular biology, biotechnology, and clinical diagnostics. Its ability to separate and analyze charged biomolecules makes it a cornerstone technique in research and applied sciences.