Gel electrophoresis | Khan Academy

Overview of DNA Fragment Analysis

  • The length and size of DNA fragments are critical for various biological analyses.

  • DNA fragments are typically very small, making physical handling impractical.

  • A typical large fragment (e.g., 5,000 base pairs) is about 1-2 micrometers long when fully stretched out.

Technique: Gel Electrophoresis

  • Gel electrophoresis is a technique used to separate DNA, RNA, and proteins based on their size.

  • Definition:

    • Involves using a gel and an electric charge to facilitate the migration of macromolecules through the gel.

    • "Phoresis" indicates movement or migration.

  • Gel Composition:

    • Commonly involves agarose gel, a polysaccharide derived from seaweed.

    • Gel is set in a buffer solution that maintains pH stability during electrophoresis.

Electrophoresis Setup

  • Samples from DNA vials are placed into wells within the gel.

  • The buffer solution helps protect and maintain DNA integrity during the process.

  • An electric current is applied, with the negative electrode at the well side and the positive electrode at the opposite end.

  • DNA has an overall negative charge due to its phosphate backbone, causing it to migrate towards the positive electrode.

Migration of DNA Fragments

  • The distance a DNA fragment migrates depends on its size—smaller fragments move farther through the gel matrix.

  • Agarose gel acts as a sieve: smaller fragments can navigate through the mesh easier than larger ones.

  • Over time, as DNA migrates, bands will form, indicating different sizes of DNA fragments.

  • Analysis of Bands:

    • Shorter bands indicate smaller DNA molecules, while longer bands suggest larger molecules.

DNA Ladder for Size Estimation

  • A DNA ladder is a standard set of DNA fragments used for comparison during gel electrophoresis.

  • Each band corresponds to a known number of base pairs (e.g., 5,000 bp, 1,500 bp, 500 bp).

  • By comparing unknown samples' migration distance with the ladder, one can estimate their size.

Visualization of DNA Fragments

  • DNA is not visible on its own due to its small size; visualization requires staining.

  • Ethidium Bromide:

    • Commonly used intercalating agent that binds to DNA and fluoresces under UV light.

  • As DNA migrates through the gel, it can be visualized using UV illumination, showing bands of DNA based on size.

Conclusion

  • Gel electrophoresis allows the measurement of DNA fragment lengths indirectly through size-dependent migration behavior.

  • This technique is widely utilized in genetics and molecular biology for analysis and manipulation of DNA.