Electrophoresis is a laboratory technique that separates molecules, including DNA fragments, based on their physical properties. This method is widely used in molecular biology for various applications, such as DNA analysis, gene mapping, and forensic science.
DNA’s Electrical Properties
DNA molecules possess an inherent negative electrical charge, a characteristic that is fundamental to their movement within an electric field. This negative charge originates from the phosphate groups present in the DNA’s sugar-phosphate backbone. Each nucleotide in a DNA strand contains a phosphate group, which carries a negative charge at physiological pH.
When DNA is placed in an electric field, these negatively charged molecules are attracted towards the positive electrode, also known as the anode. Conversely, they are repelled by the negative electrode, or cathode. The strength of the electric field directly influences the speed at which these charged DNA molecules move.
Assembling the Electrophoresis Setup
DNA electrophoresis requires several key components. A porous, jelly-like matrix called an agarose gel forms the medium through which the DNA fragments will migrate. The concentration of agarose in the gel can be adjusted, influencing the pore size and thus the separation range for DNA fragments. This gel is typically cast within an electrophoresis chamber, a specialized container designed to hold the gel and the surrounding buffer solution.
A running buffer, a conductive solution, fills the chamber, ensuring the flow of electrical current and maintaining a stable pH environment. This buffer allows the electrical current to pass through the gel, enabling the movement of charged DNA molecules. A power supply is connected to the electrophoresis chamber, providing the necessary electric current and establishing distinct positive and negative poles across the gel. Finally, DNA samples, often mixed with a loading dye for visibility, are carefully pipetted into small indentations, or wells, cast at one end of the agarose gel.
The Separation Mechanism
Once the power supply is activated, an electric field is established across the agarose gel. Since DNA fragments are negatively charged, they begin to migrate from the wells, positioned near the negative electrode, towards the positive electrode at the opposite end of the gel. The agarose gel acts as a molecular sieve, impeding the movement of DNA fragments through its intricate network of pores.
Smaller DNA fragments can navigate through these pores more easily and encounter less resistance, allowing them to travel faster and further through the gel. In contrast, larger DNA fragments experience greater friction and resistance as they attempt to pass through the same pores. This increased resistance causes larger fragments to move at a slower pace and consequently travel shorter distances within the gel during a given time. Therefore, the rate at which DNA fragments travel through the gel is inversely proportional to their size.
After a sufficient period, DNA fragments of similar size will accumulate at distinct positions within the gel, forming visible bands. These bands represent populations of DNA molecules that are approximately the same length, sorted from smallest to largest along the gel.
Making DNA Fragments Visible
DNA fragments are not visible to the naked eye within the agarose gel, necessitating a visualization step after electrophoresis. To achieve this, a fluorescent dye is commonly used to stain the DNA. A widely used dye, historically, has been ethidium bromide, which intercalates, or inserts itself, between the base pairs of the DNA double helix.
Upon exposure to ultraviolet (UV) light, the ethidium bromide-DNA complex fluoresces. Due to safety concerns regarding ethidium bromide, safer alternatives such as SYBR Green or GelRed are now frequently employed for DNA staining. These dyes also bind to DNA and fluoresce under UV or blue light, allowing for clear visualization and photography of the results. To estimate the sizes of the separated DNA fragments, a DNA ladder—a mixture of DNA fragments of known lengths—is run in a separate lane on the same gel.