Gel electrophoresis is a technique used to separate macromolecules such as DNA, RNA, and proteins. Both DNA and RNA molecules are separated based on their size while proteins are separated based on both size and charge. Agarose gel electrophoresis is the technique used to separate both DNA and RNA. From 100 bp to 25 kb DNA fragments can be separated by agarose gel electrophoresis. Generally, DNA are positively-charged molecules since they possess negative charges in their phosphate groups. Thus, DNA migrates towards the positive electrode during gel electrophoresis.
Key Areas Covered
1. What is Gel Electrophoresis
– Definition, Agarose Gel Electrophoresis, PAGE
2. How Does Gel Electrophoresis Separate DNA Fragments
– Principle of Separation of DNA
Key Terms: Agarose Gel Electrophoresis, Migrated Distance, DNA, PAGE, Pores, Positive Electrode, Size
What is Gel Electrophoresis
Gel electrophoresis is a technique used to separate fragments of macromolecules such as DNA, RNA, and proteins based on their size and charge. Both DNA and RNA possess equal negative charge throughout the molecule due to the presence of negatively-charged phosphate groups. Hence, both DNA and RNA migrates towards the positive electrode under an electric field. In addition, agarose gel electrophoresis is the technique used to separate DNA and RNA based on their size. The separation of DNA fragments by gel electrophoresis is shown in figure 1.
Figure 1: Separated DNA Fragments
The gel electrophoretic technique used to separate proteins is polyacrylamide gel electrophoresis (PAGE). The proteins used in this technique are separated based on their size and charge. PAGE can be used to separate DNA fragments with base-pair-level differences as well since the separation power of PAGE is higher than that of the agarose gel electrophoresis.
How Does Gel Electrophoresis Separate DNA Fragments
During Agarose gel electrophoresis, the DNA samples are mixed with the loading dye and are loaded on the wells of the agarose gel. The loading buffer contains tracking dyes that visualize the movement of the DNA sample on the gel. Then, an electric field is applied to both ends of the gel. The DNA sample migrates toward the positive electrode. The speed of migration on the electric field depends on the size of the DNA fragment. DNA molecules with a large number of base pairs migrate slowly while molecules with fewer base pairs migrate quickly through the gel. Therefore, gel electrophoresis allows the separation of DNA fragments based on their size. This produces a series of DNA fragments with sizes in the descending order. The relationship between the distance of migration and the size of the DNA fragment is shown in figure 2.
Figure 2: Relationship Between the Distance Migrated and the Size of the DNA Fragment
The agarose gel contains equal-sized pores through which the DNA fragments migrate. Therefore, small DNA fragments migrate quickly through the pore but, large DNA fragments take some time to migrate through them. After running a considerable distance, the agarose gel is visualized under UV. Since the agarose gel is added with a DNA staining substances under UV called ethidium bromide, DNA fragments are entangled with the stain, enabling the visualization. For the determination of the size of the DNA fragment, the samples are run along with a ladder that contains a series of DNA fragments with known size.
Conclusion
Gel electrophoresis is a technique used to separate DNA, RNA or protein molecules based on their size and charge. Agarose gel electrophoresis is the widely-used technique for the separation of DNA based on the size of the molecule. During the migration of DNA molecules through the pores of the agarose gel, they are separated based on the size.
Reference:
1. “Gel electrophoresis.” Khan Academy, Available here.
Image Courtesy:
1. “DNA fragmendid etiidiumbromiidiga värvitud agaroosgeelis” By Rainis Venta – Own work (CC BY-SA 3.0) via Commons Wikimedia
2. “Gel Electrophoresis” By Mckenzielower – Own work (CC BY-SA 4.0) via Commons Wikimedia
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