Chapter 2. Molecular Biology I
General Purpose
This pre-lab will present some of the general concepts related to some of the techniques used in the fields of microbiology and molecular biology and how these techniques relate to understanding the relationship between DNA, genes, and traits expressed by an organism.
Learning Objectives
General Purpose
- Develop a basic understanding of some of the techniques used in the field of molecular biology.
Conceptual
- Gain an understanding of electrophoresis.
Procedural
- Gain proficiency using gel electrophoresis.
Background Information
One of the procedures involved in the manipulation of DNA fragments is gel electrophoresis. Electrophoresis involves the separation of charged molecules using an electric field. Molecules are attracted to the electrode that has the charge which is opposite to the charge on the molecule (i.e., negatively charged molecules will move to the positive electrode). The charge of nucleic acid is negative due to the presence of phosphate groups in the backbone of the molecule (Figure 11-3).
In gel electrophoresis the DNA molecules move through a semisolid matrix (agarose). This matrix of long molecules acts like a three-dimensional sieve which the DNA has to move through as it travels to the positive electrode. As a DNA molecule moves through the matrix it undergoes random collisions with the molecules of the matrix. The larger the DNA molecules, the more collisions the molecules have with the matrix. Each collision effectively slows the DNA molecule in its journey toward the positive electrode. The end result is the larger a DNA molecule is, the slower it moves through the gel. If size is the only factor at work, then the distance a fragment of DNA moves in the gel would be inversely proportional to the size (or length) of the DNA molecule. However, another force of physics is also involved: the closer two oppositely charged objects are, the greater the attraction between them. The effect this has with respect to gel electrophoresis is that the closer a DNA molecule gets to the positive electrode, the faster the DNA molecule moves. Therefore, the small DNA molecules that experience few collisions with the matrix begin moving faster the farther into the gel they migrate. The final result of all these interactions is that the distance a DNA molecule migrates in the gel matrix is inversely proportional to the log10 of the size (or length) of the DNA molecule.
The following URL shows an animation of agarose gel electrophoresis:
View the following video for a discussion of the concepts involved in the process of gel electrophoresis.
Pre-Lab Quiz
Proceed to the Pre-Lab Quiz