Molecular biology is
".... not so much a technique as an approach, an approach from the viewpoint of the so-called basic sciences with the leading idea of searching below the large-scale manifestations of classical biology for the corresponding molecular plan. It is concerned particularly with the forms of biological molecules and ..... is predominantly three-dimensional and structural - which does not mean, however, that it is merely a refinement of morphology - it must at the same time inquire into genesis and function" W.T. Astbury [Nature 190, 1124 (1961)]
Molecular biology is the study of biology at a molecular level. The field overlaps with other areas of biology, particularly genetics and biochemistry. Molecular biology cheifly concerns itself with understanding the interactions between the various systems of a cell, and learning how these interactions are regulated.
Over the last 40 years, molecular biologists have learned to characterise, isolate, and manipulate the molecular components of cells and organisms. These components include DNA, the repository of genetic information; RNA, a close relative of DNA whose functions range from serving as a temporary working copy of DNA to actual structural and enzymatic functions as well as a functional and structural part of the translational apparatus; and proteins, the major structural and enzymatic type of molecule in cells.
One of the most basic techniques of molecular biology to study protein function is expression cloning. In this technique, DNA coding for a protein of interest is cloned (using PCR and/or restriction enzymes into a plasmid (known as an expression vector). This plasmid may have special promoter elements to drive production of the protein of interest, and may also have anitbiotic resistance markers to help follow the plasmid.
This plasmid can be inserted into either bacterial or animal cells. Introduding DNA into bacterial cells is called transformation, and can be effected by several methods, including electroporation, microinjection and chemically. Introducing DNA into eukaryotic cells, such as animal cells, is called transfection. Several different transfection technqiues are available, including calcium phosphate transfection, liposomes, and proprietary transfection reagents such as Fugene. DNA can also be introduced into cells using viruses as a carrier. In such cases, the technique is called viral transduction, and the cells are said to be transduced.
In either case, DNA coding for a protein of interest is now inside a cell, and the protein is now being expressed.. A variety of systems, such as inducible promoters and specific cell-signaling factors, are available to help express the protein of interest at high level. Large quantities of a protein can then be extracted from the bacterial or eukaryotic cell. The protein can be tested for enzymatic activity under a variety of situations, the protein may be crystallized so its tertiary structure can be studied, or, in the pharmaceutical industry, the activity of new drugs against the protein can be studied.
The polymerase chain reaction is an extremely versatile technique for copying DNA and RNA. In brief, PCR allows a single DNA to be copied (millions of times), and altered in predetermined ways. For example, PCR can be used to introduce restriction enzyme sites, or mutate a particular piece of DNA. PCR can also be used to find a particular gene in a cDNA library.
Gel electrophoresis is one of the principle tools of molecular biology. The basic principle is that DNA, RNA, and proteins can all be seperated using an electric field. In agarose gel electrophoresis, DNA and RNA can be seperated based on size by running the DNA through an agarose gel. Proteins can be seperated based on size using an SDS-PAGE gel. Proteins can also be seperated based on their electric charge, using what is known as an isoelectric gel.
Western Blotting and Immunochemistry
Antibodies to any protein can be created by injecting small amounts of protein into an animal such as a mouse, rabbit, sheep, or donkey. These antibodies can be used for a variety of analytical and preprative techniques.
In Western blotting, proteins are first seperated by size, on an SDS page gel. The proteins on the gel are then tranferred to a PVDF or nitrocellulose membrane. This membrane can then be probed with solutions of antibodies. Antibodies that bind to the protein of interest can then be visualized by a variety of techniques, including chemoluminescence or radioactivity.
Antibodies can also be used to purify proteins. Antibodies to a protein are generated, and then bind to the protein. Another protein can then be used to pull down the antibodies, bringing the first protein with it.
Molecular biology is the study of gene structure and function at the molecular level. This includes :
- cell biology (structures and components of the cell)
- DNA and chromosome structure
- Protein biosynthesis (transcription from DNA to RNA, translation from RNA into protein)
- Protein structure and diversity