Techniques of molecular biology

Since the late 1950s and early 1960s, molecular biologists have learned to characterize, 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.

Polymerase chain reaction (PCR)

The polymerase chain reaction is an extremely versatile technique for copying DNA. In brief, PCR allows a single DNA sequence to be copied (millions of times), or altered in predetermined ways. For example, PCR can be used to introduce restriction enzyme sites, or to mutate (change) particular bases of DNA. PCR can also be used to determine whether a particular DNA fragment is found in a cDNA library. PCR has many variations, like reverse transcription PCR (RT-PCR) for amplification of RNA, and, more recently, real-time PCR (qPCR) which allow for quantitative measurement of DNA or RNA molecules.

Gel electrophoresis

Gel electrophoresis is one of the principal tools of molecular biology. The basic principle is that DNA, RNA, and proteins can all be separated using an electric field. In agarose gel electrophoresis, DNA and RNA can be separated based on size by running the DNA through an agarose gel. Proteins can be separated based on size using an SDS-PAGE gel. Proteins can also be separated based on their electric charge, using what is known as an isoelectric gel.

Southern blotting

Named after its inventor, biologist Edwin Southern, the Southern blot is a method for probing for the presence of a specific DNA sequence within a DNA sample. DNA samples before or after restriction enzyme digestion are separated by gel electrophoresis and then transferred to a membrane by blotting via capilliary action. The membrane can then be probed using a DNA probe labeled using a complement of the sequence of interest. Most original protocols used radioactive labels, however now non-radioactive alternatives are available. Southern blotting is less commonly used in laboratory science due to the capacity of using PCR to detect specific DNA sequences from DNA samples. However, these blots are still used for some applications, such as measuring transgene copy number in transgenic mice, or in the engineering of gene knockout embryonic stem cell lines.

Northern blotting

The Northern blot is used to study the expression patterns a specific type of RNA molecule as relative comparison among of a set of different samples of RNA. It is essentially a combination of denaturing RNA gel electrophoresis, and a blot. In this process RNA is separated based on size and is then transferred to a membrane that is then probed with a labeled complement of a sequence of interest. The results may be visualized through a variety of ways depending on the label used, however, most result in the revelation of bands representing the sizes of the RNA detected in sample. The intensity of these bands is related to the amount of the target RNA in the samples analyzed. The procedure is commonly used to study when and how much gene expression is occurring by measuring how much of that RNA is present in different samples. It is one of the most basic tools for determining at what time certain genes are expressed in living tissues.

Western Blotting

Antibodies to most proteins can be created by injecting small amounts of the protein into an animal such as a mouse, rabbit, sheep, or donkey (polyclonal antibodies)or produced in cell culture (monoclonal antibodies). These antibodies can be used for a variety of analytical and preparative techniques. In Western blotting, proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as SDS-PAGE (for Sodium Dodecyl Sulphate Poly-Acrylamide Gel Electrophoresis). The proteins in the gel are then transferred to a PVDF, nitrocellulose, nylon or other support membrane. This membrane can then be probed with solutions of antibodies. Antibodies that specifically bind to the protein of interest can then be visualized by a variety of techniques, including coloured products, chemiluminescence, or autoradiography. Analogous methods to western blotting can also be used to directly stain specific proteins in cells and tissue sections. However, these immunostaining methods are typically more associated with cell biology than molecular biology. The terms "Western" and "Northern" are jokes: The first blots were with DNA, and since they were done by Ed Southern, they came to be known as Southerns. I don't think Patricia Thomas, inventor of the RNA blot, which became known as a "Northern", actually used the term. To carry the joke further, one can find reference in the literature to "southwesterns" (Protein-DNA interactions) and "farwesterns" (Protein-Protein interactions). Arrays A DNA array is a collection of spots attached to a solid support such as a microscope slide; each spot contains one or more DNA molecules. Arrays make it possible to put down a large number of very small (100 micrometre diameter) spots on a single slide; if each spot has a DNA molecule that is complementary to a single gene, one can analyze the expression of every gene in an organism in a single experiment. For instance, the common baker's yeast, Saccharomyces cerevisiae, contains about 7000 genes; with a microarray, one can measure quantitatively, how each gene is expressed, and how that expression changes, for example, with a change in temperature. There are many different ways to fabricate microarrays; the most common are silicon chips, microscope slides with spots of ~ 100 micrometre diameter, custom arrays, and arrays with larger spots on porous membranes (macroarrays). Arrays can also be made with molecules other than DNA. For example, an antibody array can be used to determine what proteins or bacteria are present in a blood sample.

Molecular medicine

Molecular medicine is a new scientific discipline in British universities. Combining contemporary medical studies with the field of biochemistry, it offers a bridge between the two subjects. At present only a handful of universities offer the course to undergraduates. With a degree in this discipline the graduate is able to pursue a career in medical sciences, scientific research, laboratory work and postgraduate medical degrees.