Molecular biology



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)]

Established in the 1930s, 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 chiefly concerns
itself with understanding the interactions between the various systems of a
cell, and learning how these interactions are regulated.

In recent years the study of gene structure and function has been the most
prominent sub-field of molecular biology.

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.

Expression cloning

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
antibiotic resistance markers to help follow the plasmid.

This plasmid can be inserted into either bacterial or animal cells.
Introducing 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, liposome transfection,
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 can now be 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 levels. 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.

PCR

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), or altered in predetermined ways. For example, PCR can
be used to introduce restriction enzyme sites, or to mutate a particular
bases of DNA. PCR can also be used to determine whether a particular DNA
fragment is found in a cDNA library.

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.

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 separated by size, on a thin gel
sandwiched between two glass plates. The gels, called an SDS-PAGE (for
Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis). The proteins on
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 chemoluminescence
or radioactivity.

Antibodies can also be used to purify proteins. Antibodies to a protein are
generated and are often then coupled to "beads". After the antibody has
bound to the protein of interest, this antibody-protein complex can be
separated from all other proteins by centrifugation. During centrifugation,
the beads, to which the antibody is coupled, will pellet (bringing the
protein of interest down with it) whereas all other proteins will remain in
the solution. Alternatively, antibodies coupled to a solid support matrix
like Sephadex or Sepharose beads, for example, can be used to remove a
protein of interest from a complex solution. After washing unbound and
non-specifically bound materials away from the "beads", the protein of
interest is then eluted from the matrix, usually by adding a solution with a
high salt concentration, or by varying the pH of the solution in which the
matrix is contained. The beads can either be suspended in solution (batch
processing) or packed into a tube (column processing).

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