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Molecular biology

(molecularbiology)





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 thevarious systems of a cell, including the interrelationship of DNA, RNA and protein synthesis and learning how these interactionsare regulated.

Writing in Nature , W.T. Astbury described molecularbiology as:

"... not so much a technique as an approach, an approach from the viewpoint of the so-called basic sciences with the leadingidea of searching below the large-scale manifestations of classical biology for the corresponding molecular plan. It is concernedparticularly with the forms of biological molecules and ..... is predominantly three-dimensional and structural - which does notmean, however, that it is merely a refinement of morphology - it must at the same time inquire into genesis and function"[Nature 190, 1124 (1961)]

Contents

Relationship to other "molecular-scale" biological sciences

Researchers in molecular biology use specific techniques native to molecular biology (see Techniques section later inarticle), but increasingly combine these with techniques and ideas from genetics , biochemistry and biophysics . There is not a hard-line between these disciplines as there once was. The following figure is aschematic that depicts one possible view of the relationship between the fields:


  • Biochemistry is the study of molecules (e.g. proteins ) in the absence ofthe rest of the organism . Biochemists take an organism or cell and dissect it into its molecular components, such as enzymes , lipids and DNA, and reconstitute them in testtubes (in vitro).
  • Genetics is the study of the effect of genetic differences on organisms. Often this can be inferred by the absenceof a normal component (e.g. one gene ). The study of " mutants " - organisms which lack one or more functional components with respect to the so-called " wild type " or normal phenotype . Genetic interactions such as epistasis can often confound simple interpretations of such "knock-out" studies.
  • Molecular biology is the study of molecular underpinnings of the process of replication, transcription andtranslation of the genetic material . The central dogma of molecular biology where genetic material is transcribed into RNA and then translated into protein, despite being an oversimplified picture ofmolecular biology, still provides a good starting point for understanding the field. This picture, however, is undergoingrevision in light of emerging novel roles for RNA .

Much of the work in molecular biology is quantitative, and recently much work has been done at the interface of molecularbiology and computer science in bioinformatics and computational biology . As of the early 2000s , the study of gene structure and function, molecular genetics , has been amongst the most prominent sub-field of molecular biology.

Increasingly many other fields of biology focus on molecules, either directly studying their interactions in their own rightsuch as in cell biology and developmental biology , or indirectly, where the techniques of molecular biology are used to inferhistorical attributes of populations or species , as in fields in evolutionary biology such as population genetics and phylogenetics . There is also a long tradition of studying biomolecules "from the ground up" in biophysics .

Techniques of molecular biology

Since the late 1950s and early 1960s ,molecular biologists have learned to characterise, isolate, and manipulate the molecular components of cells and organisms. Thesecomponents 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 structuraland 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, DNAcoding 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 productionof 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 areavailable, including calcium phosphate transfection, liposometransfection , and proprietary transfection reagents such as Fugene. DNA can also be introduced into cells using viruses as acarrier. 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 ofsystems, such as inducible promoters and specific cell-signaling factors, are available to help express the protein of interestat high levels. Large quantities of a protein can then be extracted from the bacterial or eukaryotic cell. The protein can betested for enzymatic activity under a variety of situations, the protein may be crystallized so its tertiary structure can bestudied, or, in the pharmaceutical industry, the activity of new drugs against the protein can be studied.

Polymerase chain reaction (PCR)

Main article: Polymerase chainreaction

The polymerase chain reaction is an extremelyversatile technique for copying DNA. In brief, PCR allows a single DNA sequence to be copied (millions of times), or altered inpredetermined ways. For example, PCR can be used to introduce restriction enzyme sites, or to mutate (change) particular bases ofDNA. PCR can also be used to determine whether a particular DNA fragment is found in a cDNA library .

Gel electrophoresis

Main article: Gel electrophoresis

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

Western blotting and immunochemistry

Main article: Western blot

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. These antibodies can be used for a variety of analytical and preprative techniques.

In Western blotting , proteins are first separated by size, in a thin gelsandwiched between two glass plates. This technique is called SDS-PAGE (for Sodium Dodecyl Sulfate Poly-Acrylamide GelElectrophoresis). The proteins in the gel are then transferred to a PVDF, nitrocellulose, nylon or other support membrane. Thismembrane can then be probed with solutions of antibodies. Antibodies that specifically bind to the protein of interest can thenbe 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 proteinsby centrifugation. During centrifugation, the beads, to which the antibody is coupled, will pellet (bringing the protein ofinterest down with it) whereas all other proteins will remain in the solution. Alternatively, antibodies coupled to a solidsupport matrix like Sephadex or Sepharose beads, for example, can be used to remove a protein of interest from a complexsolution. After washing unbound and non-specifically bound materials away from the "beads", the protein of interest is theneluted from the matrix, usually by adding a solution with a high salt concentration, or by varying the pH of the solution inwhich the matrix is contained. The beads can either be suspended in solution (batch processing) or packed into a tube (columnprocessing).

History

Molecular biology was established in the 1930s , the term was first coined by Warren Weaver in 1938 however. Warren wasdirector of Natural Sciences for the RockefellerFoundation at the time and believed that biology was about to undergo a period of significant change given recent advances infields such as X-ray crystallography . He thereforechanneled significant amounts of (Rockefeller Institute) money into biological fields.

Further reading

  • Keith Roberts, Martin Raff, Bruce Alberts, Peter Walter, Julian Lewis and Alexander Johnson, Molecular Biology of theCell, 4th Edition, Routledge, March, 2002, hardcover, 1616 pages, 7.6 pounds, ISBN 0815332181

Related topics

Notable molecular biologists

See also



General subfields within biology

Anatomy | Bioinformatics | Botany | Ecology | Evolutionary biology | Genetics | Marine biology | Human biology | Cellbiology | Microbiology | Molecular biology | Biochemistry | Origin oflife | Paleontology | Physiology | Taxonomy | Xenobiology | Zoology



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