Molecular Biology

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Molecular biology is the branch of biology that deals with the molecular basis of biological activity. Molecular biology overlaps with other areas of biology and chemistry, particularly genetics and biochemistry. Molecular biology chiefly concerns itself with understanding the interactions between the various systems of a 1) ___, including the interactions between the different types of DNA, RNA and protein biosynthesis as well as learning how these interactions are regulated.

 

Writing in Nature in 1961, William Astbury described molecular biology as: “?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.

 

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Schematic relationship between biochemistry, genetics and molecular biology.

 

Researchers in molecular biology use specific techniques native to molecular biology but increasingly combine these with techniques and ideas from genetics and 2) ___. There is not a defined line between these disciplines. The figure above is a schematic that depicts one possible view of the relationship between the fields:

 

1. Biochemistry is the study of the chemical substances and vital processes occurring in living organisms. Biochemists focus heavily on the role, function, and structure of biomolecules. The study of the chemistry behind biological processes and the synthesis of biologically active molecules are examples of biochemistry.

 

2. Genetics is the study of the effect of genetic differences on organisms. This can often be inferred by the absence of 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. 3) ___ interactions (epistasis) can often confound simple interpretations of such “knockout“ studies.

 

3. Molecular biology is the study of molecular underpinnings of the processes of replication, transcription, translation, and cell function. The central dogma of molecular biology where genetic material is transcribed into RNA and then translated into protein, despite being an oversimplified picture of molecular 4) ___ biology, still provides a good starting point for understanding the field. This picture, however, is undergoing revision 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 molecular biology and computer science in bioinformatics and computational biology. As of the early 2000s, the study of gene structure and function, molecular genetics, has been among the most prominent sub-field of 5) ___ biology. Increasingly many other loops of biology focus on molecules, either directly studying their interactions in their own right such as in cell biology and developmental biology, or indirectly, where the techniques of molecular biology are used to infer historical 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.

 

Since the late 1950s and early 1960s, molecular biologists have learned to characterize, isolate, and manipulate the molecular components of cells and 6) ___. 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). A vector has 3 distinctive features: an origin of replication, a multiple cloning site (MCS), and a selective marker (usually antibiotic resistance). The origin of replication will have promoter regions upstream from the replication/transcription start site. This plasmid can be inserted into either bacterial or 7) ___ cells. Introducing DNA into bacterial cells can be done by transformation (via uptake of naked DNA), conjugation (via cell-cell contact) or by transduction (via viral vector). Introducing DNA into eukaryotic cells, such as animal cells, by physical or chemical means is called transfection.

 

Several different transfection techniques are available, such as calcium phosphate transfection, electroporation, microinjection and liposome transfection. DNA can also be introduced into eukaryotic cells using viruses or bacteria as carriers, the latter is sometimes called bactofection and in particular uses Agrobacterium tumefaciens. The plasmid may be integrated into the genome, resulting in a stable transfection, or may remain independent of the genome, called transient transfection. 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 8) ___ 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.

 

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.

 

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 by means of an electric field and size. In agarose 9) ___ electrophoresis, DNA and RNA can be separated on the basis of size by running the DNA through an agarose gel. Proteins can be separated on the basis of size by using an SDS-PAGE gel, or on the basis of size and their electric charge by using what is known as a 2D gel electrophoresis.

 

Macromolecule blotting and probing

The terms northern, western and eastern blotting are derived from what initially was a molecular biology joke that played on the term Southern blotting, after the technique described by Edwin Southern for the hybridisation of blotted DNA. Patricia Thomas, developer of the RNA blot which then became known as the northern blot, actually didn’t use the term. Further combinations of these techniques produced such terms as southwesterns (protein-DNA hybridizations), northwesterns (to detect protein-RNA interactions) and farwesterns (protein-protein interactions), all of which are presently found in the literature.

 

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 (restriction endonuclease) digestion are separated by gel electrophoresis and then transferred to a membrane by blotting via capillary action. The membrane is then exposed to a labeled DNA probe that has a complement base sequence to the sequence on the DNA of interest. Most original protocols used radioactive labels, however non-radioactive alternatives are now available.

 

These blots are still used for some applications, however, such as measuring transgene copy number in transgenic mice, or in the engineering of gene knockout embryonic stem cell lines. A DNA array is a collection of spots attached to a solid support such as a microscope slide where each spot contains one or more single-stranded DNA oligonucleotide fragment. Arrays make it possible to put down large quantities of very small (100 micrometre diameter) spots on a single slide. Each spot has a DNA fragment molecule that is complementary to a single DNA sequence (similar to Southern blotting). 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.

 

In molecular biology, procedures and technologies are continually being developed and older technologies abandoned. For example, before the advent of DNA gel electrophoresis (agarose or polyacrylamide), the size of DNA molecules was typically determined by rate sedimentation in sucrose gradients, a slow and labor-intensive technique requiring expensive instrumentation; prior to sucrose gradients, viscometry was used.

 

Aside from their historical interest, it is often worth knowing about older technology, as it is occasionally useful to solve another new problem for which the newer technique is inappropriate.

 

History of molecular biology

While molecular biology was established in the 1930s, the term was coined by Warren Weaver in 1938. Weaver was the director of Natural Sciences for the Rockefeller Foundation at the time and believed that biology was about to undergo a period of significant change given recent advances in fields such as X-ray crystallography. He therefore channeled significant amounts of (Rockefeller Institute) money into biological fields.

 

Clinical research and medical therapies arising from molecular biology are partly covered under gene therapy. The use of molecular biology or molecular cell biology approaches in medicine is now called molecular medicine. Molecular biology also plays important role in understanding formations, actions, regulations of various parts of cells which can be used efficiently for targeting new 10) ___, diagnosis of disease, physiology of the Cell.

 

ANSWERS: 1) cell, 2) biochemistry; 3) Genetic; 4) biology; 5) molecular; 6) organisms; 7) animal; 8) protein; 9) gel; 10) drugs

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