Molecular biology

Molecular biology /məˈlɛkjʊlər/ is a branch of biology that concerns the molecular basis of biological activity between biomolecules in the various systems of a cell, including the interactions between DNA, RNA, proteins and their biosynthesis, as well as the regulation of these interactions. 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. Molecular biology /məˈlɛkjʊlər/ is a branch of biology that concerns the molecular basis of biological activity between biomolecules in the various systems of a cell, including the interactions between DNA, RNA, proteins and their biosynthesis, as well as the regulation of these interactions. Writing in Nature in 1961, William Astbury described molecular biology as: Researchers in molecular biology use specific techniques native to molecular biology but increasingly combine these with techniques and ideas from genetics and biochemistry. There is not a defined line between these disciplines. This is shown in the following schematic that depicts one possible view of the relationships between the fields: Much of molecular biology is quantitative, and recently much work has been done at its interface with computer science in bioinformatics and computational biology. In the early 2000s, the study of gene structure and function, molecular genetics, has been among the most prominent sub-fields of molecular biology. Increasingly many other areas of biology intersecting with molecular Biology, focus on molecules, either directly studying interactions in their own right such as in cell biology and developmental biology, or indirectly, where molecular techniques 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. One of the most basic techniques of molecular biology to study protein function is molecular cloning. In this technique, DNA coding for a protein of interest is cloned using polymerase chain reaction (PCR), and/or restriction enzymes into a plasmid (expression vector). A vector has 3 distinctive features: an origin of replication, a multiple cloning site (MCS), and a selective marker usually antibiotic resistance. Located upstream of the multiple cloning site are the promoter regions and the transcription start site which regulate the expression of cloned gene.This plasmid can be inserted into either bacterial or animal 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. The plasmid may be integrated into the genome, resulting in a stable transfection, or may remain independent of the genome, called transient transfection. 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. Polymerase chain reaction (PCR) is an extremely versatile technique for copying DNA. In brief, PCR allows a specific DNA sequence to be copied or modified in predetermined ways. The reaction is extremely powerful and under perfect conditions could amplify one DNA molecule to become 1.07 billion molecules in less than two hours. The PCR technique can be used to introduce restriction enzyme sites to ends of DNA molecules, or to mutate particular bases of DNA, the latter is a method referred to as site-directed mutagenesis. 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, quantitative PCR which allow for quantitative measurement of DNA or RNA molecules. 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 gel electrophoresis, DNA and RNA can be separated on the basis of size by running the DNA through an electrically charged 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. 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. 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. Southern blotting is less commonly used in laboratory science due to the capacity of other techniques, such as PCR, to detect specific DNA sequences from DNA samples. 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.