Please read:A personal appeal fromWikipedia founder Jimmy Wales[Hide][Show]Wikipedia Forever Our shared knowledge. Our shared treasure. Help us protect it.[Show]Wikipedia Forever Our shared knowledge. Our shared treasure. Help us protect it.Molecular biologyFrom Wikipedia, the free encyclopediaJump to: navigation, searchMolecular biology is the study of biology at a molecular level. The field overlapswith other areas of biology and chemistry, particularly genetics and biochemistry.Molecular biology chiefly concerns itself with understanding the interactionsbetween the various systems of a cell, including the interactions between DNA, RNAand protein biosynthesis as well as learning how these interactions are regulated.Writing in Nature in 1961, William Astbury described molecular biology asnot 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-scalemanifestations of classical biology for the corresponding molecular plan. It isconcerned particularly with the forms of biological molecules and [...] ispredominantly three-dimensional and structural—which does not mean, however, thatit is merely a refinement of morphology. It must at the same time inquire intogenesis and function.[1]•[edit] Relationship to other "molecular-scale" biological sciences Schematic relationship between biochemistry, genetics, and molecular biologyResearchers in molecular biology use specific techniques native to molecularbiology (see Techniques section later in article), but increasingly combine thesewith techniques and ideas from genetics and biochemistry. There is not a definedline between these disciplines. The following figure is a schematic that depictsone possible view of the relationship between the fields:•Biochemistry is the study of the chemical substances and vital processesoccurring in living organisms. Biochemists focus heavily on the role, function,and structure of biomolecules. The study of the chemistry behind biologicalprocesses and the synthesis of biologically active molecules are examples of
biochemistry.•Genetics is the study of the effect of genetic differences on organisms.Often this can be inferred by the absence of a normal component (e.g. one gene).The study of "mutants" – organisms which lack one or more functional componentswith respect to the so-called "wild type" or normal phenotype. Geneticinteractions (epistasis) can often confound simple interpretations of such "knock-out" studies.•Molecular biology is the study of molecular underpinnings of the process ofreplication, transcription and translation of the genetic material. The centraldogma of molecular biology where genetic material is transcribed into RNA and thentranslated into protein, despite being an oversimplified picture of molecularbiology, still provides a good starting point for understanding the field. Thispicture, 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 hasbeen done at the interface of molecular biology and computer science inbioinformatics and computational biology. As of the early 2000s, the study of genestructure and function, molecular genetics, has been amongst the most prominentsub-field of molecular biology.Increasingly many other loops of biology focus on molecules, either directlystudying their interactions in their own right such as in cell biology anddevelopmental biology, or indirectly, where the techniques of molecular biologyare used to infer historical attributes of populations or species, as in fields inevolutionary biology such as population genetics and phylogenetics. There is alsoa long tradition of studying biomolecules "from the ground up" in biophysics.[edit] Techniques of molecular biologySince the late 1950s and early 1960s, molecular biologists have learned tocharacterize, isolate, and manipulate the molecular components of cells andorganisms. These components include DNA, the repository of genetic information;RNA, a close relative of DNA whose functions range from serving as a temporaryworking copy of DNA to actual structural and enzymatic functions as well as afunctional and structural part of the translational apparatus; and proteins, themajor structural and enzymatic type of molecule in cells.For more extensive list on protein methods, see protein methods.For more extensive list on nucleic acid methods, see nucleic acid methods.[edit] Expression cloningMain article: Expression cloningOne of the most basic techniques of molecular biology to study protein function isexpression cloning. In this technique, DNA coding for a protein of interest iscloned (using PCR and/or restriction enzymes) into a plasmid (known as anexpression vector). This plasmid may have special promoter elements to driveproduction of the protein of interest, and may also have antibiotic resistancemarkers to help follow the plasmid.This plasmid can be inserted into either bacterial or animal cells. IntroducingDNA 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 orchemical means is called transfection. Several different transfection techniquesare available, such as calcium phosphate transfection,electroporation,microinjection and liposome transfection. DNA can also be introduced intoeukaryotic cells using viruses or bacteria as carriers, the latter is sometimescalled bactofection and in particular uses Agrobacterium tumefaciens. The plasmidmay be integrated into the genome, resulting in a stable transfection, or mayremain independent of the genome, called transient transfection.In either case, DNA coding for a protein of interest is now inside a cell, and theprotein can now be expressed. A variety of systems, such as inducible promotersand specific cell-signaling factors, are available to help express the protein ofinterest at high levels. Large quantities of a protein can then be extracted fromthe bacterial or eukaryotic cell. The protein can be tested for enzymatic activity
under a variety of situations, the protein may be crystallized so its tertiarystructure can be studied, or, in the pharmaceutical industry, the activity of newdrugs against the protein can be studied.[edit] Polymerase chain reaction (PCR)Main article: Polymerase chain reactionThe 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), oraltered in predetermined ways. For example, PCR can be used to introducerestriction enzyme sites, or to mutate (change) particular bases of DNA, thelatter is a method referred to as "Quick change". PCR can also be used todetermine whether a particular DNA fragment is found in a cDNA library. PCR hasmany variations, like reverse transcription PCR (RT-PCR) for amplification of RNA,and, more recently, real-time PCR (QPCR) which allow for quantitative measurementof DNA or RNA molecules.[edit] Gel electrophoresisMain article: Gel electrophoresisGel electrophoresis is one of the principal tools of molecular biology. The basicprinciple is that DNA, RNA, and proteins can all be separated by means of anelectric field. In agarose gel electrophoresis, DNA and RNA can be separated onthe basis of size by running the DNA through an agarose gel. Proteins can beseparated on the basis of size by using an SDS-PAGE gel, or on the basis of sizeand their electric charge by using what is known as a 2D gel electrophoresis.[edit] Macromolecule blotting and probingThe terms northern, western and eastern blotting are derived from what initiallywas a molecular biology joke that played on the term Southern blotting, after thetechnique described by Edwin Southern for the hybridisation of blotted DNA.Patricia Thomas, developer of the RNA blot which then became known as the northernblot actually didn't use the term[2]. Further combinations of these techniquesproduced such terms as southwesterns (protein-DNA hybridizations), northwesterns(to detect protein-RNA interactions) and farwesterns (protein-proteininteractions), all of which are presently found in the literature.[edit] Southern blottingMain article: Southern blotNamed after its inventor, biologist Edwin Southern, the Southern blot is a methodfor probing for the presence of a specific DNA sequence within a DNA sample. DNAsamples before or after restriction enzyme digestion are separated by gelelectrophoresis and then transferred to a membrane by blotting via capillaryaction. The membrane is then exposed to a labeled DNA probe that has a complementbase sequence to the sequence on the DNA of interest. Most original protocols usedradioactive labels, however non-radioactive alternatives are now available.Southern blotting is less commonly used in laboratory science due to the capacityof other techniques, such as PCR, to detect specific DNA sequences from DNAsamples. These blots are still used for some applications, however, such asmeasuring transgene copy number in transgenic mice, or in the engineering of geneknockout embryonic stem cell lines.[edit] Northern blottingMain article: northern blotThe northern blot is used to study the expression patterns of a specific type ofRNA molecule as relative comparison among a set of different samples of RNA. It isessentially a combination of denaturing RNA gel electrophoresis, and a blot. Inthis process RNA is separated based on size and is then transferred to a membranethat is then probed with a labeled complement of a sequence of interest. Theresults 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 RNAdetected in sample. The intensity of these bands is related to the amount of thetarget RNA in the samples analyzed. The procedure is commonly used to study whenand how much gene expression is occurring by measuring how much of that RNA ispresent in different samples. It is one of the most basic tools for determining at
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