Molecular biology /məˈlɛkjʊlər/ concerns the molecular disciplines. The figure to the right is a schematic that de- basis of biological activity between the various systems of picts one possible view of the relationship between the a cell, including the interactions between DNA, RNA and fields: and their biosynthesis, as well as the regulation of these interactions. Writing in Nature in 1961, William • Astbury described molecular biology as: Biochemistry is the study of the chemical substances and vital processes occurring in live organisms. Biochemists focus heavily on the role, function, and "...not so much a technique as an approach, structure of biomolecules. The study of the chem- an approach from the viewpoint of the so- istry behind biological processes and the synthe- called basic sciences with the leading idea sis of biologically active molecules are examples of of searching below the large-scale manifesta- biochemistry. tions of classical biology for the correspond- ing molecular plan. It is concerned particu- larly with the forms of biological molecules • Genetics is the study of the effect of genetic differ- and [...] is predominantly three-dimensional ences on organisms. This can often be inferred by and structural—which does not mean, how- the absence of a normal component (e.g. one gene). ever, that it is merely a refinement of morphol- The study of "mutants" – organisms which lack one ogy. It must at the same time inquire into gen- or more functional components with respect to the esis and function.”[1] so-called "wild type" or normal phenotype. Genetic interactions (epistasis) can often confound simple interpretations of such "knockout" studies. 1 Relationship to other biological • Molecular biology is the study of molecular sciences 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 , despite being an oversimplified picture of molecular biology, still provides a good start- ing 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 quantita- tive, and recently much work has been done at the in- terface of molecular biology and computer science in 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 molecular biology. Increasingly many other loops of biology focus on molecules, either directly studying their interactions in Schematic relationship between biochemistry, genetics and their own right such as in cell biology and developmental molecular biology biology, or indirectly, where the techniques of molec- ular biology are used to infer historical attributes of Researchers in molecular biology use specific tech- populations or species, as in fields in evolutionary biol- niques native to molecular biology but increasingly com- ogy such as population genetics and phylogenetics. There bine these with techniques and ideas from genetics and is also a long tradition of studying biomolecules “from the biochemistry. There is not a defined line between these ground up” in biophysics.

1 2 2 TECHNIQUES OF MOLECULAR BIOLOGY

2 Techniques of molecular biology crystallized so its tertiary structure can be studied, or, in the pharmaceutical industry, the activity of new drugs Since the late 1950s and early 1960s, molecular biolo- against the protein can be studied. gists have learned to characterize, isolate, and manipulate the molecular components of cells and organisms. These components include DNA, the repository of genetic in- 2.2 Polymerase chain reaction (PCR) formation; RNA, a close relative of DNA whose func- tions range from serving as a temporary working copy of Main article: Polymerase chain reaction DNA to actual structural and enzymatic functions as well as a functional and structural part of the translational ap- paratus, the ribosome; and proteins, the major structural Polymerase chain reaction is an extremely versatile tech- and enzymatic type of molecule in cells. nique for copying DNA. In brief, PCR allows a spe- cific DNA sequence to be copied or modified in prede- For more extensive list on , see protein termined ways. The reaction is extremely powerful and methods. For more extensive list on nucleic acid meth- under perfect conditions could amplify 1 DNA molecule ods, see nucleic acid methods. to become 1.07 Billion molecules in less than 2 hours. The PCR technique can be used to introduce restriction enzyme sites to ends of DNA molecules, or to mutate (change) particular bases of DNA, the latter is a method 2.1 Molecular cloning referred to as site-directed mutagenesis. PCR can also be used to determine whether a particular DNA frag- Main article: Molecular cloning ment is found in a cDNA library. PCR has many vari- ations, like reverse transcription PCR (RT-PCR) for am- plification of RNA, and, more recently, quantitative PCR One of the most basic techniques of molecular biology which allow for quantitative measurement of DNA or to study protein function is molecular cloning. In this RNA molecules. 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 distinc- tive features: an origin of replication, a multiple cloning 2.3 site (MCS), and a selective marker (usually antibiotic re- sistance). The origin of replication will have promoter Main article: Gel electrophoresis regions upstream from the replication/transcription start site. Gel electrophoresis is one of the principal tools of molec- This plasmid can be inserted into either bacterial or an- ular biology. The basic principle is that DNA, RNA, and imal cells. Introducing DNA into bacterial cells can proteins can all be separated by means of an electric field be done by transformation (via uptake of naked DNA), and size. In agarose gel electrophoresis, DNA and RNA conjugation (via cell-cell contact) or by transduction (via can be separated on the basis of size by running the DNA viral vector). Introducing DNA into eukaryotic cells, through an electrically charged agarose gel. Proteins can such as animal cells, by physical or chemical means is be separated on the basis of size by using an SDS-PAGE called transfection. Several different transfection tech- gel, or on the basis of size and their electric charge by niques are available, such as calcium phosphate transfec- using what is known as a 2D gel electrophoresis. tion, electroporation, microinjection and liposome trans- fection. DNA can also be introduced into eukaryotic cells using viruses or bacteria as carriers, the latter 2.4 Macromolecule blotting and probing is sometimes called bactofection and in particular uses Agrobacterium tumefaciens. The plasmid may be inte- The terms northern, western and eastern blotting are de- grated into the genome, resulting in a stable transfection, rived from what initially was a molecular biology joke or may remain independent of the genome, called tran- that played on the term Southern blotting, after the tech- sient transfection. nique described by for the hybridisa- In either case, DNA coding for a protein of interest is now tion of blotted DNA. Patricia Thomas, developer of the inside a cell, and the protein can now be expressed. A RNA which then became known as the northern variety of systems, such as inducible promoters and spe- blot, actually didn't use the term.[2] Further combinations cific cell-signaling factors, are available to help express of these techniques produced such terms as southwest- the protein of interest at high levels. Large quantities of erns (protein-DNA hybridizations), northwesterns (to de- a protein can then be extracted from the bacterial or eu- tect protein-RNA interactions) and farwesterns (protein- karyotic cell. The protein can be tested for enzymatic protein interactions), all of which are presently found in activity under a variety of situations, the protein may be the literature. 2.5 Microarrays 3

2.4.1 Southern blotting In western blotting, proteins are first separated by size, in a thin gel sandwiched between two glass plates in Main article: a technique known as SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The pro- Named after its inventor, biologist Edwin Southern, the teins in the gel are then transferred to a polyvinylidene Southern blot is a method for probing for the presence of fluoride (PVDF), , nylon, or other sup- a specific DNA sequence within a DNA sample. DNA port membrane. This membrane can then be probed samples before or after restriction enzyme (restriction en- with solutions of antibodies. Antibodies that specif- donuclease) digestion are separated by gel electrophore- ically bind to the protein of interest can then be vi- sis and then transferred to a membrane by blotting via sualized by a variety of techniques, including colored capillary action. The membrane is then exposed to a products, chemiluminescence, or autoradiography. Of- labeled DNA probe that has a complement base se- ten, the antibodies are labeled with enzymes. When a quence to the sequence on the DNA of interest. Most chemiluminescent substrate is exposed to the enzyme it original protocols used radioactive labels, however non- allows detection. Using western blotting techniques al- radioactive alternatives are now available. Southern blot- lows not only detection but also quantitative analysis. ting is less commonly used in laboratory science due to Analogous methods to western blotting can be used to di- the capacity of other techniques, such as PCR, to de- rectly stain specific proteins in live cells or tissue sections. tect specific DNA sequences from DNA samples. These However, these methods, such as FISH, blots are still used for some applications, however, such are used more often in cell biology research. as measuring transgene copy number in transgenic mice, or in the engineering of gene knockout embryonic stem cell lines. 2.4.4 Eastern blotting Main article: Eastern blot 2.4.2 Northern blotting The Eastern blotting technique is used to detect post- Main article: translational modification of proteins.[3] Proteins blotted on to the PVDF or nitrocellulose membrane are probed The northern blot is used to study the expression patterns for modifications using specific substrates. of a specific type of RNA molecule as relative compari- son among a set of different samples of RNA. It is essen- tially a combination of denaturing RNA gel electrophore- 2.5 Microarrays sis, and a blot. In this process RNA is separated based on size and is then transferred to a membrane that is then Main article: DNA microarray probed with a labeled complement of a sequence of in- terest. The results may be visualized through a variety of A DNA microarray is a collection of spots attached ways depending on the label used; however, most result to a solid support such as a microscope slide where in the revelation of bands representing the sizes of the each spot contains one or more single-stranded DNA RNA detected in sample. The intensity of these bands is oligonucleotide fragment. Arrays make it possible to put related to the amount of the target RNA in the samples down large quantities of very small (100 micrometre di- analyzed. The procedure is commonly used to study when ameter) spots on a single slide. Each spot has a DNA frag- and how much gene expression is occurring by measur- ment molecule that is complementary to a single DNA ing how much of that RNA is present in different sam- sequence (similar to Southern blotting). A variation of ples. It is one of the most basic tools for determining at this technique allows the gene expression of an organ- what time, and under what conditions, certain genes are ism at a particular stage in development to be qualified expressed in living tissues. (expression profiling). In this technique the RNA in a tissue is isolated and converted to labeled cDNA. This cDNA is then hybridized to the fragments on the array 2.4.3 Western blotting and visualization of the hybridization can be done. Since multiple arrays can be made with exactly the same posi- Main article: tion of fragments they are particularly useful for compar- ing the gene expression of two different tissues, such as a Antibodies to most proteins can be created by inject- healthy and cancerous tissue. Also, one can measure what ing small amounts of the protein into an animal such as genes are expressed and how that expression changes with a mouse, rabbit, sheep, or donkey (polyclonal antibod- time or with other factors. For instance, the common ies) or produced in cell culture (monoclonal antibodies). baker’s yeast, Saccharomyces cerevisiae, contains about These antibodies can be used for a variety of analytical 7000 genes; with a microarray, one can measure qualita- and preparative techniques. tively how each gene is expressed, and how that expres- 4 7 FURTHER READING sion changes, for example, with a change in temperature. about to undergo a period of significant change given re- There are many different ways to fabricate microarrays; cent advances in fields such as X-ray crystallography. He the most common are silicon chips, microscope slides therefore channeled significant amounts of (Rockefeller with spots of ~ 100 micrometre diameter, custom ar- Institute) money into biological fields. rays, and arrays with larger spots on porous membranes (macroarrays). There can be anywhere from 100 spots to more than 10,000 on a given array. 4 Clinical significance Arrays can also be made with molecules other than DNA. For example, an antibody array can be used to determine Clinical research and medical therapies arising from what proteins or bacteria are present in a blood sample. molecular biology are partly covered under gene ther- apy . The use of molecular biology or molecular cell biology approaches in medicine is now called molecular 2.6 Allele-specific oligonucleotide medicine. Molecular biology also plays important role in understanding formations, actions, regulations of various Allele-specific oligonucleotide (ASO) is a technique that parts of cells which can be used efficiently for targeting allows detection of single base mutations without the new drugs, diagnosis of disease, physiology of the Cell. need for PCR or gel electrophoresis. Short (20-25 nu- cleotides in length), labeled probes are exposed to the non-fragmented target DNA. Hybridization occurs with 5 See also high specificity due to the short length of the probes and even a single base change will hinder hybridization. The • Central dogma of molecular biology target DNA is then washed and the labeled probes that didn't hybridize are removed. The target DNA is then • Genetic code analyzed for the presence of the probe via radioactivity or fluorescence. In this experiment, as in most molecular • Genome biology techniques, a control must be used to ensure suc- • cessful experimentation. The Illumina As- Molecular microbiology say is an example of a method that takes advantage of the • Molecular modeling ASO technique to measure one base pair differences in sequence. • Protein interaction prediction

• Protein structure prediction

2.7 Antiquated technologies • Proteome

In molecular biology, procedures and technologies are continually being developed and older technologies aban- doned. For example, before the advent of DNA gel elec- 6 References trophoresis (agarose or polyacrylamide), the size of DNA molecules was typically determined by rate sedimentation [1] Astbury, W.T. (1961). “Molecular Biology or Ultra- in sucrose gradients, a slow and labor-intensive technique structural Biology?" (PDF). Nature 190 (4781): 1124. doi:10.1038/1901124a0. PMID 13684868. Retrieved requiring expensive instrumentation; prior to sucrose gra- 2008-08-04. dients, viscometry was used. Aside from their historical interest, it is often worth [2] Thomas, P.S. (1980). “Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose”. knowing about older technology, as it is occasionally use- PNAS 77 (9): 5201–5. doi:10.1073/pnas.77.9.5201. ful to solve another new problem for which the newer ISSN 1091-6490. PMC 350025. PMID 6159641. technique is inappropriate. [3] Thomas S, Thirumalapura N, Crossley EC, Ismail N, and Walker DH (2009). Antigenic protein modifications in Ehrlichia. Parasite Immunology 31, 296-303. 3 History

Main article: History of molecular biology 7 Further reading

While molecular biology was established in the 1930s, • Cohen, S.N., Chang, A.C.Y., Boyer, H. & Heling, the term was coined by Warren Weaver in 1938. Weaver R.B. Construction of biologically functional bacte- was the director of Natural Sciences for the Rockefeller rial plasmids in vitro. Proc. Natl. Acad. Sci. 70, Foundation at the time and believed that biology was 3240 – 3244 (1973). 5

• Rodgers, M. The Pandora’s box congress. Rolling Stone 189, 37 – 77 (1975). • Keith Roberts, Martin Raff, Bruce Alberts, Peter Walter, Julian Lewis and Alexander Johnson, Molec- ular Biology of the Cell

• 4th Edition, Routledge, March, 2002, hard- cover, 1616 pages, 7.6 pounds, ISBN 0-8153- 3218-1 • 3rd Edition, Garland, 1994, ISBN 0-8153- 1620-8 • 2nd Edition, Garland, 1989, ISBN 0-8240- 3695-6

8 External links

• Biochemistry and Molecular Biology at DMOZ 6 9 TEXT AND IMAGE SOURCES, CONTRIBUTORS, AND LICENSES

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