File Name: tools and techniques of molecular biology .zip
The following points highlight the top sixteen techniques used in cell biology. Some of the techniques are: 1. Immunofluorescence Microscopy 2.
Ion-Exchange Chromatography 3. Affinity Chromatography 4. Partition and Adsorption Chromatography 5. Gel Filtration Chromatography 6. Radioactive Tracer Technique 7. Radioimmunoassay RIA 8. Enzyme Immunoassay 9. Spectroscopy and Others. Immunofluorescence is another slightly modified technique used to study cells under fluorescence microscopy to locate the distribution of the antigen in the cells.
In this case the first antibody is un-labelled and the second antibody is made against IgGs of the organisms in which the first antibody is made. This secondary antibody is coupled to some fluorochrome such as Fluorescein Isothiocyanate, Rhodamine etc. The microfilaments, microtubules and intermediate filaments can be studied to have an idea about the cytoskeletal structure of the cell Fig. This technique is useful to study many structures at higher resolution in the electron microscope top.
Antibodies to many cellular proteins are available in the market from different firms like Amershain, Dako, Sigma etc. Primary antibodies may be monoclonal or polyclonal. Secondary antibodies are generally coupled with fluorochrome for immunofluorescence studies. The main instrument needed for the immunofluorescence studies is the fluorescence microscope with automatic photomicrograph system.
Confocal microscopy is being recently used for detailed studies of the structures in or near the nucleus and also for round cells to obtain greater resolutions at different levels in the cell. In this method, molecules are separated on the basis of differences in charge.
They may carry positive or negative charge. The charge showed by these compounds depend on the pH of the solution. Cation exchangers are negatively charged and so they can attract positively charged molecules. Anion exchangers are positively charged, so they can bind negatively charged molecules.
Tire commercial ion-exchangers are made of porous polystyrene beads. The co-polymerisation of styrene is made with varying proportions of divinylbenzene and styrene.
The most common example is Dowex 50, Sephadex etc. When the samples to be separated are passed through a column, molecules with opposite charge will bind while the other molecules with the same charge of the medium used in the column and some unwanted materials will pass through Fig. As the desired compound is retained in the column, this technique is sometimes known as Sorption chromatography. The bind molecules can be eluted by increasing the concentration of the buffer or by changing the pH of the buffer.
Sometimes the unwanted molecules are also retained within the column—thus eluting the desired substances. Some examples of ion- exchangers used in biology have been shown in Table 8. The selection of strong and weak exchanger depends on the stability over pH and the effect of pH on charge.
Generally, cationic buffers like Tris, Pyridine and Alkyl amines are used with anion exchangers and anionic buffers like Acetate, Barbiturate and Phosphate are used with cation exchangers. Two types of elution can be made, i.
When the gradient of buffer is passed through the column it is called Gradient elution. The separation of amino acids is generally performed using strong acid cation exchanger. Gradient elution method helps in the sequential elution of amino acids. The acidic amino acids come out first followed by the neutral amino acids like Glycine and Valine.
This is followed by basic amino acids like Arginine and Lysine. This principle is also followed in the Amino acid Analyser. Proteins are separated generally through weakly acidic or basic exchangers.
The elution of proteins takes place on the basis of their isotonic points. In this case some ligand molecule i. On passing the molecules through the column, only those materials that form a specific bond or complex with the ligand will be retained and all others will pass through the column Fig. The main principle of this method is that the compound to be purified is passed through the column containing some immobilised ligand, and the desired compound will bind to the ligand.
The ligand is the substrate in case of enzymes. Instead of column, this method can be utilised on nitrocellulose membranes for the purification of single-stranded DNA molecules. When the enzyme is to be purified, the ligand used is generally the substrate and, for the separation of proteins, specific antibodies are used. The bound-molecules can be eluted by increasing the ionic strength of the buffer.
Thus this type of chromatography is highly specific and versatile. This affinity chromatography is very useful for the purification and separation of macromolecules of very small amount.
It is a common practice to separate many substances by shaking the substance in two immiscible liquid phases in a separating funnel. When a substance is shaken in the solvent it will partition with the formation of two phases. If one phase is allowed to move the substance will also move on the basis of its partition coefficient. The substance will move rapidly if it likes the mobile phase while, if it prefers the stationary phase, it will move slowly.
The substance will move at a varying speed depending on the intensity and characteristics of adsorption and solubility in the solvent used for separation. Adsorption chromatography can be performed either in the column or on the thin layer of the matrix. Hydroxyapatite Calcium phosphate is used in the column to separate proteins, nucleic acids etc. For mobile phases, different organic solvents may be used, depending on the polarity of the compounds to be resolved.
In case of paper chromatography, the paper is the support. Water is used to moist the paper and this hydrated phase is the stationary phase. The organic solvent which is used in the chromatography is the mobile phase which moves rapidly through the aqueous phase. In Gas chromatography, the partitioning takes place between a liquid and a gas phase. Here the gas is the mobile phase and the nonvolatile liquid that coats the matrix substances of a column is the stationary phase.
This partitioning depends on the temperature and the gradual increase in temperature helps more and more of the substance to come out in the mobile gas phase from the stationary phase. This method is widely used for the qualitative and quantitative analyses of large number of compounds. The principle for the separation is the difference in the partitioning of the volatilized compounds between the liquid and gas phases.
During the passage of the substance through the column a detector is attached with a chart recorder, which scan the peak as the substance passes through the detector. This method is also known as exclusion or permeation chromatography.
Varying properties of the matrix axe obtained by crosslinking the poly-dextran with other compounds. Sephadex and polyacrylamide gels are used as swollen beads. Many gels are available in the market as Superfine, Fine, Medium and Coarser. The coarser bead shows fast flow rate but with poorer resolution.
Fine and Superfine beads are used for analytical work and the coarse one for preparative work. When the molecules of different sizes and pores are passed through a column, molecules larger than pores of gel will pass through it. Molecules of small pore size will enter the beads and flows are retarded in the column. It is also used for the isolation of ribosomal proteins. The easiest method for monitoring cellular events and their localisation in the cell is the use of radioactive isotopes.
They release high energy electron or beta particles during their radioactive decay. Radioactive decay is a spontaneous process and its rate varies with the source. The number of atoms disintegrating at any time is proportional to the number of atoms present in the isotope at that time. Conveniently, it is expressed as half-life which is defined as the time taken for the activity to fall from any value to half that value.
The half- life of some important isotopes is shown in the table 8. According to SI system, the unit of radioactivity is Becquerel Bq , which is one disintegration per second. But the most commonly used unit is the curie Ci which is measured as the number of nuclear disintegrations per second as compared to that of 1 gm of Radium, i. The disintegrations measured by the Counter are referred to as Counts. For any biological research, macromolecules of the cell are made radioactive by administering the radioactive compounds to tissues or cells and then the fate of the radioactive compound can be monitored.
For the study of DNA synthesis in the various types of cells or tissues, the use of H 3 is necessary. The radioactive substance is added in the solution where cells or tissues are exposed for a certain period of time, either cells are fixed at regular intervals or aliquots are removed at various time intervals and the radioactivity is noted.
The radioactivity may be measured through Liquid Scintillation Counter or the location of the radioactivity in different positions of the cell, can be noted through Radioactive Tracer Technology, called Autoradiography. In this method labelled cells are fixed and squashed on a slide or can be spread on a slide. Then a thin layer of Special Auto radiographic stripping film is placed over it and is kept in the dark for exposure. Instead of film, sometimes the slide is coated with photographic emulsions for autoradiography and is kept in the dark.
During storage of slides in the dark, the emission of beta particles from the radioactive substance activates Silver halide crystals of film or emulsion. After a few weeks, the slides are developed like the photographic film, which show the activated silver crystals as black spots under the light microscope. The auto radiographic technique is also applicable to electron microscopy where cells or tissues are placed on a grid instead of a slide.
Liverworts occupy a basal position in the evolution of land plants, and are a key group to address a wide variety of questions in plant biology. Marchantia polymorpha is a common, easily cultivated, dioecious liverwort species, and is emerging as an experimental model organism. The haploid gametophytic generation dominates the diploid sporophytic generation in its life cycle. Genetically homogeneous lines in the gametophyte generation can be established easily and propagated through asexual reproduction, which aids genetic and biochemical experiments. Owing to its dioecy, male and female sexual organs are formed in separate individuals, which enables crossing in a fully controlled manner. Reproductive growth can be induced at the desired times under laboratory conditions, which helps genetic analysis.
Molecular biology methods have tremendous value not only in the investigation of basic scientific questions, but also in application to a wide variety of problems affecting the overall human condition. Disease prevention and treatment, generation of new protein products, and manipulation of plants and animals for desired phenotypic traits are all applications that are routinely addressed by the application of molecular biology methods. Because of the wide applicability of these methods, they are rapidly becoming a pervasive--some would argue invasive--aspect of our technologically based society. The public concerns that address the application of these methods should be addressed by informed public discussion and debate. While scientists can be extremely critical of the quality, interpretation, and significance of experimental results, they have a rather remarkable tendency to be non-judgmental of the relative social merits of many applications of scientific research. It remains a public responsibility to be sufficiently well-informed to critically assess the merits of applied science research and participate in a communal decision-making process regarding the extent to which a new technology will be allowed to affect society. Abstract Molecular biology methods have tremendous value not only in the investigation of basic scientific questions, but also in application to a wide variety of problems affecting the overall human condition.
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