Basic Concept of Biotechnology 23

  Tools and Techniques in Biotechnology   The fundamental unit of cell, genetic material, decides the type of cell and its behavior. The study with reference to nucleic acid, expression pattern of genes, proteins and other final products, formation of metabolites and their types gives a reflection about the cell and its function. Advancement in biotechnological methods has made these studies approachable and applicable. Since, biotechnology is the application of biological organisms, system or processes to manufacturing and service industries related to animals, plants, microbes and environment, here we are exploring the diverse tools and techniques involved in genomics, transcriptomic and proteomics. The genomic approaches facilitate to study the genetic material, locate the position of genes, chromosome paintings and hybridization techniques. The expression pattern of these genes studied by RNA isolation, qRT PCR, differential display techniques and recent methods like SAGE,...

  BASIC TOOLS IN THE BIOTECHNOLOGY   Biotechnology is an interdisciplinary science that borrows scientific instruments commonly used in chemistry, biochemistry, genetics, and physics laboratories. Very few instruments are specificallydesigned for biotechnology. Those that are unique to biotechnology were developed for the specific needs of particular research studies. A trip to a biotechnology laboratory would seem very much like a visit to any other science laboratory. This is also true for large facilities that produce biotechnology products. The machinery is used in many other industries. However, biotechnology instruments are focused on analyzing, manipulating, or manufacturing the chemicals that make up organisms. The major chemicals of interest in biotechnology are biological molecules called nucleic acids and proteins. Each instrument mentioned in this chapter can be found in most biotechnology industrial settings. Research laboratories are usually limited to particular...

  Measurement of Volume   A)       Erlenmeyer flasks  –Erlenmeyer flasks are used primarily toprepare solutions prior to an accurate volume adjustment. Although there are volumetric markings on these flasks, they are not calibrated and should not be relied upon for exact volume measurements.   B)        Beakers  –Beakers are also used for preparing solutions,especially when a pH adjustment requires access to the solution by a pH probe. The volumetric markings on beakers are also not reliable.   C)        Graduated cylinders  –Graduated cylinders are calibrated withsufficient accuracy for most volume measurements when preparing solutions. For example, the calibration of most 100 mL graduated cylinder can be relied upon to accurately measure to within +/- 0.6 mL.   D)       Volumetric flasks  –Volumetric flasks are used to measure aspec...

  Measurement of Weight Instruments for weighing materials are called balances, and most laboratories have more than one type of balance, depending on the amount of material being measured and the degree of accuracy required.   a.          Mechanical balances –  Mechanical balances weigh an object ona pan hanging from a beam that has a counterbalanced weight. We do not use mechanical balances in our lab.   b.          Electronic balances –  Electronic balances have replaced mostmechanical balances due to their greater accuracy and ease of operation. They are easier to use because they usually have a digital readout, and weighing dishes can be tared to read zero mass before using. Most balances used for preparation of solutions have a sensitivity of +/- 0.01 g, but electronic analytical balances can be sensitive to +/- 0.1 mg or less. Electronic balances require routine maintenance an...

  Measurement of pH Most solutions prepared in the biological laboratory must have a carefully controlled pH. Buffers are prepared by adjustment to a specific pH with strong acid and base solutions, using a meter to monitor the pH. A pH meter is a volt meter that measures the electrical potential between two electrodes. One electrode is in contact with your solution, and the other is in contact with a reference solution. Usually both of these electrodes are combined in a single pH probe that you place in your solution. These meters can read to the nearest 0.1 pH unit, but require frequent calibration with reference buffers of known pH.

  Measurement of light Solutions are often analyzed in the biotechnology lab by measuring how the solutes interact with light. A spectrophotometer measures the amount of light that is absorbed by a solution at a specific wavelength or over a range of wavelengths. If you know a wavelength at which a specific substance absorbs light, you can calculate the amount of that substance in a solution from the measured absorbance of that solution at that wavelength.   A.     VIS spectrophotometer  –A visible (VIS) spectrophotometermeasures absorbance of light in the visible region of the spectrum (wavelength of about 400-700 nm). A small vessel called a cuvette, which is generally plastic or glass and which usually has an internal diameter of 1.0 cm, is filled with the solution and placed in the spectrophotometer for measurements.   B.     UV/VIS spectrophotometer  –An ultraviolet/visible (UV/VIS)spectrophotometer can also measure absorbance ...

Solution Preparation Solution preparation involves mixing liquids and dissolving solids in liquids. There are many specialized devices in addition to balances, volume measuring devices, and pH meters involved in these processes.   A.     Magnetic stirrers  –Magnetic stirrers come in the form of a box witha magnet inside attached to a motor that spins the magnet. When a vessel containing a magnetic stir bar is on top of the magnetic stirrer, the stir bar spins and stirs the contents of the vessel.   B.      Vortex mixer  –A vortex mixer rotates the bottom of a tube rapidly;  setting up a vortex in the liquid that rapidly mixes the contents.

  Microbiological techniques   Specialized equipment is required to isolate, transfer, and grow up cultures of microbes and tissues in the laboratory.   A.   Autoclaves  –Autoclaves are machines that achieve a high internaltemperature and pressure and are used to sterilize solutions and glassware. The kitchen pressure cooker achieves the same results and can be used instead of an autoclave.   B.   Cell Culture Hood  –A biological safety or cell culture hood filterssmall particles out of the air in order to avoid contamination of cultures or sterile media. The filters are similar to those used to decontaminate air for operating rooms in hospitals or clean rooms used in the semiconductor industry.   C.   Fermentors  –Fermentors are used to grow up a large quantity of cellswith automatically controlled pH and levels of oxygen and other nutrients.   D.   Since most cells are generally too small to be seen with the naked eye, m...

  Preparation of biological samples for analysis   There are many pieces of equipment that are used to prepare biological samples for analysis.   A.     Sorvall-type centrifuge  –A Sorvall-type centrifuge, or preparativecentrifuge, has a balanced rotor that holds vessels and spins them at high speed, up to 20,000 rpm. This will cause most insoluble particles such as cells and many subcellular components to rapidly form a pellet at the bottom of the vessel. Rotors are available that hold vessels as small as a few milliliters to as large as a liter. These centrifuges are often refrigerated so that heat-sensitive compounds are not damaged during centrifugation. We do not have one of these in our lab.   B.      Tabletop centrifuge  –A tabletop, or clinical, centrifuge is generallynot refrigerated and spins at a much slower speed than a preparative centrifuge. Rotors for clinical centrifuges generally hold tubes with a capacity...

  Separation of macromolecules   Since there are thousands of different macromolecules in each cell, purification of a specific one from all the others requires powerful separation techniques, such as chromatography and electrophoresis. Both of these approaches take advantage of physical and chemical properties that differ between the individual macromolecules.   A.   Gel electrophoresis  –In gel electrophoresis, the macromolecules areplaced in a solid matrix, called a gel, which is under a liquid buffer. An electric field is applied to this system, and since biological macromolecules carry ionic charges, they will be attracted towards one pole of the electric field and repelled by the opposite. Thus, macromolecules characteristically migrate in either direction in the field. The migration speed is determined by the charge-to-mass ratio of the macromolecule.   Ø             In a flat gel, also called a ...

  Manipulation of Nucleic Acids   To analyze DNA some of the specialized pieces of equipment are:   A.     Thermal cycler  –A thermal cycler is a machine that is used foramplification of a specific section of DNA by PCR (polymerase chain reaction). The machine cycles through several temperatures, which allows an enzyme called DNA polymerase to use chemicals in solution to build DNA molecules identical to a template provided.   B.     Electroporator  –An electroporator is used to discharge a high-voltage, high-amperage pulse of electricity of very short duration through a cuvette containing suspended cells to disrupt their plasma membranes, allowing DNA to be introduced.   C.     Real-time PCR  –A real-time PCR machine amplifies and measuresthe production of amplicons in one step. It is a thermal cycler and fluorescent analyzer in one instrument and is usually computer-controlled. You do not have to load...

  Techniques of Biotechnology and Innovations The techniques used in modern biotechnology will be briefly highlighted in this section. A basic knowledge of their fundamental principles and applications is important for understanding the way biotechnology is used to benefit life. The techniques used in modern biotechnology can be divided into three major categories: genomics, proteomics, and metabolomics. Genomics is described as the study or use of genes and their functions. It involves techniques that investigate or make use of DNA. Proteomics is the study or use of the structure and function of proteins. It includes the various ways that proteins work individually and interact with each other inside cells. Metabolomics is the study or use of specific cellular processes carried out inside and outside of a cell. It includes the interaction of the cell with other cells (physionomics) and with environmental factors (environomics). Biotechnology techniques can also be classified into ...

  ISOLATION OF NUCLEIC ACID (DNA) Fundamentally all living organisms are either unicellular or multicellular, which can grow, reproduce, process the information, respond to stimuli and process the several other chemical /biological/physical process. These all processes are governed by genetic information stored in the genome of an organism. Either prokaryotic of eukaryotic organism’s their genome is made of deoxyribonucleic acid  (DNA). DNA is duplex of polynucleotide strands and it consisting of genes, promoter non-genic region and regulatory elements. To study this information and to characterize the genome of an organism, it is essential to isolate the genome (DNA) from their respective organism. The total genetic materials vary from organism to organism and within the genera also. The DNA isolation methods more or less remain the same irrespective of size of the genome. However, the predominant factors like age of the sample and tissue type influence the isolation efficien...