Biotechnology: Principles and Processes PDF

Biotechnology: Principles and Processes INTRODUCTION y The term biotechnology is of recent Definition origin, but the discipline is very old. Humans had started using Biotechnology: The integration of microorganisms as early as 50...

Biotechnology: Principles and Processes INTRODUCTION y The term biotechnology is of recent Definition origin, but the discipline is very old. Humans had started using Biotechnology: The integration of microorganisms as early as 5000 BC for natural science and organisms, cells, making wine, vinegar, etc. Some of these parts thereof, and molecular analogues processes have become so common in for products and services. our homes that we use them in our daily works. y Biotechnology is derived from two words — biology and technology. It is a field in which the biological agents and their components have been extracted and used for the generation of useful products. It is a vast technique which has improved and enlarged the scope of modifying organisms and their products for the humans and animals. y The European Federation of Biotechnology has given a definition of biotechnology that takes into view the traditional as well as modern molecular biotechnology. The definition given by EFB is “The integration of natural science and organisms, cells, parts “The rewards for biotechnology are thereof, and molecular analogues for tremendous – to solve disease, eliminate poverty, age gracefully.” products and services”. y Biotechnology is multi-disciplinary and George M. Church uses biology, chemistry, biochemistry, Biotechnology: Principles and Processes genetics, molecular biology, immunology, cell tissue culture and physiology for Previous Year’s Question the process of biotechnology. Introduction of foreign genes for PRINCIPLES OF BIOTECHNOLOGY improving genotype is y Genetic engineering and Bioprocessing (1) tissue culture are the two core techniques that have led to (2) vernalization the development of biotechnology. (3) genetic engineering ⚪ Genetic engineering (4) biotechnology ⚪ Bioprocessing 1.  Definition Recombinant DNA or rDNA: DNA molecule ligated into a vector and then inserted into a host organism to produce new genetic combinations. PROCESS OF RECOMBINANT DNA TECHNOLOGY Biotechnology: Principles and Processes 2.  CONSTRUCTION OF FIRST ARTIFICIAL RECOMBINANT DNA MOLECULE y Plasmids are extra-chromosomal Definition material present in the bacteria. It can be physically separated and can Plasmid: Autonomously replicating replicate independently of the bacterial extra-chromosomal material present chromosome. It provides resistance to in the bacteria that provides antibiotic the bacteria against certain antibiotics. resistance to the bacteria. y First Artificial Recombinant DNA Molecule was constructed by Stanley Cohen and Herbert Boyer in 1972. y Stanley Cohen and Herbert Boyer in 1972 isolated antibiotic resistant gene by cutting a fragment of DNA from a plasmid. This antibiotic resistant gene fragment was then integrated with the Previous Year’s Question plasmid DNA. Biotechnology: Principles and Processes y They cut the gene of antibiotic Genetic engineering is possible, resistance from a plasmid with the help because of a restriction enzyme and joined it to (1) we can cut DNA at specific sites the plasmid of Salmonella typhimurium by endonucleases like DNase with the help of DNA ligase. (2) restriction endonucleases purified y This newly formed DNA having fragments from bacteria can be used in-vitro of antibiotic resistance is known as the (3) the phenomenon of transduction in recombinant DNA. bacteria is well understood y The plasmid of Salmonella typhimurium (4) we can see DNA by electron with the gene of antibiotic resistance microscope. acts as a vector and is transferred into the bacterium Escherichia coli. 3.  y The plasmid was replicated in the bacteria E. coli with the help of the enzyme DNA polymerase and made multiple copies of the recombinant DNA. STEPS INVOLVED IN THE PROCESS OF RECOMBINANT DNA TECHNOLOGY Biotechnology: Principles and Processes Isolation of the DNA y DNA is the genetic material of many organisms. In some organisms, it is the RNA. Keywords y In the process of biotechnology, the fragment of DNA with the desired Š Genetic Engineering characteristics is first to be isolated Š Bioprocessing from the source cell. The cell could be Š European Federation of a plant, animal, bacterial or fungal cell. Biotechnology y The DNA needs to be separated from Š Recombinant DNA the cell and the other macromolecules Š Plasmid and then purified. 4. y To cut the cell we need to lyse it with certain enzymes depending upon the type of cell from which the DNA needs to be isolated. ⚪ Bacterial Cell-Lysozyme ⚪ Fungal Cell-Chitinase ⚪ Plant Cell-Cellulase y The DNA removed from the cell now need to separated from RNA, proteins and other macromolecules. y RNA is removed by treating with ribonuclease while proteins are removed by treating with proteases. y Chilled ethanol is now added to Definition the soluble DNA and purified DNA Spooling: The precipitated DNA precipitates out. molecules is swirled with the glass rod y The DNA is now removed by spooling. or a spooling stick and separated from the solution. Biotechnology: Principles and Processes Restriction Enzymes y Steward Linn and Werner Arber isolated two enzymes that restricted the growth of bacteriophage in E.coli. y One restricted the growth of the bacteriophage by adding a methyl group to the DNA while the other cut the 5.  DNA. The enzyme which cut the DNA is known as restriction enzyme. These Previous Year’s Question enzymes are placed under the category of Nucleases. Which of the following is not correctly y Restriction enzymes are of four broad matched for the organism and its cell categories wall degrading enzyme? ⚪ Type I-These enzymes cleave at (1) Algae - Methylase random sites far away from the (2) Fungi - Chitinase restriction sites. Thus they are not (3) Bacteria - Lysozyme used in the recombinant technology. (4) Plant cells - Cellulase ⚪ Type II-These enzymes cut DNA at specific sites, close to or within their recognition sequences. They are used in biotechnology. Many of these restriction enzymes have been identified from a large number of bacterial species. Biotechnology: Principles and Processes ⚪ Type III-These enzymes also cleave at sites a short distance from a recognition site far away from the restriction sites. Rack your Brain ⚪ Type IV-These enzymes cleave only methylated DNA. Restriction endonuclease is obtained ⚪ Type V-These enzymes use guide from ______. RNAs to target specific non- palindromic sequences present on invading organisms. 6. y Restriction enzymes recognize specific Gray Matter Alert!!! locations in the DNA strand where it makes the cut. The sequence at which the DNA strands recognize the Blunt ends or non-cohesive specific site for cutting is known at the ends: A straight cut by the recognition sequence. restriction enzymes generates y These recognition sequences are usually strands that terminate in a base palindromic sequences. pair. It contains no unpaired DNA y Palindromic sequences are those strand fleeting at the end of DNA. sequences in which the sequence is These blunt ends are not desirable read same in the forward and backward in biotechnology as the yield is lower directions. For example, MALAYALAM with blunt ends. y In the DNA strand, sequence of the nucleotides when read from 5′ to 3′ in one strand and 3′ to 5′ in the other strand read the same. 5′—GAATTC—3′ 3′—CTTAAG—5′ Biotechnology: Principles and Processes 7.  y The restriction enzyme cuts the strand of DNA a little away from the center of the strand but between the same nucleotide sequences. y This results in single stranded unpaired bases at the cut ends. These are known Previous Year’s Question as sticky ends or cohesive ends. y These sticky ends form hydrogen bonds There is a restriction endonuclease with their complementary cut parts. called EcoRI. What does “co” part in When the DNA having the desired it stand for? gene and the plasmid DNA are cut by (1) colon the same restriction enzyme then the (2) coelom fragments will have the same sticky (3) coenzyme ends. (4) coli Nomenclature of the Enzymes y The restriction enzymes isolated from the bacteria are named according to the source from which they have been isolated. The rules of nomenclature are— Biotechnology: Principles and Processes 8.  Table. Sources of Different Restriction Enzymes Enzyme Name Genus Species Strain BamH I Bacillus amyloliquefaciens H Sal I Streptomyces albus - Pst I Providencia stuartii - Pvu I Proteus vulgaris - Cla I Caryophanon latum - HinD III Haemophilus influenzae DSM 11121 Gel Electrophoresis y The process of separation of DNA, RNA or protein fragments under the presence of electric field is known as gel electrophoresis. The DNA segments need to be separated into different fragments according to their sizes and Biotechnology: Principles and Processes then the desired fragment needs to be separated. y Materials required: Desired DNA fragment to be cut, agarose gel, gel plate, ethidium bromide y Principle involved: DNA being negatively Rack your Brain charged moves towards the positive electrode i.e. anode under the influence of electric field. What is the principle on which gel y Procedure: Electrophoresis is performed electrophoresis works? in a gel matrix. 9. Biotechnology: Principles and Processes Keywords Definition Š Nuclease Elution: The separated DNA fragments Š Recognition sequence are cut from the agarose gel and Š Palindrome sequence extracted from it. Š Gel electrophoresis Š Elution 10.  Biotechnology: Principles and Processes Cloning Vectors or Cloning Vehicle Previous Year’s Question y Cloning vectors are structures that help in making multiple copies of the desired Agarose extracted from sea weeds is DNA fragments. used in y These vectors can replicate within the (1) spectrophotometry host bacterial cell. Some of them are (2) tissue culture present naturally in the bacterial cell, (3) PCR while in others they can be introduced (4) gel electrophoresis artificially. 11.  CHARACTERISTIC FEATURES OF CLONING VEHICLES (VECTORS) y A vector is a DNA molecule that has the ability to replicate inside the host cell. y A vector can be inserted with the foreign Rack your Brain desired DNA fragments for cloning. y To act as a vector and to facilitate Name the technique used for separating cloning a vector must have the following the DNA fragments in a laboratory? characteristics– ⚪ Origin of Replication or ori Biotechnology: Principles and Processes  It is a set of sequence which is present on the vector from which the replication of the DNA starts.  The desired foreign DNA which is isolated from the organism needs to be integrated with the vector at the origin of replication or near Definition to the origin of replication of a vector, then it can replicate the Copy Number: It is the average number desired DNA fragment during its of copies of the plasmids made per own replication. It also controls host cell. the copy number. 12. ⚪ Restriction Sites or Recognition Sites Definition  Each vector should have only one recognition site of a particular Transformation: The uptake of DNA enzyme. fragment by the cell and integration  If it has more than one recognition into its own chromosomal DNA and site for a particular enzyme expressing the trait of the incorporated then managing the vector DNA DNA. would be difficult as it would be cut at different positions and thus integration of the new DNA would be difficult. ⚪ Selectable Markers  Vectors should have selectable marker which helps in identifying and eliminating the non-recombinant or non-transformants from the recombinants or the transformants.  Transformation is the process through which a piece of DNA is introduced into the bacteria so that it can be integrated with Previous Year’s Question the vector present inside the bacteria.  Usually genes encoding A selectable marker is used to resistance for antibiotics like (1) help in eliminating the non- ampicillin, chloramphenicol, transformants, so that the transformants can be regenerated Biotechnology: Principles and Processes tetracycline or kanamycin are considered as useful selectable (2) identify the gene for a desired trait markers. in an alien organism ⚪ Molecule Weight – Low (3) select a suitable vector for ⚪ Sites for different restriction transformation in a specific crop enzymes – Several sites for a large (4) mark a gene on a chromosome for number of restriction enzymes. isolation using restriction enzyme 13. Biotechnology: Principles and Processes TYPES OF VECTORS 14.  Plasmids y They are the most commonly used Previous Year’s Question cloning vectors. They are circular double-stranded DNA molecule that The plasmid pBR322 used in occurs as an extra-chromosomal biotechnology is material in the bacteria. Naturally they (1) Yeast are self replicating usually one or two (2) M13 phase copies of the plasmid are present in a (3) parasite cell. The size of plasmid may range from (4) cloning vehicle 1 to 200 bps. y Plasmids can be of two types- single copy plasmid or multiple copy plasmid. ⚪ Single copy plasmid maintains one plasmid per host while multiple copy plasmids can present in large numbers inside the host. y Plasmid vector is isolated from the bacterial cell. Rack your Brain y Foreign DNA carrying the desired gene for a particular characteristic is then integrated with the DNA of the vector Name the type of host cells suitable and joined together. for the introduction of an alien DNA. y The newly formed recombinant DNA is now inserted into the bacterial cell for cloning. y One of the earliest vectors that has been constructed artificially is the pBR322. p stands for plasmid, B is for Boliver, R is for Rodriquez, initials of the scientist Biotechnology: Principles and Processes who developed pBR322. The numerical 322 distinguishes this plasmid from the other plasma that developed in the Previous Year’s Question same lab. y pBR322 is the most popular and widely Which of the following is not a feature used plasmid which has 4361 base pairs of the plasmids? in length. Its entire sequence is known. (1) Transferable y It has two selectable markers (2) Single-stranded tetracycline and ampicillin resistant (3) Independent replication genes and many different recognition (4) Circular structure sites for restriction enzymes. The 15.  presence of the restriction sites helps in easy selection for the recombinant pBR322. y Insertion of the DNA fragment into the plasmid at the Pst I or Pvu I, places the DNA insert within the gene ampicillin and thus makes it non-functional. y Bacterial cell containing such a recombinant pBR322 will be able to grow in the presence of tetracycline but will not grow in ampicillin. y Similarly when DNA insert is placed in the restriction enzyme BamH I and Sal I, the gene coding for tetracycline resistance becomes non-functional. y This is a cumbersome process and requires multiple plating. To avoid this process which takes a lot of time we insertional inactivation is favoured which helps us to identify the transformants from the non-transformants easily by the inactivation of a gene by insertion of a DNA sequence within it. Identification of Recombinants y Once a recombinant DNA molecule has been introduced into the vector and the vector is introduced into the bacterial cell it is important to isolate Biotechnology: Principles and Processes the transformants from the non- transformants. y This can be done by growing the bacterial cell on antibiotics and thus this helps us to identify the transformers and separate Rack your Brain them from the non-transformers. y Let’s understand this with the example of pBR322. What will happen if a foreign DNA y It may happen that the bacteria E coli is ligated at BamH I site of PBR322 may get inserted with a normal vector plasmid? which does not have the recommend 16.  DNA inserted into it. So we need to select those bacteria which have the recombinant vector inserted into it. y This can be done with the help of a procedure called replica plating. y After trying to transform pBR322 plasmid with the desired DNA, the host cell is first grown on agarose gel containing antibiotic ampicillin. y Here we assume in this case that the recombinant DNA has ligated with the tetracycline resistance gene. y Colonies will develop on the single plate. In the plate both the bacterial cells having the vectors with the recombinant DNA and the ones not having the recombinant DNA would multiply. y The role of the recombinant plasmid is to help the host cell to multiply in the presence of antibiotic which otherwise would not be able to do. y A petri plate containing solid media with antibiotic tetracycline is kept carefully under aseptic conditions. To this, a circular piece of velvet is aligned and pressed onto the colony containing ampicillin plate (master plate). y With the same alignment it is pressed Biotechnology: Principles and Processes onto the tetracycline plate. y Overnight, only colonies not containing the insert will grow. No colonies which Previous Year’s Question have the insert at tetracycline position will grow. A gene whose expression helps to y The colonies which have the insert can identify transformed cell is known as easily be selected by comparing the two (1) vector plates. (2) plasmid y The above described process is a time (3) structural gene taking one and so another process of (4) selectable marker insertional inactivation is used. 17.  y In this process a recombinant DNA is inserted into the coding sequence Previous Year’s Question of b galactosidase. This bring about insertional inactivation. Selectable marker is used to y If the plasmid in the bacteria does (1) eliminate the non-transformants not have an insert the presence of (2) identify the gene for a desired trait chromogenic substrate gives blue (3)  select a suitable vector for coloured colonies. transformation in a specific crop y If the plasmid in the bacteria have an (4) mark a gene on a chromosome for insert then the colonies do not produce isolation using restriction enzyme any colour and these are the colonies that are recombinant colonies. Bacteriophage y Bacteriophages are viruses that attack bacteria. y They follow lytic or lysogenic cycle. In the lysogenic cycle, the phage DNA integrates with the bacterial chromosome and multiply. y The two bacteriophages that have been used are lambda and M13 phage ⚪ Lambda phage-The phage has 48,502 base pairs and it is double-stranded, linear DNA and has single-stranded protruding cohesive ends of 12 bases.  The genome is linear in the phage head but in the E.coli, the two cohesive ends anneal to form Biotechnology: Principles and Processes a circular molecule. The sealed cohesive sites are known as COS sites. ⚪ M13 phage - Filamentous phage having a linear DNA which converts into a double-stranded circular intermediate form within the host referred to as the Replicative Form (RF).  Advantage over plasmid-More efficient. 18.   Easy to clone large amount of DNA.  Easy to screen a large number of recombinant phage. Cosmids y It is a combination of plasmids and 200 base pairs of lambda phage containing the COS site of the lambda phage. y It also has a replication origin, unique restriction sites, a selectable marker from the plasmid. y Cosmids can accommodate 40-45 kb of DNA insert in it. Shuttle vector y They can replicate in two different species and so contain two origins of replication specific for each host. y Some can grow in two different Prokaryotic species for example in E coli and Streptomyces and some in Eukaryotic species like Yeast. Yeast Artificial Chromosome Vector (YAC) y Linear vectors that behave as yeast chromosomes. It consists of a sequence Biotechnology: Principles and Processes for replication, one or two selectable markers, Telomeric sequences at both Previous Year’s Question the ends that protect from exonuclease activity. Which vector can clone only a small y It was used in human genome project. fragment of DNA? (1) Bacterial artificial chromosome Bacterial Artificial Chromosome (2) Yeast artificial chromosome Vectors (BAC) (3) Plasmid y They have sequence of replication of (4) Cosmid origin. It controls the stability to maintain 1-2 copies of the vectors per cell. y It can accommodate inserts up to 500 kb. It contains a selectable marker and 19.  cloning sites, and genes for replication and maintenance of the F plasmid. y It was used in human genome project. y Phasmid-They consist of linear lambda genome consisting of DNA replication. y Transposons-First observed by Mc Clintock in maize. Units of DNA that can move from one DNA to another DNA and hence are mobile. They are also called as mobile genes or jumping genes. Animal Vectors y DNA fragments from animals are cloned in E.coli and then integrated with different viruses to be introduced into the host animal cell. y The ability of viruses to adsorb to cells, introduce their DNA and replicate the DNA , have made them ideal to transfer foreign DNA into eukaryotic cells. y Adenoviruses and Papillomavirus have been used to clone genes in mammals. Retroviral vectors are used for cloning genes in mammalian cells. y Retrovirus is disarmed of its harmful Previous Year’s Question DNA and then in its place the desired DNA fragment is inserted to be integrated with the host. Which one of the following is commonly Biotechnology: Principles and Processes used in transfer of foreign DNA into Plant Vectors crop plants? y Two plasmids namely Ti and Ri are (1) Meloidogyne incognita present in Agrobacterium tumefaciens (2) Agrobacterium tumefaciens and A. rhizogenes respectively and are (3) Penicillium expansum considered as natural genetic engineer (4) Trichoderma for the plants. y Ti is a tumour inducing plasmid while Ri is a root inducing plasmid. y Ti plasmid is around 200kb size. The process of infection by A. tumefaciens is because of the transfer 20.  of a small part of Ti into the plant cell Rack your Brain genome. The DNA sequence is known as T-DNA i.e. transferred DNA. y vir region regulates the transfer of Why DNA cannot pass through the cell T-DNA into the plant cell. membrane? y A.tumefaciens produces nitrogenous compounds called opines like octopines or nopaline which are used by the plasmid as their carbon and nitrogen source. While A. rhizogene produces either agropine or mannopine. Opine catabolism region produces enzymes required for the utilization of opines by Agrobacterium. y Both the plasmids have the genes for Indole acetic acid and cytokinin which help in rapid division of the cells. COMPETENT HOST Vector Mediated Gene Transfer y The recombinant DNA now needs to be incorporated into a cell which can be a bacteria, plant or animal cell. y DNA is hydrophilic and it is not possible for it to pass through the membranes. y The bacterial cell is treated first with high concentration of calcium ions to increase the efficiency of the membrane to intake DNA. y The cells are then incubated on ice bath Biotechnology: Principles and Processes and then given a heat treatment at 42°C and again put on ice bath. y This helps the bacteria in taking up the DNA. Direct Gene Transfer y Introduction of DNA into the plant cell Rack your Brain or animal cell directly without the use of a biological agent is known as direct gene transfer. The spontaneous uptake Why is it important to make bacterial of DNA by the plant or animal cell is less cell competent? and thus there are different ways to increase the uptake. 21.  y The various method of direct gene transfer is– Keywords ⚪ Chemical method ⚪ Electroporation Š Ori ⚪ Biolistic Š Selectable marker ⚪ Microinjection Š Insertional Inactivation ⚪ Macroinjection Š Ti Plasmid ⚪ Lipofection Š Microinjection y Chemical Method Š Biolistics  Polyethylene glycol, polyvinyl alcohol is used to increase the intake of DNA by the plant cell.  The plant protoplast is placed in a medium rich in Mg2+. To this, the plasmid DNA with the desired gene is added.  After this, Polyethylene glycol is Rack your Brain added and pH is maintained at 8.  The protoplast is given a five Name the natural genetic engineer of minute heat treatment and then plants. placed on ice before the addition of DNA.  This increases the frequency of transformation.  After some period of incubation the polyethylene glycol is reduced and calcium ions are increased. Previous Year’s Question  This helps in increasing the Biotechnology: Principles and Processes transformation frequency. Electroporation procedure involves y Electroporation (1) fast passage of food through sieve  The cells are exposed to electric pores in phloem elements with the impulse which induces transient help of electric stimulation pores in the plasmalemma that (2) opening of stomatal pores during helps in the intake of the DNA. night by artificial light  Plant protoplast is suspended (3) making transient pores in the in an ionic medium containing cell membrane to introduce gene the recombinant plasmid. The contruct mixture is subjected to voltage (4) purification of saline water with the and thus help in the uptake of help of a membrane system DNA. 22.   The following two ways of electroporation can be used–  Low voltage long pulse-For this way 300-400Vcm-1 is used.  High Voltage short pulse-For this way 1000-1500Vcm-1 is used.  Electroporation has been used in intact plant cells too.  Electroporation has been used in Tobacco, Petunia, Maize, Rice and Wheat. y Biolistics or Gene gun  1-2 mm Tungsten or gold particle coated with DNA is used for transformation.  These particles are accelerated at high velocities and thus this enables it to enter into the plant cell. y Lipofection  The desired DNA is introduced into the cell with the help of liposomes. This method can be used for animals as well as the plants. y Microinjection  The DNA solution is injected directly into the cell with the help of glass micropipettes. Biotechnology: Principles and Processes  Protoplasts should be used as it does not interfere with the use of microinjection but if an intact cell is used then cell wall interferes with the process of microinjection.  The protoplast is held on a glass slide coated with polylysine or held under the suction by a micropipette and then the DNA is injected.  It is a time taking process. 23.  ⚪ Macroinjection  In this process, plasmid DNA Definition is injected into the lumen of the inflorescence and using a Macroinjection: The process of injecting hypodermic syringe. the plasmid DNA into the lumen of the inflorescence with a hypodermic OBTAINING THE FOREIGN GENE syringe. PRODUCT y The recombinant DNA is inserted into the bacterial cell, plant cell or animal cell. Then the foreign DNA is multiplied. y The recombinant DNA should produce the desired protein. y After the cloning of the gene, optimum conditions should be maintained for the production of the proteins. y If a protein-encoding gene is expressed in a heterologous host then it is known Previous Year’s Question as recombinant protein. y The cells can be grown at a small scale in the laboratory.But in this case, the Biolistics (gene gun) is suitable for amount of proteins produced will be (1) disarming pathogen vectors less. (2) transformation of plant cells y To ensure the production of large scale (3)  constructing recombinant DNA by of proteins, large vessels known as joining with vectors bioreactors are used. (4) DNA fingerprinting ⚪ Bioreactors or fermenters  Large vessels in which the raw materials are biologically Biotechnology: Principles and Processes converted into the products by the microbes, plant and animal cells or enzymes are known as bioreactors.  Bioreactors can be aerated, Definition stirred for mixing of the medium. They can be made contamination- Recombinant Protein: If a protein free and replacement of used encoding gene is expressed in a medium takes place. heterologous host then it is known as  Usually the most commonly used recombinant protein. bioreactor is the stirring type. They are of two types- 24.  Simple stirred tank bioreactor Sparged stirred tank bioreactor  Simple stirred-tank bioreactor- These are simple type and consists of a stirrer which help in mixing and even distribution of oxygen throughout the reactor.  It is usually cylinderical or has a curved base to help in mixing the contents.  Arrangements are provided to maintain temperature, pH, foam and delivery of oxygen to the vessel.  Sparged stirred-tank bioreactor- In it, air is supplied in the form of air bubbles. The surface Previous Year’s Question area for oxygen transfer is increased. Stirred-­tank bioreactors have been  Fermentation of the raw designed for materials is carried out in (1) purification of product the bioreactors. Two types of (2) addition of preservatives to the fermentation can take place product namely batch fermentation and (3) availability of oxygen throughout continuous fermentation. the process  In batch fermentation nutrients, (4) ensuring anaerobic conditions in raw materials are put in a the culture vessel closed chamber and not changed once the fermentation Biotechnology: Principles and Processes starts.  In continuous fermentation, the nutrients and raw material are added continuously and products are removed as they are made. DOWN STREAMING y After the product has been made in the bioreactor, it needs to be processed before it is marketed. 25.  y The process includes separation and purification of the product before it is Definition marketed and it is together known as Downstream Processing: Separation downstreaming. and purification of the product is y After down streaming process many known as downstream processing. clinical trials need to be done so that the product is checked before making it commercial. GENE AMPLIFICATION THROUGH Biotechnology: Principles and Processes POLYMERASE CHAIN REACTION y The polymerase chain reaction (PCR) technique, developed by Kary Previous Year’s Question Mullis. It generates upto billion copies of the desired DNA (or PCR and restriction fragment length RNA) segment. The PCR process is polymorphism are the methods for completely. (1) study of enzymes y The PCR is carried out in vitro. It utilizes (2) genetic transformation the following: (3) DNA sequencing ⚪ a DNA containing the desired (4) genetic fingerprinting segment of gene. 26.  ⚪ two nucleotide primers (about 20 Rack your Brain bases long) ⚪ the four deoxyribose nucleotide triphosphates, dTTP (Deoxythy- What is the source of thermostable midine triphosphate), dCTP (deox- DNA polymerase? ycytidine triphosphate), dATP (de- oxyadenosine triphosphate) and dGTP (deoxyguanosine triphosphate) ⚪ a heat stable DNA polymerase, e.g., Taq (isolated from the bacterium Thermus aquaticus) Procedure of PCR y It consists of the following steps- Denaturation y The mixture containing the DNA is first heated to temperature between 90–98°C. y This brings about DNA denaturation. The hydrogen bonds between the base pairs break and this leads to the separation of the DNA strands. y Each single strand then acts as a template for DNA synthesis. y The duration of this step in the first cycle of PCR is usually 2 min. Annealing Biotechnology: Principles and Processes y The mixture is now cooled to a temperature that permits annealing of the primer to the complementary sequences in the DNA. y Two oligonucleotide primers anneal to each of the single-stranded template DNA. y These sequences are located at the 3′ ends of the two strands of the desired segment. This step is called annealing. y The primer concentration is kept very high relative to that of the template 27.  DNA, primer-template hybrid formation is greatly favoured. Primer Extension y The final step is extension. y Taq DNA polymerase (of a thermophilic bacterium Thermus aquaticus) synthesizes the DNA region between the primers, using dNTPs (deoxyribose nucleoside triphosphates) and Mg2+. y DNA polymerase synthesizes the complementary strands utilizing 3′ OH of the primers. y The primers are extended towards each other so the DNA segment lying between the two primers are copied. y The duration of primer extension is usually 2 min at 72°C. y The original template sequence will be copied during the second and all the other subsequent cycles. y At each cycle, both new and old strands anneal to the primers and serve as templates for DNA synthesis. Thus at the end of each cycle, the number of copies of the desired segment becomes twice the number present at the end of the previous cycle. Biotechnology: Principles and Processes y After PCR cycles, the amplified DNA segment is purified by gel electrophoresis and used for the desired purpose. Rack your Brain What is the advantage of PCR over the normal method of using plasmid for gene cloning? 28. Applications of Polymerase Chain Reaction Biotechnology: Principles and Processes 29. Biotechnology: Principles and Processes Summary 30.  Summary 31. Biotechnology: Principles and Processes  Solved Exercise Q1 Two bacteria found to be very useful in genetic engineering experiments are (1) Nitrobacter and Azotobacter (2) Rhizobium and Diplococcus (3) Nitrosomonas and Kliebsiella (4) Escherichia and Agrobacterium A1 (4) Both act as vectors for cloning the desired DNA and Agrobacterium is used for DNA transfer. Q2 Restriction endonucleases are (1) used for in vitro DNA synthesis (2) used in genetic engineering (3) synthesized by bacteria (4) present in mammalian cells for degradation of DNA A2 (2) Used for cutting the DNA fragments. Q3 Genetic engineering is possible, because (1) we can cut DNA at specific sites by endonucleases like DNase I (2) restriction endonucleases purified from bacteria can be used in vitro (3) the phenomenon of transduction in bacteria is well underwood (4) we can see DNA by electron microscope Biotechnology: Principles and Processes A3 (2) Restriction nucleases help in cutting DNA at specific sites and removing DNA with desired gene. Q4 The restriction enzymes are used in genetic engineering, because (1) they can cut DNA at specific base sequence (2) they are nucleases that cut DNA at variable sites (3) they can degrade harmful proteins (4) they can join different DNA fragments 32. A4 (1) They can cut DNA at specific sites and thus help in further process of genetic engineering. Q5 Which of the following organelles is related with genetic engineering? (1) Mitochondria (2) Plasmids (3) Golgi bodies (4) Lysosomes A5 (2) Extra-chromosomal structure that helps in cloning DNA Q6 Plasmid has been used as vector because (1) it is circular DNA which have capacity to join to eukaryotic DNA (2) it can move between prokaryotic and eukaryotic cells (3) both ends show replication (4) it has antibiotic resistance gene A6 (1) It can join to any eukaryotic DNA and hence helps in making copies of it. Q7 The process of replication in plasmid DNA, other than initiation, is con- trolled by (1) mitochondrial gene Biotechnology: Principles and Processes (2) plasmid gene (3) bacterial gene (4) none of these A7 (3) The plasmid gene has the ability to control the initiation while other steps of replication are controlled by the bacterial gene. 33.  Q8 Which of the following is related to genetic engineering? (1) Heterosis (2) Mutation (3) Plastid (4) Plasmid A8 (4) It acts as a cloning vector. Q9 Elution process is involved in (1) PCR (2) Gel Electrophoresis (3) Separation of DNA from the cell (4) Replication of the plasmid A9 (2) Separation of desired DNA fragment from agarose gel is known as Elution. Q10 The linking of antibiotic resistance gene with the plasmid vector became possible with (1) DNA polymerase (2) exonucleases (3) DNA ligase (4) endonucleases Biotechnology: Principles and Processes A10 (3) It helps in joining strands together. 34.