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NEET BOTANY VOL-II CH-11: BIOTECHNOLOGY PRINCIPLES AND PROCESSES NARAYANA GROUP 123 11 BIOTECHNOLOGY PRINCIPLES AND PROCESSES Principles and process of Biotechnology: Genetic engineering (Recombinant DNA technology).  Introduction  Principles of Biotechnology  Tools of recombinant DNA technology  Restriction enzymes  Nomenclature  Cleavage patterns  Cloning Vectors  Competent Host  Processes of Recombinant DNA Technology  Isolation of the Genetic Material (DNA)  Cutting of DNA at Specific Locations  Separation and isolation of DNA fragments  Insertion of isolated gene into a suitable vector  Amplification of Gene of Interest using PCR  Insertion of Recombinant DNA into the Host Cell / Organism  selection of transformed host cells  Obtaining the Foreign Gene Product  Downstream Processing C O N T E N T S NEET SYLLABUS
CH-11: BIOTECHNOLOGY PRINCIPLES AND PROCESSES NEET BOTANY VOL- II 124 NARAYANA GROUP INTRODUCTION * Biotechnology is an application to industry of advance made in the techniques and instrument of research in the biological science * The origin of biotechnology lies deep in human history. * It is use of living organism or substances obtained from them in industrial processes * Natural science became anthropocentric since the days of Rene Descartes, a french philosopher of 17th century. * Major utility of the biological world is as a source of food. * Herbert Boyer Performed studies on a couple of restriction enzymes of the E. coli bacterium with especially useful properties. * Boyer observed that these enzymes have the capability of cutting DNA strands in a particular fashion, which left what has become known as ‘sticky ends’ on the strands. * These clipped ends made pasting together pieces of DNA a precise exercise. * Cohen had been studying small ringlets of DNA called plasmids which float about freely in the cytoplasm of certain bacterial cells and replicate independently from the coding strand of DNA. * Cohen had developed a method of removing the plasmids from the cell and then reinserting them in other cells. * Combining this process with that of DNA splicing enabled Boyer and Cohen to recombine segments of DNA in desired configurations and insert the DNA in bacterial cells, which could then act as manufacturing plants for specific proteins. * Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans. * In this sense, making curd, bread or wine, which are all microbe mediated processes, could also be considered as a form of biotechnology. * Further, many other processes/techniques are also included under biotechnology * For example, in vitro fertilisation leading to a ‘test-tube’ baby, synthesizing a gene and using it, developing a DNA vaccine or correcting a defective gene, are all part of biotechnology * The European Federation of Biotechnology (EFB) has given a definition of biotechnology that combines both the traditional view and the modern molecular emphasis. The definition given by EFB is as follows: * “The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services”. * This means biotechnology is a science which utilizes properties and uses of microorganisms or exploits cells and the cell constituents at the industrial level for generating useful products essential to life and human welfare PRINCIPLES OF BIOTECHNOLOGY Among many, the two core techniques that enabled birth of modern biotechnology are A) Genetic Engineering: Techniques to alter the chemistry of genetic material (DNA or RNA), to introduce these into host organisms and thus change the phenotype of the host organism. B) Tissue culture: Maintenance of sterile (microbial contamination free) atmosphere in chemical engineering processes to enable growth of only the desired microbe or eukaryotic cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes etc.,
NEET BOTANY VOL-II CH-11: BIOTECHNOLOGY PRINCIPLES AND PROCESSES NARAYANA GROUP 125 CONCEPTUAL DEVELOPMENT OF THE PRINCIPLES OF GENETIC ENGINEERING: * Sexual reproduction provides opportunities for variations and formation of unique combinations of genetic setup, some of which may be beneficial to the organism as well as the population. * Asexual reproduction preserves the genetic information, while sexual reproduction permits variation. * Traditional hybridization procedures used in plant and animal breeding results in the appearance of undesirable genes with desired genes. This is natural recombination of genes which occurs during meiotic crossing over. * In contrast, the techniques of genetic engineering which include creation of recombinant DNA, use of gene cloning and gene transfer, overcome this limitation and permits us to isolate and introduce only one or a set of desirable genes without introducing undesirable genes into the target organism. * For the multiplication of any alien piece of DNA in an organism it needs to be a part of a chromosome (s) which has a specific sequence known as ‘origin of replication’(ori).  Thus, an alien piece of DNA, linked with ori, can replicate and multiply itself in the host organism. This can also be called as cloning or making multiple identical copies of any template DNA. CLONE: A collection of genetically identical cells or organisms derived from a common parent where all members have similar genetic composition. Construction of first recombinant DNA : * The construction of the first recombinant DNA emerged from the possibility of linking a gene encoding antibiotic resistance with a native plasmid (autonomously replicationg circular extra - chromosomal DNA) of Salmonella typhimurium. * Stanley N. Cohen and Herbert Boyer accomplished this in 1972 by isolating the antibiotic resistant gene by cutting out a piece of DNA from a plasmid which was responsible for conferring antibiotic resistance. * The cutting of DNA at specific locations became possible with the discovery of the so called ‘molecular scissors’ - restriction enzymes * The cut piece of DNA was then linked with the plasmid DNA. These plasmid DNAs act as vectors to transfer the piece of DNA attached to it. * A plasmid can be used as vector to deliver an alien piece of DNA into the host organism. * The linking of antibiotic resistance gene with the plasmid vector became possible with the enzyme DNA ligase, which acts on cut DNA molecules and joins their ends. * This makes a new combination of circular autonomously replicating DNA created invitro and is known as recombinant DNA. * When this DNA is transferred into Escherichia coli, a bacterium closely related to Salmonella, it could replicate using the new host’s DNA polymerase enzyme and make multiple copies. * The ability to multiply copies of antibiotic resistance gene in E.coli. was . called cloning of antibiotic resistance gene in E.coli * There are three basic steps in genetically-modifying an organism (GMO) (i) Identification of DNA with desirable genes (ii) Introduction of the identified DNA into the host (iii) Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.
CH-11: BIOTECHNOLOGY PRINCIPLES AND PROCESSES NEET BOTANY VOL- II 126 NARAYANA GROUP EVALUATE YOURSELF - 1 1. Autonomously replicating circular extra chromosomal DNA of prokaryotic cell is called 1) Satellite DNA 2) Plasmid 3) Recombinant DNA 4) Nucleo id 2. Name the enzyme that promotes sealing or joining of the sticky ends of vector: (1) DNA ligase (2) Primase (3) RNAase (4) Restriction endonuclease 3 .In living organism during replication of DNA, the process is initiated by 1) RNA primer 2) DNA primer 3) RNA & DNA primers 4) None of them 4. The specific sequence recognised by ‘mo- lecular scissors ‘ is called (1) Isomer (2) Isobar (3) Misnomar (4) palindrome 5. Introduction of foreign genes for improving genotype is called 1) Biotechnology 2) Tissue culture 3) Genetic engineering 4) Both (1) and (3) 6. The linking of antibiotic resistance gene with plasmid vector became possible with 1) DNA polymerase 2) Exonuclease 3) DNA ligase 4) Endonuclease 7. Chemical knives of molecular biology are 1) Restriction endonucleases 2) Transcriptase 3) Reverse transcriptase 4) Ligase 8. Plasmids are extra-chromosomal genetic material found in 1) Algae 2) Yeast (Episomal Plasmid) 3) Bacteria 4) Both 2 & 3 9. Who is given the credit for constructing first artificial recombinant DNA ? 1) Hargobind Khorana 2) Stanley Cohen and Herbert Boyer 3) Linus Pauling 4) Arber and Nathans 10. Which of the following is not related to biotechnology 1)Integration of natural science & organisms (Microbes to plants & animals) 2)Techniques to change the chemistry of DNA 3)Maintenance of sterile ambience to maxi- mum growth of the desired DNA 4)Introducing undesirable genes into the target organism for multiplication 11. Which of the following is the most ac- cepted definition of biotechnology by European Federation of Biotechnology (EFB)? 1)Maintenance of sterile ambience for enabling growth of desired microbe eukary- otic cell in large quantities 2)Technique of using live organisms or enzyme from organisms to produce products and processes useful to animals 3)Process which use genetically engineered animals only on a large scale for benefit of mankind 4)The integration of natural science and organisms cells, parts thereof and molecular analogies for products and services 12. The technique of removing plasmids from cells and then reinserting them in to other cells was developed by. 1) Herbert Boyer 2) Stanley Cohen3) Nathens 4) W.M.Stanley 13. All the following statements about Stanley Cohen and Herbert Boyer are correct but one is wrong. Which one is wrong? 1)They discovered recombinant DNA (r- DNA) technology which marked the birth of modern biotechnology 2)They first produced, healthy sheep clone, a Finn Dorset lamb, Dolly, from the differen- tiated adult mammary cells 3)They invented genetic engineering by combining a piece of foreign DNA containing a gene from a bacterium with a bacterial (E.coli) plasmid using the enzyme restriction endonuclease 4)They isolated the antibiotic resistance gene by cutting out a piece of DNA from a plasmid which was responsible for conferring antibi- otic resistance