1. Monday, 09/05/2011 1 BIOTECHNOLOGY LECTURE BY DR.SRINIVASREDDY PATIL M.Sc Zoology (Gold Medalist).,Ph.D (Reproductive Physiology)., M.Ed ., PGDBA.,FMSPI sahanarsha_patil@yahoo.com

2. Monday, 09/05/2011 2 SYNOPSIS INTRODUCTION GENETIC ENGINEERING #INTRODUCTION # TOOLS USED IN GENETIC ENGINEERING, # VECTORS,ENZYMES AND HOST CELL. RECOMBINENT DNA TECHNOLGY AND ITS APPLICATION. INSULIN SYNTHESIS TO BE USED AS AN EXAMPLE. A BRIEF ACCOUNT OF # DNA FINGER PRINTING. # GENE THERAPY. # HUMAN GENOME PROJECT. # MONOCLONAL ANTIBODIES. HAZARDS AND SAFE GAURDS OF GENETIC ENGINEERING.

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4. Monday, 09/05/2011 4 Biotechnology is the technological employment of microorganisms, plant cells, animal cells their components or biological processes to generate products and services useful to human beings.

5. Monday, 09/05/2011 5 OLD BIOTECHNOLOGY It is use of natural strains of microorganisms and cell lines for obtaining useful products.This type of biotechnology has been exploited by human beings for the past few thousand years in obtaining products such as curd,wine,vinegar,bread,cheese,etc.Pasteur,Kuhne and Buchner were able to find out that fermentation process was accomplished by micro-organisms and their enzymes. PRODUCTS OF OLD BIOTECHNOLOGY Fermented foods, Curd, Cheese and Butter,Organic Acids, Alcoholic Beverages, Industrial solvents, Curing of Beverages,Enzymes, Dextrin, Vitamins, Antibiotics, Tissue Culture, Vaccines, Steroids and old biotechnology is also used in sewage treatment,biogas generation and development of better strains of biofertilizers.

6. Monday, 09/05/2011 6 Even though biotechnology developed as a unique branch of science only in the recent centuries, the application from this knowledge was familiar to man prior to 600 B.C. For examples, Sumerians were skilled is distilling different types of beer.

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8. Monday, 09/05/2011 8 MODERN BIOTECHNOLOGY It is the use of genetically engineered microorganisms,plant and animal cells in developing production technologies that not only enhance their productivity but also use new products. The organisms,which received genes from other sources through recombinant DNA technology are called transgenics.The first commercial exploitation of transgenic microorganism was the production of novel pharmaceutical proteins.Human insulin(humulin) is being commercially produced by transgenic Escherichia coli in which human insulin gene had been introduced. APPLICATION OF BIOTECHNOLOGY Micro-organisms: Biofertiliser, Vaccine production, Antibiotic production, Vitamins,OrganicBioinsecticide. Animal cells: Vaccine production,Animal proteins (including antibodies.) Plant cells : Micropropagation, haploidplants, somaclonal variants

9. Monday, 09/05/2011 9 PRODUCTS OF MODERN BIOTECHNOLOGY Insulin , Growth Hormone, Other Hormones. Interferons:They are glycoproteins which stimulate synthesis of antiviral proteins that inhibit synthesis of viral proteins and RNAs.They are synthesized by cultured leucocytes,fibroblasts and lymphocytes inoculated with viruses. Antisense Therepy.Antisense RNAs are used to keep certain genes silent. Gene Therapy.The defective genes can now be replaced by healthy genes for overcoming hereditary discorders. Other Drugs.Recombinant DNA technology has helped in synthesis of a number of drugs like tissue plasminogenactivator,erythropoeitin,lung surfactant protein,factor VIII, epidermal growth factor,etc. Transgenic Crops.Almost all types of crop plants are being genetically modified for improving yield,resistance to particular pests of pathogens,herbicide resistance and other functions,ex. BT Cotton(against Bollowotm), BT Corn(against hornworm larvae,against over-ripening ) Golden Rice(with vitamin A), Potato(with additional protein content),etc. Transgenic Animals.Transgenic pigs can be used in providing vital material and organs to human beings.Animals can also be modified to yield biochemicals like plasminogen activator in milk of goat or blood clotting factor VIII in sheep.

10. Monday, 09/05/2011 10 GENETIC ENGINEERING It is branch of biotechnology which is specialized to manipulate DNA for producing and modified sequences creating changed cells, tissues and organs. AIM OF GENETIC ENGINEERING #Unraveling molecular events in biological processes. #Cloning genes. #Cloning organisms. #Producing transgenic or genetically modified organisms-animals,plants and microbes. #Obtain genetically modified foods.

11. Monday, 09/05/2011 11 #Synthesize desired human gene products. #Obtain pharmacologically and therapeutically useful products. #To replace deleterious genes with normal or useful genes in gene therapy so as to treat incurable hereditary discorders. #DNA fingerprinting. #Study human genome in details so as to know the number and functioning of each gene. Two important discoveries helped in the development of genetic engineering. Plasmids and restriction endonucleases. Cohen and Boyer(1973) were the first to produce recombinant DNA by introducing a foreign gene into plasmid of Escherichia coli.

12. Monday, 09/05/2011 12 TOOLS OF GENETIC ENGINEERING 1. Source of Donor DNA. The separated cells are broken by homogenization and the nuclei separated through centrifugation.Nuclei are then ruptured to collect DNA.DNA segments are separated through gel electrophoresis. 2. Vector(Cloning Vector).It is carrier of passenger DNA which can undergo independent replication to increase desired genes.Cloning vehicles are of four types-plasmids,viruses,cosmids and artificial chromosomes. (i) Plasmids.They are small extranuclear circular DNAs which carry extrachromosomal genes in bacteria and some fungi.They replicate independently.The best known vectors which are also available commercially are pBR322 and pUC-18.

13. Monday, 09/05/2011 13 The characteristic of an ideal plasmid are(i) Presence of minimum amount of its own DNA.(ii) Recognition sites for restricitionendounclease.(iii)Presence of at least two markers with recognition site being present in one of the two markers.(iv)Relaxed replication control so that the recombinant plasmid is capable of forming several copies. (ii) Viruses.Bacterial viruses or bacteriophages(ex. lambda I virus) are used as clones for introducing desired genes into bacterium. Virus carrying a foreign gene becomes harmless and therefore, proves a good vector. (iii) Cosmids.They are plasmids in which phage lambda cos sites have been inserted.Acosmid can be packed in the phage coat.It is useful in carrying large DNA fragment.

14. Monday, 09/05/2011 14 3.Enzymes. Two major types of enzymes used in genetic engineering are restriction endonucleases and ligases. (i)Restriction Endonucleases (RENs) Restriction endonuleases are enzymes that are specialized to cut DNA at specific sites in the regions having plaindromic sequences. (ii)DNA Ligase. The enzyme is used in ligation or union of vector DNA and passenger DNA.It produces recombinant DNA. 4. Host Cells. Common host microbes used in biotechnology are Yeast,Bacillussubtilis and Escherichia coli.

15. Monday, 09/05/2011 15 #Synthesize desired human gene products. #Obtain pharmacologically and therapeutically useful products. #To replace deleterious genes with normal or useful genes in gene therapy so as to treat incurable hereditary discorders. #DNA fingerprinting. #Study human genome in details so as to know the number and functioning of each gene. Two important discoveries helped in the development of genetic engineering. Plasmids and restriction endonucleases. Cohen and Boyer(1973) were the first to produce recombinant DNA by introducing a foreign gene into plasmid of Escherichia coli.

16. Monday, 09/05/2011 16 #Synthesize desired human gene products. #Obtain pharmacologically and therapeutically useful products. #To replace deleterious genes with normal or useful genes in gene therapy so as to treat incurable hereditary discorders. #DNA fingerprinting. #Study human genome in details so as to know the number and functioning of each gene. Two important discoveries helped in the development of genetic engineering. Plasmids and restriction endonucleases. Cohen and Boyer(1973) were the first to produce recombinant DNA by introducing a foreign gene into plasmid of Escherichia coli.

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18. Monday, 09/05/2011 18 RECOMBINANT DNA TECHNOLOGY 1. DNA Fragments. Donor individual having desired genes or DNA fragments is selected. Appropriate DNA fragments obtained by rupturing the nuclei.The same are identified by Southern blotting. 2. Restriction Endonuclease.It is selected on the basis of availability of restriction sites on the two ends of DNA fragment or gene. 3. Cloning Vectors. It is selected on the basis of requirement. Introducing of desired gene in an organism to improve its function. 4. Formation of Recombinant or rDNA. Both the passenger and vehicle DNAs are treated separately with the same restriction endonuclease.It produces complementary sticky ends.Self ligation is checked by use of alkaline phosphatase.The two types of DNAs (passenger and vehicle)are now joined together with the help of DNA ligase. The union produce rDNA.

19. Monday, 09/05/2011 19 5. Gene Transfer.It is carried out by two methods,vector transfer and direct transfer. (i) VetorTransfer.rDNA is present in the form of plasmid,virus,cosmid or artificial chromosome.It is introduced into host cell by adding it into culture of plasmid free bacteria or animal cells. Once inside the host cell,the recombinant DNA begins to multiply and form the desired product. (ii) Direct or VectorlessTransfer.The desired gene as well as recombinant DNA can be passed into plant,animal or human cells through (a) Microinjection by means of micropirpettes. (b) Particle or gene gun where tungsten or gold particles coated with desired genes are bombarded into the cells with great force.Instead special sprays are also used for this.

20. Monday, 09/05/2011 20 INSULIN SYNTHESIS Insulin is pancreatic hormone essential for maintenance of glucose-glycogen balance in the body .In its deficiency,the glucose content of blood rises while the cells fail to take up glucose from blood.It results in diabetes mellitus . The easiest method is to take out insulin gene from healthy human leucocytes and use it in preparation of recombinant DNA. As natural human genes are split genes, they do not express their effect in bacteria.Two DNA sequences were prepared by for the two chains,A and B of insulin.

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22. Monday, 09/05/2011 22 Plasmids of Escherichia coli(ex.pUC-18) and insulin gene are treated with the same restriction endonuclease.It produces sticky ends in both.The two are joined together by DNA ligase.It produces recombinant DNA in the form plasmid carrying the insulin gene. The bacteria are treated with antibiotic for which the recombinant plasmid is carrying the resistance factor. The genetically engineered bacteria are first multiplied or cloned.They are then introduced in a sterilized bioreactor or fermenter having the growth medium.As the bacteria increase in number a part of population is harvested. Insulin is extracted from the harvested bacteria.

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26. Monday, 09/05/2011 26 APPLICATION OF RECOMBINANT DNA TECHNOLOGY 1. Study of molecular events. 2. Gene maps.rDNA technology can be employed to make gene maps. 3.Development stages.A development stage can be stopped,delayed or quickened through manipulation of genes. 4. Antisense therapy.Extra-activity of genes of a particular region can be checked by introducing specific DNA fragments.The treatment is called antisense therapy. 5. Foods with extra biochemicals.With the help of Agrobocteriumtumefaciens and viruses,genes for synthesis of various biochemicals can be introduced in plants. 6. Nitrogen fixation.Genes required for nitrogen fixation can be made part of genome of all plants. 7. Study of defective genes.The technique can be used in the study of defective genes in the foetus stage.

27. Monday, 09/05/2011 27 8. Tailor-made organisms,Usefulplants,animals and microbes can be tailor-made to suit varied human needs. 9. Medical diagnosis of diseases.Short segments of single stranded DNA with attached fluorescent or radioactive marker are being used as probes for identification of infectious hepatitis,HIV,cysticfibrosis,musculardystrophy,etc. 10. Genetic discorders.DNA of prospective genetic disorder parents can be studied thoroughly so as to fully determine their genotypes and know the chances of producing defective children.The parents can than be properly counseled. 11. Recombinant products.A number of biochemicals,drugs and vaccines are being prepared through recombinant DNA technology. 12. Gene Therapy.It is the replacement of a defective gene by a normal healthy and functional gene.Gene therapy can overcome the effects of various genetic disorders like sickle-cell anaemia,colourblindness,alkaptonuria,muscular dystrophy.

28. Monday, 09/05/2011 28 PRODUCTS OF RECOMBINANT DNA TECHNOLOGY 1. Human Insulin(Humulin) -Treatment of diabetes type.I., 2 .Human Growth Hormone(HGH) - Replacement or augmentation of deficient hormone in short stature persons. 3. Calcitonin - Treatment of rickets. 4. Chorionic Gonadotropin - Treatment of infertility. 5. Blood Clotting Factors VIII&IX - Replacement of clotting factor missing in patients with haemophilia A or B. 6. Tissue Plasminogen Activator - Dissolving blood clots after heart attack and stroke. 7. Erythropoietin - Dissolving blood clots after heart attack and stroke. 8. Platelet Growth Factor - Simulation of wound healing. 9. Interferon(a,b and g) - Treatment of viral infections and cancer. 10. Interleukins - Enhancing activity of immune system. 11. Vaccines - For preventing diseases like herpes,hepatitisB, influenza,pertussis,etc.

29. Monday, 09/05/2011 29 DNA Finger Printing DNA finger printing or DNA profiling is the technique used for determining nucleotide sequences of certain areas of DNA which are unique to each individual . While skin finger prints can be altered through plastic surgery, there is no known method to alter DNA finger prints. It remains the same in very cell, tissue and organ of a person. Discovery : DNA finger printing was discovered by Jefferys et al in 1985.It was accepted in the law of court in 1986. Principle : DNA contains non-cistronichypervariable repeat minisatellite sequences commonly called variable number tandem repeats or VNTRs.Each VNTR has 11-60 base pairs surrounded by conserved restriction sites.

30. Monday, 09/05/2011 30 Technique Source of DNA :White blood corpuscles,blood,semen,saliva,vaginal swab cells,cells from hair root etc, are used as a source of DNA for finger printing.The amount of DNA required can be met by 1 microgram of tissue or 1,00,000 cells. Extraction of DNA : DNA source is exposed to high speed refrigerated ultracentrifugation.It separates the nuclei and breaks the same to free DNA. DNA Amplification : If the amount of DNA is small,the extracted DNA is amplified through PCR (polymerase chain reaction).It produces numerous copies of DNA. Fragmentation:With the help of site recognizing restriction enzymes,DNA is cut to separate VNTRs. Separation of VNTRs :Chopped DNA is exposed to electrophoresis over agarose polymer gel.It separates DNA fragments.The separated VNTRs can be recognized by staining them with dye that becomes fluorescent when illuminated with UV radiations. Single Stranded DNA :VNTRs are treated with alkaline chemicals to split them into single stranded DNAs.

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32. Monday, 09/05/2011 32 Southern Blotting:The separated VNTR single stranded sequences are transferred to nitrocellulose or nylon membrane placed over a gel.The procedure is called Southern blotting after the name of its inventor E.M.Southern. DNA Probes :They are small radioactive synthetic DNA segments of known sequences of nitrogen bases. Hybridisation :Nylon sheet or nitrocellulose is immersed in a bath to which DNA probes are added. The probes get attached to single stranded VNTRs having complementary nucleotide sequences. Exposure to X-ray Films :The nylon membrane containing the radioactive DNA probes and VNTRs is exposed to X-ray film.The hybridized radioactive VNTRs appear as dark bands.The film gives us DNA prints or DNA profiles :The prints are compared and relationship found.

33. Monday, 09/05/2011 33 APPLICATIONS Identification :DNA finger printing is a sure method of identification of criminals involved in various types of crime including rape and murder. Paternity –Maternity Disputes. The method can provide reliable information as to real biological father,mother or offspring. Close Relations.DNA finger printing can establish the closeness of relation of an intending immigrant. Human Lineage. It provides information as to human lineage and relationship with various apes. Migrations.DNA finger printing can identify racial groups,theirorigin,historical migrations and invasions. Specific Alleles. Certain ethnic groups have specific alleles that provide the group resistance or proneness to various disease. It is useful in gene therapy.

34. Monday, 09/05/2011 34 GENE THERAPY It is a therapeutic treatment of defective heredity by introduction of normal healthy and functional genes. However,it has not been possible to introduce healthy genes in an ovum,sperm or zygote.Therefore,gene therapy is applied to somatic cells where the defect occurs. Success has been achieved in a few cases, ex. ADA related SCID, cystic fibrosis, atherosclerosis. Major requirement of gene therapy is the isolation of the desired gene, its amplification, a retrovirus for removal of viral genes and insertion of desired gene. SCID or severe combined immuno deficiency is a hereditary defect in which infants lack antibody formation due to impairment of components of immune system(T-lymphocytes and B-lymphocytes).Children suffering from SCID can die just for ordinary diseases,vaccination and blood transfusion.In 25% of SCID cases, patients have a defective autosomal gene for synthesis of enzyme adenosine deaminase or ADA.

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37. Monday, 09/05/2011 37 GENOMICS It is the study of genomes through analysis, sequencing and mapping of genes and non-cistronic areas along with the study of their functions. The term was coined by Thomas Roderick in 1986.Human body has some 100 trillion cells grouped into over 260 types. Every cell contains the same number of chromosomes and the same kind of genomes.

38. Monday, 09/05/2011 38 HUMAN GENOME PROJECT (26.06.2000) The human genome contains about 3.2. billion base pairs.Fruitfly Drosophila melanogaster has 132 million base pairs.This is 1/24 of the total length of the human genome. Based upon complexity of human machinery it was believed that the human genome carries more than 1,00,000 genes.However,HGP has found out that human genome carries between 30,000-40,000 genes. Eukaryotes contain mostly split genes having long non-coding areas or introns.Further not more than 10% of the genome contains coding areas or exons. Part of DNA which contains repeated sequences is called satellite DNA. They include transposons and Alu elements. Minisatellite sequences are 11-60 base pairs long hypervariable repeat sequences.They are VNTRs used in DNA finger printing . Simple sequence repeats (SSR) have 5-8 pairs are called junked DNA. The length of different human genes varies widely.Generally it is over a few thousand base pairs in length B-globin or insulin genes are less than 10 kilo base pairs long.The longest gene of human body is that of Duchenne Muscular Dystrophy on’X’ chromosome.It is 24,000 kilo base pair long.The smallest gene is that of TDF (Testis Determining Factor), a holandricgene,present on ‘Y’ chromosome.It is only 14 base pairs long.

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40. Monday, 09/05/2011 40 MONOCLONAL ANTIBODIES Monoclonal antibodies are chemically and immunologically pure and homogeneous antibodies against particular antigen which are produced through clonal culture of hybridomas. Hybridoma is a somatic cell hybrid formed by fusion of a normal lymphocyte with a tumourcell.TIt was developed by Kohler and Milstein(1974). The antigen against which antibody is required is injected in to a mouse.After an interval,its B-lymphocytes start producing the antibody against the antigen.B-lymphocytes are taken out from the spleen of the animal.They are made to fuse with myeloma or bone marrow cancer cells.The fusion products are hybridoma cells.

41. Monday, 09/05/2011 41 APPLICATIONS OF HYBRIDOMA TECHNOLOGY Monoclonal antibodies are highly specific and efficient in treating a disease.They can even differentiate two related diseases. Monoclonal antibodies are given for developing temporary immunity against a disease. A major use of monoclonal antibodies is to attach them to cytotoxic drugs for destroying cancerous cells without harming the healthy cells. Monoclonal antibodies are useful for immune suppression for transplants. They are modified by attaching them to catalytic groups in bringing about chemical transformation as in ELISA. Immunofluorescnce assays and radio immuno assays.

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43. Monday, 09/05/2011 43 Hazards and Safeguards of Genetic Engineering Proteins.Genes introduced in various organisms operate through synthesis of polypeptides. Antibiotic resistance. It can give rise to more harmful mutants. Genetic pollution.A number of genes are being introduced into genetically modified crops without evaluating their effects on biotic environment. Super weeds. The transgenic crops can themselves become persistent weeds. Damage to environment.GM crops are likely to damage the environment. Human organs.Replaceable human organs like kidneys,liver,heart,pancreasetc.can best be obtained only from autografts and isografts.Will it be ethical to grow human body. Human cloning.It is possible to produce clone of any individual. Recreation. Dinosaurs, our primate ancestors and many other peculiar living beings of the past can be created with the complete extraction of DNA from their fossils.Such creations can endanger the present ecological balance. Gene effects. Gene PS3 is anticancer gene but it starts precipitating ageing effect after certain period of activity. Microbes.Microbes may be created which are resistant to all known medicines. Animal sufferings.Animals used to produce biochemicals. Integrity of species.With the introduction of gene into various organisms,the integrity of species is violated. Therefore, appropriate safeguards will have to be built before venturing into various potentialities of genetic engineering as the same are full of hazards.