RECOMBINANT DNA TECHNOLOGY - Presentation

1. RECOMBINANT DNA TECHNOLOGY

2. DEFINITION OF RECOMBINANT DNA TECHNOLOGY: • RECOMBINANT DNA TECHNOLOGY, ALSO KNOWN AS GENETIC ENGINEERING, IS A TECHNIQUE THAT ALLOWS DNA FROM TWO DIFFERENT SPECIES TO BE COMBINED INTO ONE MOLECULE. THIS IS ACHIEVED BY CUTTING THE DNA INTO SMALL FRAGMENTS, COMBINING IT WITH DNA FROM ANOTHER ORGANISM, AND THEN INSERTING IT INTO A HOST ORGANISM, WHERE IT CAN REPLICATE AND FUNCTION LIKE NORMAL DNA.

3. BRIEF HISTORY AND SIGNIFICANCE OF RECOMBINANT DNA TECHNOLOGY: • THE CONCEPT OF RECOMBINANT DNA TECHNOLOGY WAS FIRST INTRODUCED IN THE 1970S. IT HAS SINCE REVOLUTIONIZED THE FIELD OF GENETICS AND MOLECULAR BIOLOGY, ALLOWING SCIENTISTS TO MANIPULATE GENES IN WAYS THAT WERE PREVIOUSLY IMPOSSIBLE. THIS TECHNOLOGY HAS BEEN INSTRUMENTAL IN NUMEROUS SCIENTIFIC BREAKTHROUGHS AND HAS APPLICATIONS IN VARIOUS FIELDS SUCH AS MEDICINE, AGRICULTURE, AND INDUSTRY. FOR EXAMPLE, IT HAS ENABLED THE PRODUCTION OF GENETICALLY MODIFIED ORGANISMS (GMOS), GENE THERAPY, AND THE SYNTHESIS OF BIOPHARMACEUTICALS.

4. THE ROLE OF SWISS MICROBIOLOGIST WERNER ARBER IN THE DISCOVERY OF RESTRICTION ENZYMES: • WERNER ARBER, A SWISS MICROBIOLOGIST, PLAYED A SIGNIFICANT ROLE IN THE DEVELOPMENT OF RECOMBINANT DNA TECHNOLOGY. IN 1962, HE DISCOVERED RESTRICTION ENZYMES, PROTEINS THAT CAN CUT DNA AT SPECIFIC SEQUENCES. THIS DISCOVERY, FOR WHICH HE WAS AWARDED THE NOBEL PRIZE IN PHYSIOLOGY OR MEDICINE IN 1978, LAID THE FOUNDATION FOR THE DEVELOPMENT OF RECOMBINANT DNA TECHNOLOGY. RESTRICTION ENZYMES ARE NOW A FUNDAMENTAL TOOL IN GENETIC ENGINEERING, ALLOWING SCIENTISTS TO CUT AND PASTE DNA SEQUENCES, THEREBY CREATING RECOMBINANT DNA.

5. Isolation of Genetic Material: The DNA is extracted from the cell of an organism. • Cutting of DNA at Specific Locations: Restriction enzymes are used to cut the DNA at specific sequences. • Amplification of Gene of Interest using PCR: The Polymerase Chain Reaction (PCR) is used to amplify the gene of interest. • Insertion of Recombinant DNA into the Host Organism: The recombinant DNA is inserted into the host organism using vectors. • Obtaining the Foreign Gene Product: The host organism is cultured and as it multiplies, the foreign gene product is obtained. • Process Involved in Recombinant DNA Technology: Recombinant DNA Technology involves several steps:

6. ROLE OF THE HOST ORGANISM IN RECOMBINANT DNA TECHNOLOGY: • THE HOST ORGANISM PLAYS A CRUCIAL ROLE IN RECOMBINANT DNA TECHNOLOGY. ONCE THE RECOMBINANT DNA IS INSERTED INTO THE HOST, THE HOST’S CELLULAR MACHINERY IS USED TO REPLICATE THE RECOMBINANT DNA AND EXPRESS THE GENE OF INTEREST. THIS ALLOWS THE PRODUCTION OF THE PROTEIN ENCODED BY THE FOREIGN GENE. COMMONLY USED HOST ORGANISMS INCLUDE BACTERIA (LIKE E. COLI), YEAST, AND MAMMALIAN CELLS.

7. PROCESS OF INTEGRATING A RECOMBINANT GENE INTO THE HOST’S GENOME: THE INTEGRATION OF A RECOMBINANT GENE INTO THE HOST’S GENOME INVOLVES SEVERAL STEPS:

8. FORMATION OF RECOMBINANT DNA: THE GENE OF INTEREST IS INSERTED INTO A VECTOR (LIKE A PLASMID) TO FORM RECOMBINANT DNA.

9. TRANSFORMATION: THE RECOMBINANT DNA IS INTRODUCED INTO THE HOST ORGANISM IN A PROCESS CALLED TRANSFORMATION.

10. INTEGRATION: THE RECOMBINANT DNA INTEGRATES INTO THE HOST’S GENOME. THIS CAN OCCUR NATURALLY OR CAN BE FACILITATED USING TECHNIQUES LIKE ELECTROPORATION.

11. SELECTION: CELLS THAT HAVE SUCCESSFULLY INTEGRATED THE RECOMBINANT DNA ARE SELECTED USING MARKERS (LIKE ANTIBIOTIC RESISTANCE GENES).

12. EXPRESSION: THE HOST ORGANISM EXPRESSES THE FOREIGN GENE, PRODUCING THE DESIRED PROTEIN.

13. ROLE OF ENZYMES IN RECOMBINANT DNA TECHNOLOGY: • ENZYMES PLAY A CRUCIAL ROLE IN RECOMBINANT DNA TECHNOLOGY. THEY ARE USED TO CUT AND JOIN DNA FRAGMENTS, FACILITATING THE CREATION OF RECOMBINANT DNA.

14. Endonucleases: These enzymes cut DNA strands at specific points within the DNA molecule. Restriction endonucleases are a type of endonuclease that recognize specific DNA sequences and cut at those sites. • Exonucleases: These enzymes remove nucleotides from the ends of DNA molecules. They are often used to create blunt ends on DNA fragments for ligation. • 2. Types of Enzymes Used: Endonucleases and Exonucleases:

15. FUNCTION OF RESTRICTION ENZYMES IN DETERMINING THE LOCATION OF GENE INSERTION: • RESTRICTION ENZYMES, A TYPE OF ENDONUCLEASE, RECOGNIZE SPECIFIC SEQUENCES IN THE DNA CALLED RESTRICTION SITES AND MAKE CUTS AT THESE LOCATIONS. THIS ALLOWS A GENE OF INTEREST TO BE INSERTED AT A SPECIFIC LOCATION WITHIN THE DNA MOLECULE. THE CHOICE OF RESTRICTION ENZYME DETERMINES THE LOCATION OF GENE INSERTION.

16. ROLE OF VECTORS IN CARRYING AND INTEGRATING THE DESIRED GENE: • VECTORS ARE DNA MOLECULES USED AS A VEHICLE TO ARTIFICIALLY CARRY FOREIGN GENETIC MATERIAL INTO ANOTHER CELL, WHERE IT CAN BE REPLICATED AND/OR EXPRESSED. A VECTOR CONTAINING FOREIGN DNA IS TERMED RECOMBINANT DNA. THE FOUR MAJOR TYPES OF VECTORS ARE PLASMIDS, VIRAL VECTORS, COSMIDS, AND ARTIFICIAL CHROMOSOMES.

17. 5. COMMON VECTORS USED IN RECOMBINANT DNA TECHNOLOGY: PLASMIDS AND BACTERIOPHAGES:

18. PLASMIDS: PLASMIDS ARE SMALL, CIRCULAR, DOUBLE-STRANDED DNA MOLECULES THAT ARE DISTINCT FROM A CELL’S CHROMOSOMAL DNA. THEY NATURALLY EXIST IN BACTERIAL CELLS, AND THEY ALSO OCCUR IN SOME EUKARYOTES. OFTEN, THE GENES CARRIED IN PLASMIDS PROVIDE BACTERIA WITH GENETIC ADVANTAGES, SUCH AS ANTIBIOTIC RESISTANCE.

19. BACTERIOPHAGES: BACTERIOPHAGES ARE VIRUSES THAT INFECT BACTERIA. THEY HAVE THE ABILITY TO CARRY BACTERIAL DNA FROM ONE BACTERIAL CELL TO ANOTHER IN A PROCESS CALLED TRANSDUCTION. BACTERIOPHAGES, LIKE PLASMIDS, ARE USED TO MAKE RECOMBINANT DNA FOR USE IN BACTERIAL CELLS. THEY ARE PARTICULARLY USEFUL WHEN LARGER PIECES OF DNA NEED TO BE INSERTED.

20. CONSTRUCTION OF GENE LIBRARY: • A GENE LIBRARY IS A COLLECTION OF DNA FRAGMENTS THAT REPRESENT THE ENTIRE GENOME OF AN ORGANISM. IT IS CREATED BY CUTTING THE ORGANISM’S DNA INTO FRAGMENTS USING RESTRICTION ENZYMES, AND THEN INSERTING THESE FRAGMENTS INTO VECTORS SUCH AS PLASMIDS OR BACTERIOPHAGES. THESE VECTORS ARE THEN INTRODUCED INTO HOST CELLS (USUALLY BACTERIA), WHICH REPLICATE AND PRODUCE MANY COPIES OF THE INSERTED DNA.

21. SPECIFIC GENE SCREENING FROM LIBRARIES: • ONCE A GENE LIBRARY HAS BEEN CONSTRUCTED, THE NEXT STEP IS TO IDENTIFY AND ISOLATE THE SPECIFIC GENE OF INTEREST. THIS IS DONE USING A PROCESS CALLED GENE SCREENING. THERE ARE SEVERAL METHODS FOR GENE SCREENING, INCLUDING HYBRIDIZATION, PCR, AND DNA SEQUENCING. THESE METHODS ALLOW RESEARCHERS TO IDENTIFY THE SPECIFIC GENE THEY ARE INTERESTED IN AMONG THE THOUSANDS OR EVEN MILLIONS OF GENES IN THE LIBRARY.

22. CHROMOSOME WALKING AND GENE CLONING: • CHROMOSOME WALKING IS A METHOD USED TO IDENTIFY AND SEQUENCE GENES IN A CHROMOSOME. IT INVOLVES STARTING AT A KNOWN LOCATION ON THE CHROMOSOME AND THEN “WALKING” ALONG THE CHROMOSOME, SEQUENCING EACH GENE AS IT IS ENCOUNTERED. ONCE THE GENE OF INTEREST HAS BEEN IDENTIFIED AND SEQUENCED, IT CAN BE CLONED USING RECOMBINANT DNA TECHNOLOGY. THIS INVOLVES INSERTING THE GENE INTO A VECTOR, INTRODUCING THE VECTOR INTO A HOST CELL, AND THEN ALLOWING THE HOST CELL TO REPLICATE THE GENE.

23. INVESTIGATION OF DNA POLYMORPHISM BY RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) TECHNIQUE: • RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) IS A TECHNIQUE USED TO ANALYZE THE GENETIC DIVERSITY AND RELATEDNESS OF DIFFERENT INDIVIDUALS WITHIN A SPECIES. IT INVOLVES USING RANDOM PRIMERS TO AMPLIFY DNA FRAGMENTS FROM THE GENOME. THE RESULTING PATTERN OF AMPLIFIED FRAGMENTS, OR “RAPD PROFILE,” IS UNIQUE TO EACH INDIVIDUAL. BY COMPARING THE RAPD PROFILES OF DIFFERENT INDIVIDUALS, RESEARCHERS CAN DETERMINE THE LEVEL OF GENETIC DIVERSITY WITHIN A POPULATION AND THE DEGREE OF RELATEDNESS BETWEEN INDIVIDUALS.

24. THIS TECHNIQUE IS OFTEN USED IN POPULATION GENETICS, CONSERVATION BIOLOGY, AND BREEDING PROGRAMS.

25. 1. APPLICATIONS IN BIOLOGY, MEDICINE, AND AGRICULTURE: RECOMBINANT DNA TECHNOLOGY HAS A WIDE RANGE OF APPLICATIONS IN VARIOUS FIELDS:

26. BIOLOGY: IT IS USED IN THE STUDY OF GENE FUNCTION AND REGULATION. BY CREATING RECOMBINANT DNA, SCIENTISTS CAN STUDY HOW GENES ARE REGULATED AND HOW THEY FUNCTION IN NORMAL AND DISEASE STATES.

27. MEDICINE: IT HAS REVOLUTIONIZED THE FIELD OF MEDICINE BY ENABLING THE PRODUCTION OF BIOPHARMACEUTICALS SUCH AS INSULIN, GROWTH HORMONES, AND VACCINES. GENE THERAPY, WHICH USES RECOMBINANT DNA TO TREAT GENETIC DISORDERS, IS ANOTHER SIGNIFICANT APPLICATION IN MEDICINE.

28. AGRICULTURE: IN AGRICULTURE, IT IS USED TO CREATE GENETICALLY MODIFIED ORGANISMS (GMOS) WITH DESIRABLE TRAITS SUCH AS PEST RESISTANCE, DROUGHT TOLERANCE, AND IMPROVED NUTRITIONAL CONTENT.

29. ROLE OF RECOMBINANT DNA TECHNOLOGY IN GENETIC ENGINEERING: • RECOMBINANT DNA TECHNOLOGY IS A FUNDAMENTAL TOOL IN GENETIC ENGINEERING. IT ALLOWS SCIENTISTS TO MANIPULATE AN ORGANISM’S GENOME BY INTRODUCING, ELIMINATING OR ALTERING SPECIFIC GENES. THIS CAN RESULT IN ORGANISMS WITH NEW ABILITIES OR TRAITS, SUCH AS BACTERIA THAT CAN PRODUCE HUMAN INSULIN OR CROPS THAT ARE RESISTANT TO PESTS.

30. HOW RECOMBINANT DNA TECHNOLOGY IS USED TO PRODUCE NEW GENETIC COMBINATIONS: • RECOMBINANT DNA TECHNOLOGY ALLOWS FOR THE CREATION OF NEW GENETIC COMBINATIONS BY ENABLING THE TRANSFER OF GENES BETWEEN ORGANISMS THAT WOULD NOT NORMALLY INTERBREED. THIS IS DONE BY ISOLATING THE GENE OF INTEREST FROM ONE ORGANISM, INSERTING IT INTO A VECTOR, AND THEN INTRODUCING THE VECTOR INTO THE HOST ORGANISM. THE HOST ORGANISM THEN EXPRESSES THE FOREIGN GENE, RESULTING IN A NEW GENETIC COMBINATION.

31. THIS HAS BEEN USED TO CREATE A VARIETY OF GENETICALLY MODIFIED ORGANISMS WITH NEW TRAITS, SUCH AS BACTERIA THAT CAN DEGRADE OIL SPILLS OR CROPS THAT ARE RESISTANT TO CERTAIN PESTS.

32. IMPACT OF RECOMBINANT DNA TECHNOLOGY ON SCIENTIFIC RESEARCH

33. RECOMBINANT DNA (RDNA) TECHNOLOGY HAS HAD A PROFOUND IMPACT ON SCIENTIFIC RESEARCH. IT HAS ENABLED RESEARCHERS TO ISOLATE GENES FROM ANY ORGANISM, ALIVE OR DEAD1. THIS TECHNOLOGY HAS LED TO THE DEVELOPMENT OF A WIDE RANGE OF THERAPEUTIC PRODUCTS AND HAS HAD AN IMMEDIATE EFFECT IN THE FIELDS OF MEDICAL GENETICS AND BIOMEDICINE2. IT HAS APPLICATIONS IN VARIOUS DOMAINS OF OUR LIVES, INCLUDING MEDICAL AND DIAGNOSTICS, PHARMACEUTICALS, AGRICULTURE, INDUSTRY, AND ENVIRONMENTAL SCIENCES3. FOR INSTANCE, THE YEAR 1982 MARKED THE APPROVAL OF THE FIRST RECOMBINANT HUMAN INSULIN—HUMULIN BY THE US FDA.

34. APPROXIMATELY 275 RECOMBINANT DRUGS/VACCINES HAVE BEEN APPROVED AROUND THE WORLD FOR HUMAN AILMENTS SO FAR3. GENETICALLY MODIFIED PLANTS WITH RESISTANCE TO DISEASES, CAPABLE OF TOLERATING PHYSICAL OR CHEMICAL STRESS, AND FLEXIBILITY TO ADAPT ALONG WITH IMPROVED PRODUCTION DOMINATE THE AGRICULTURE SECTOR

35. FUTURE PROSPECTS OF RECOMBINANT DNA TECHNOLOGY

36. THE FUTURE PROSPECTS OF RECOMBINANT DNA TECHNOLOGY ARE PROMISING. THE GLOBAL RECOMBINANT DNA TECHNOLOGY MARKET IS PROJECTED TO GROW SUBSTANTIALLY IN THE COMING YEARS, BACKED BY INCREASING DEMAND FOR BIOLOGICS AND BIOPHARMACEUTICALS ACROSS MAJOR WORLD REGIONS4. EMERGING TECHNOLOGIES PROMISE EVEN GREATER POSSIBILITIES, SUCH AS ENABLING RESEARCHERS TO SEAMLESSLY STITCH TOGETHER MULTIPLE DNA FRAGMENTS AND TRANSFORM THE RESULTING PLASMIDS INTO BACTERIA, IN UNDER TWO HOURS

37. IN THE NEAR FUTURE, MOLECULAR CLONING WILL LIKELY SEE THE EMERGENCE OF A NEW PARADIGM, WITH SYNTHETIC BIOLOGY TECHNIQUES THAT WILL ENABLE IN VITRO CHEMICAL SYNTHESIS OF ANY IN SILICO-SPECIFIED DNA CONSTRUCT5. THESE ADVANCES SHOULD ENABLE FASTER CONSTRUCTION AND ITERATION OF DNA CLONES, ACCELERATING THE DEVELOPMENT OF GENE THERAPY VECTORS, RECOMBINANT PROTEIN PRODUCTION PROCESSES, AND NEW VACCINES5.

38. RECOMBINANT DNA TECHNOLOGY IS LIKELY TO ALSO HAVE PROFOUND EFFECTS ON SOCIETY, INCLUDING BETTER HEALTH THROUGH IMPROVED DISEASE DIAGNOSIS, MUCH BETTER UNDERSTANDING OF HUMAN GENE VARIATION, IMPROVED DRUG AND PHARMACEUTICAL PRODUCTION, VASTLY MORE SENSITIVE AND SPECIFIC CRIME SCENE FORENSICS, AND PRODUCTION OF GENETICALLY MODIFIED ORGANISMS THAT SIGNIFICANTLY IMPROVE YIELDS AND NUTRITIONAL VALUE OF CROPS WHILE DECREASING RELIANCE ON PESTICIDES AND ARTIFICIAL FERTILIZERS6.