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R dna

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recombinant dna technology

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R dna

  1. 1. RECOMBINANT DNA TECHNOLOGY Presented by, Tripthi Saliyan I M Pharm Department of Pharmacology 1
  2. 2. DEFINITION • It is technique used in genetic engineering that involves the identification, isolation and insertion of gene of interest into a vector such as a plasmid or bacteriophage to form a Recombinant DNA molecule. 2
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  4. 4. Steps involved in the production of recombinant DNA • Isolation of specific DNA • Selection of vectors • Preparation of rDNA • Transfer of rDNA to host cell • Identification and isolation of rDNA 4
  5. 5. ISOLATION OF SPECIFIC DNA Mechanical shearing • Random fragments of source DNA can be obtained by mechanical shearing of bacterial, plant or animal cells. • Mechanical shearing caused by high speed mixing at 1500 rpm for 30 min. This gives fragments of mean size. • Short single stranded regions- termini or blunt end fragments are formed. • Sonication can reduce the length of the fragments to about 300 nucleotide pairs. • Shearing does not necessarily produce 5’ phosphate and3’ ends . Therefore the end of the fragments must be repaired. 5
  6. 6.  Restriction endonuclease digestion • Large number of restriction enzymes which recognize and cut DNA with in target sites of nucleotides are known. • Depending upon the number of target sites present, DNA may be cut into too small or too big size fragments. • Generally the same restriction enzyme is used for vector and the DNA of interest. • The digestion carried out may be light ,moderate or heavy producing from small number to large number of fragments which are reproducible. 6
  7. 7.  Reverse transcriptase method • If eukaryotic gene is to be cloned and expressed in prokaryotic cell, the difference in gene organization has to be considered. • The introns present in eukaryotic genes are transcribed into mRNA. Such precursor mRNA in eukaryotic cell undergoes post transcriptional modification and removal of introns occurs to give rise to processed mRNA. • Mature mRNA molecules from animal cell do not contain sequences complementary to introns as they are removed by processing. • These molecules then can be directly transcribed into DNA using an enzyme reverse transcriptase. • cDNA thus produced for a particular protein can be joined to appropriate vector and cloned into a host. 7
  8. 8.  Hybridization method • Hybridization method depends on the principle that on mRNA forms a complex with complementary DNA segments from which it has been transcribed. • This method is possible if protein encoding gene does not have introns. • DNA from the donor organism is first isolated. This ds DNA is treated with heat or alkali and is converted to ss DNA by denaturation . • Strands are then mixed with mRNA transcribed by the gene. mRNA pairs with cDNA portion to form DNA-RNA complex • This complex is then isolated and DNA separated from RNA. • Ss DNA thus obtained can be converted to ds DNA by DNA polymerase I. 8
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  10. 10. Separation of isolated DNA fragments • The cutting of DNA by different isolation methods, these fragments can be separated by a technique known as gel electrophoresis. 10
  11. 11. GEL ELECTROPHORESIS • Electrophoresis is a separation technique, where the compounds are separated due to varying behavior under the influence of applied electric field. • In Gel Electrophoresis, the separation is brought about through molecular sieving technique, based on the molecular electrophoresis by gel method at two or more pH values. It is parallel to charaterize the newly disovered protein molecule. 11
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  13. 13. SELECTION OF VECTORS • Vectors are used to transform DNA material into the host cell. • Types of vectors: • Cloning vectors and expression vectors • Cloning vectors: propagation of DNA inserts. • Expression vectors: production of proteins. 13
  14. 14. • Vector is a “molecular carrier” or a “molecular vehicle” . It is used as a mode of transportation or transference to insert and amplify a gene into a target genome. • Since DNA fragments are not capable of self replication in a host cell, a vector is used. 14
  15. 15. Selection of cloning vectors depends on , • Objective of cloning experiment • Ease of working • Knowledge existing about the vector • Suitability • reliability 15
  16. 16. Properties of vectors: • Able to self replicate in the host • Easy to isolate • Non toxic to host cells • Have space for foreign inserts • Have unique restriction sites for common restriction enzymes. 16
  17. 17. Practical features of DNA cloning vectors • Size • Origin of replication • Multiple cloning sites • Selectable marker genes • RNA polymerase promoter sequence • DNA sequencing primers 17
  18. 18. Plasmid cloning vectors • Plasmids are circular ,double stranded DNA molecules that exist in bacteria and in the nuclei of some eukaryotic cells. • They can replicate independently of the host cell . The size of plasmids ranges from few kb to near 100 kb • Can hold up to 10 kb fragments • Plasmids have an origin of replication , antibiotic resistance genes as markers ,and several unique restriction sites. • After culture growth the clone fragment can be recovered easily. The cells are lysed and the DNA is isolated and purified. • DNA fragment can be kept indefinitely if mixed with glycerol in a -70 degree c freezer. 18
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  20. 20. Phage cloning vectors • Fragments up to 23 kb can be accommodated by a phage vector . • Lambda is most common phage • 60% of the genome is needed for lytic pathway • Segments of the lambda DNA is removed and a stuffer fragment is put in • The stuffer fragment keeps the vector at a correct size and carries marker genes that are removed when foreign DNA is inserted into the vector. • Example: charon 4A lambda • When charon 4A lambda is intact, beta galactosidase reacts with xgal and the colonies turn blue. • When the DNA segment replaces the stuffer region , the lac5 gene is missing, which codes for beta galactosidase, no beta galactosidase is formed and the colonies are white, 20
  21. 21. Cosmid cloning vectors: • Fragments from 30 to 46kb can be accommodated by a cosmid vector. • Cosmids combine essential elements of a plasmid and lambda systems. • Cosmids are extracted from bacteria and mixed with restriction endonucleases. • Cleaved cosmids are mixed with foreign DNA that has been cleaved with the same endonucleases. • Recombinant cosmids are packaged into lambda casp5 ds. • Recombinant cosmid is injected into the bacterial cell where the rcosmids arranges into a circle and replicates as a plasmid . It can be maintained recovered just as plasmids. 21
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  23. 23. Yeast artificial chromosomes (YACs) • YACs can hold up to 500kbs • YACs are designed to replicate as plasmids in bacteria when no foreign DNA is present. Once a fragment is inserted , YACs are transferred to cells , they can replicate as eukaryotic chromosomes . • YACs contain : a yeast centromere ,two yeast telomeres, a bacterial origin of replication and bacterial selectable markers . • YAC plasmid – yeast chromosome • DNA is inserted to a unique restriction site and cleaves the plasmid with another restriction endonucleases that removes a fragment of DNA and causes YAC to become liner . Once in the cell the rYAC replicates as a chromosome, also replicating the foreign DNA. 23
  24. 24. Bacterial artificial chromosomes (BACs) • BACs can hold up to 300 kbs • The F factor of E.coli is capable of handling large segments of DNA. • RECOMBINANT BACs are introduced into E.coli by electroportation once in the cell the rBAC replicates like an F factor. 24
  25. 25. Insertion of target DNA into vector • After cutting the source DNA to generate fragment of interest , the next task of cloning is generation of recombinant DNA molecule by joining DNA fragment of interest to appropriate vector. After this joining has been done, the vector along with inserted fragment can be transferred to suitable host . Following methods are most commonly used for joining of DNA to vector. • Ligation of cohesive termini • Blunt end ligation • Homopolymer tailing • Use of linker molecules • Use of adaptor molecules 25
  26. 26. Transfer of rDNA to host cell Recombinant DNA is allowed to enter into a suitable host cell for expression of foreign DNA. The recombinant vector is mainly introduced into E.coli to select the recombinant from the unchanged vector and to obtain many copies of the DNA insert or recombinant vector. The specific method is selected for transformation, it depends on types of vectors and host cells. 26
  27. 27. ISOLATION OF RDNA The main objective of cloning experiment is to isolate the cells that contain recombinant vector from non transformed cells. Recombinant cells express the characters while the non recombinants do not express the characters or traits .Different methods are used for screening or selection of recombinats. • Direct selection Many times cloned DNA itself codes for resistance to the antibiotic ampicillin and the recombinants can be allowed to grow on minimal medium containing ampicillin . Such recombinants contain ampr gene on its plasmid vector . 27
  28. 28. • Hybrid arrested translation In this method the portion of mixture is used for in vitro translation and it serves as the control . The remaining portion of the mRNA mixture is subdivided and mixed with denatured recombinant molecule. The mixture is incubated under suitable conditions favouring annealing . The DNA insert present in a given clone is hybridize with the complementary mRNA . The mRNA mixture is used for in vitro translation and the resulting mixture of polypeptides is subjected to electrophoresis. The protein bands obtained in each sample are compared with those obtained from the control mRNA. The DNA insert causing the absence of desired protein are identified and isolated. 28
  29. 29. • Hybrid selection In hybrid selection method recombinant vectors are purified ,denatured and fixed separately to a solid support . The DNA attached to each disc is isolated separately and used for invitro translation . The resulting polypeptides are identified by electrophoresis. The identification of specific polypeptide may be facilitated by using antibodies specific to it the antibodies may be used for western blotting or RNA blotting methods. 29
  30. 30. • Colony hybridization This method is used to identify those bacterial colonies in a petri plate which contain specific DNA sequence .The bacterial colonies are replica placed or phage plaques are directly lifted on nitrocellulose filters . The filter disc is removed and put on blotting paper soaked with 0.5N NaOH solution .The alkali diffuses into filters, lyses bacterial cells and denatures their DNA .The disc is neutralized by tris amino methane HCl buffer by maintaining high concentrations of the salt. The cDNA is fixed properly by baking at 80 0c. The disc is incubated with a solution containing radioactive chemical labelled probe at suitable conditions . The probe hybridizes any bound dna that contains sequences complementary to probe. The unhybridized probe is removed by washing. Colonies that develop positive x ray image are compared with water plate and these colonies are picked up for further studies. 30
  31. 31. APPLICATIONS OF rDNA • Recombinant DNA is widely used in biotechnology, medicine and research. Today, recombinant proteins and other products that result from the use of rDNA technology are found in essentially every western pharmacy, doctor's or veterinarian's office, medical testing laboratory, and biological research laboratory. In addition, organisms that have been manipulated using recombinant DNA technology, as well as products derived from those organisms, have found their way into many farms, supermarkets, home medicine cabinets, and even pet shops, such as those that sell GloFish and other genetically modified animals. 31
  32. 32. GLOFISH 32
  33. 33. • Many additional practical applications of recombinant DNA are found in industry, food production, human and veterinary medicine, agriculture, and bioengineering. Some specific examples are identified below. • Recombinant human insulin: Almost completely replaced insulin obtained from animal sources (e.g. pigs and cattle) for the treatment of insulin- dependent diabetes. A variety of different recombinant insulin preparations are in widespread use.Recombinant insulin is synthesized by inserting the human insulin gene into E. coli, or yeast which then produces insulin for human use. 33
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  35. 35. • Recombinant human growth hormone: Administered to patients whose pituitary glands generate insufficient quantities to support normal growth and development. Before recombinant HGH became available, HGH for therapeutic use was obtained from pituitary glands of cadavers. This unsafe practice led to some patients developing Creutzfeldt–Jakob disease. Recombinant HGH eliminated this problem, and is now used therapeutically. It has also been misused as a performance enhancing drug by athletes and others. 35
  36. 36. • Recombinant hepatitis B vaccine: Hepatitis B infection is controlled through the use of a recombinant hepatitis B vaccine, which contains a form of the hepatitis B virus surface antigen that is produced in yeast cells. The development of the recombinant subunit vaccine was an important and necessary development because hepatitis B virus, unlike other common viruses such as polio virus, cannot be grown in vitro. Vaccine information from Hepatitis B Foundation 36
  37. 37. • Golden rice: A recombinant variety of rice that has been engineered to express the enzymes responsible for β-carotene biosynthesis. This variety of rice holds substantial promise for reducing the incidence of vitamin A deficiency in the world's population. Golden rice is not These crops are in common commercial use in several countries. 37
  38. 38. GOLDEN RICE 38
  39. 39. • Herbicide-resistant crops: Commercial varieties of important agricultural crops (including soy, maize/corn, sorghum, canola, alfalfa and cotton) have been developed that incorporate a recombinant gene that results in resistance to the herbicide glyphosate, and simplifies weed control by glyphosate application. These crops are in common commercial use in several countries 39
  40. 40. • Insect-resistant crops: Bacillus thuringeiensis is a bacterium that naturally produces a protein with insecticidal properties. The bacterium has been applied to crops as an insect-control strategy for many years, and this practice has been widely adopted in agriculture and gardening. Recently, plants have been developed that express a recombinant form of the bacterial protein, which may effectively control some insect predators. Environmental issues associated with the use of these transgenic crops have not been fully resolved. 40
  41. 41. Advantages of Recombinant technology: •Provide substantial quantity •No need for natural or organic factors •Tailor made product that you can control •Unlimited utilizations •Cheap •Resistant to natural inhibitors 41
  42. 42. Disadvantages of Recombinant technology: •Effects natural immune system of the body •Can destroy natural ecosystem that relies on organic cycle •Prone to cause mutation that could have harmful effects •Major international concern: manufacturing of biological weapons such as botulism & anthrax to target humans with specific genotype 42
  43. 43. References • Pharmaceutical biotechnology by Dr . Chandrakant R. Kokare 9.1-9.22 • Gene biotechnology by S.N Jogdnand 138- 150 • Internet source 43
  44. 44. 44 Thank you