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  1. 1. 1 Recombinant DNA Technology I. Natural recombination: A. Provides means of genetic exchange between closely related species: Conjugation Transformation Transduction B. Genetic Engineering: Deliberate modification of an organism's genetic information by directly changing its DNA II. Recombinant DNA Technology: A. Methods used in genetic engineering: Depend on universality of: Genetic code Mechanisms of protein synthesis B. Allows artificial recombination of DNA from very different organisms: Plants Bacteria Animals Yeast Viruses C. Foreign DNA inserted into cell: Cell then produces proteins coded for by the foreign genes D. Fairly recent aspect of bacterial genetics: Started in the late 1960's: III. Mechanisms used in recombinant DNA technology: A. Similar to processes that occur during natural recombination: During both natural recombination and the production of recombinant DNA DNA from two separate sources: Fragmented by: Normal cell enzymes Joined by: Normal cell enzymes B. Discovery of two types of enzymes allowed production of recombinant DNA: Restriction endonucleases: Cut double stranded DNA: At specific nucleotide sequences Normal function is to destroy phage DNA after its enterence into a host B363
  2. 2. 2 cell DNA ligase: Enzyme: Repairs breaks in the sugar-phosphate backbone of DNA C. Four major steps in producing recombinant DNA: Step 1. DNA for a particular phenotype identified Purified Isolated May use gene machine to: Synthesize eukaryotic genes: so that they can be expressed in prokaryotic cells: DNA sequence of: Human insulin Human growth hormone Synthesized from the amino acid sequence of the proteins Isolation actual genes from chromosomes: Difficult to do with eukaryotic genes Easily done with viral genes: Gene for surface antigen of the Hepatitis B virus isolated: Introduced into bacterium: Bacteria make the viral protein: Protein used as vaccine for hepatitis B Step 2. Purified DNA fused with other pieces of DNA to form recombinant DNA molecules: Fragmented donor DNA attached to a vector: Carrier of foreign genes: Plasmids: Contain no fertility factors Viruses Vector DNA must be: Able to replicate in host cell Stable in the host cell B363
  3. 3. 3 Restriction endonucleases and DNA ligase: Enzymes used to insert foreign DNA into vector: Restriction endonucleases: Cut DNA into fragments: Cut at specific nucleotide sequences: Each restriction endonuclease recognizes a specific sequence: Each cuts DNA at a different place Fragmented donor DNA attached to a vector that has been: Cut by the same restriction endonuclease Break double stranded DNA in regions called palindromes: Highly specific: Each cleaves DNA at a specific palindrome Protect cells from foreign DNA (viruses): Palindrome: Word, phrase, sentence, etc. Reads the same backwards or forwards: Radar Otto Eve Deed Able was I ere I saw Elba. Madam I'm Adam. A man, a plan, a canal, Panama. Straw, no too stupid a fad, I put soot on warts. Results in formation of DNA fragments with sticky ends: Sticky ends able to pair with any complementary sticky end regardless of source: ────────────────────────── DNA strand T (A A T T) C C G C A (T T A A ) G G C G ────────────────────────── DNA strand DNA molecule with palindrome. Restriction endonuclease cuts DNA between A and T on one strand and between T and C on the other. ───────────── ───────────── DNA strand TA A T T CC GC B363
  4. 4. 4 A TT A AGG CG ── ──────────────────── DNA strand Cleavage with restriction endonuclease ─ fragments with "sticky ends." Sticky ends will form hydrogen bonds with any other DNA cleaved with the same enzyme. DNA ligase: Used to join fragments into a recombinant DNA molecule: Repairs breaks in sugar phosphate backbone Reseals cuts made by restriction endonuclease: ∴DNA from any source can be joined to DNA from any other source Step 3. Recombinant DNA molecules: Inserted into a host cell Vector moved into recipient (host) cells using transformation techniques: Electroporation: Pulses of high-voltage electricity: Increase membrane permeability In E. Coli use: Cold CaCl2 treatment Increases membrane permeability Step 4. Grown as a clone: Recipient cell containing the desired foreign gene must be selected: Use genetic markers: Genes for: Pigment production Antibiotic resistance Use selective media Necessary because: Other genes may combine with the vector: Taken up by recipient cells Getting the recombined gene expressed: Foreign gene must be expressed: Recipient cell must produce large amounts of the desired B363
  5. 5. 5 product IV. Potential benefits of genetic engineering: A. Synthesis of human hormones: Insulin: Human insulin gene introduced into E. coli: Human insulin produced by E. coli used to treat diabetes Human growth hormone Human growth hormone gene introduced into E. coli: Human growth hormone produced by E. coli used to treat children with growth deficiencies B. Production of large amounts of antibiotics Production of anti-cancer drugs: Interferon C. Increase nitrogen fixing capabilities of plants D. Making animal proteins for food E. Adding photosynthetic genes to more kinds of microorganisms F. Genetic repair of hereditary defects G. Improving marketing qualities of fruits and vegetables V. Potential hazards of genetic engineering: A. Introduction of foreign DNA into bacteria could create potentially dangerous strains of bacteria: Done by accident or design: In 1974 Geneticists called a: Voluntary halt to recombinant studies to evaluate possible risks: In 1976: The National Institutes of Health Guidelines for Recombinant DNA research were approved: 1976 E. coli strain developed: Grows only in laboratory environment: Grows only at temperatures below 18oC Will not grow in humans Has cell wall defects Killed by sunlight In 1982 Guidelines relaxed: Hazards have not developed B363