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BIOTECHNOLOGY -intentional manipulation of genetic material of an organism • Deoxyribonucleic Acid (DNA) • determines the characteristics of all living organisms. • occurs in most cells of all organisms • composed of four different nucleotides in different combinations • each cell in the human body contains more than 3 BILLION letters Four bases: Adenine Thymine Guanine Cytosine 2 bonds 3 bonds • Sugar and phosphate backbone • Double helix structure (two spirals around each other) • The only difference between living organisms is the amount and order of the four nucleotide bases. Genome: the entire sequence of DNA Gene: the part of the code that corresponds to a protein *genes can be transferred from one organism to another* BIOTECHNOLOGY The intentional manipulation of genetic material of an organism Why would we want to do this? • To study cellular processes of an organism – E.g. Glowing gene from jellyfish to tobacco plant • To give one organism the trait(s) of another – E.g. Anti-freeze from fish blood into strawberries to survive through early frosts Part 1: Manipulating Bacteria: The Making of a Plasmid Plasmid: - a small circular piece of extra-chromosomal bacterial DNA, able to replicate - bacteria exchange these plasmids to share DNA - E.g. antibiotic resistance genes • Since plasmid is made of DNA it can code for genes, ex. antibiotic resistance, and can carry specific sequences of DNA • Specific DNA sequences can be recognized by enzymes called restriction endonucleases Restriction Endonucleases/Restriction Enzymes • enzymes that are able to cut doublestranded DNA into fragments at specific recognition sites in DNA sequences Ex. EcoRI: 5’-GAATTC-3’ 3’-CTTAAG-5’ • Restriction enzymes can create “sticky ends or “blunt ends” Sticky Ends • fragment end of a DNA molecule with a short single-stranded overhang Blunt Ends • fragment end of a DNA molecule with no overhang Once made, the ends can be re-joined together by other enzymes ("enzyme glue") To Make a Recombinant Plasmid: Insert 1. Cut the plasmid and the insert with the same restriction endonuclease to make complementary sticky ends. 2. Combine the sticky ends using ligase. ligase: enzyme used to join DNA together 3. Introduce the recombinant plasmid into bacteria. Making a Recombinant Plasmid Bacterial Transformation • introduction of foreign DNA into a bacterial cell • plasmid is used as a vector, a vehicle by which DNA can be introduced into host cell ---- ---+ + - ++ - ++ -+ -+-+- +- -+ ------ plasmid +++++ phospholipid bilayer - ++ -++ Ca2+ ions Following transformation bacteria are grown in medium with antibiotic… Only the bacteria that have the plasmid (and therefore the antibiotic resistance) will survive. Example plasmid: Origin of Replication: • where the plasmid starts to duplicate itself • the specific sequence MUST NOT be cut by restriction endonucleases or it won’t be able to replicate Part 2: Where do we get our insert sequence? • From someone else’s DNA – ex. fish gene in strawberries, – jellyfish gene in plants • Make it! • In order to do these things, we need a way to make many copies of the genes we want Using Bacteria as Production Factories • • • • easy to grow no ethical issues small genome easy to manipulate Making an insert: Polymerase Chain Reaction Common uses of biotechnology: 1. Making "stuff” • proteins, enzymes, medication, etc. can be produced by engineered bacteria! • Food can be altered to have new traits • Cloning (therapeutic and reproductive) 2. Genetic screening • crime cases, relationship, genetic screening, etc. 3. Gene Therapy Therapeutic cloning • used to produce tissue that is identical to the donor, to prevent rejection Reproductive Cloning • creates an organism with the same genetic material (DNA) as the original organism – an EXACT COPY of the donor Dolly the Sheep • the first cloned sheep Ex. RFLP: Restriction Fragment Length Polymorphism Comparison of different lengths of DNA fragments produced by restriction enzymes to determine genetic differences between individuals Gene therapy - desired gene is inserted into cell's nucleus using a retrovirus as a carrier - defective gene replaced by functional gene Ex. ADA deficiency - adenosine deaminase deficiency - little immunity with low chances of recovery - the T-cells of a four-year-old were removed, modified and re-inserted to fix her immune system