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Ch. 19 Part 1 Gene Technology Genetic Engineering (GE) • modification of an organism's genetic composition by artificial means • involves transfer of genes from one organism into a plant or animal of an entirely different species • Uses of GE: – understanding that changes in DNA changes in protein synthesis – Sequencing nucleotides in DNA & comparing them to different organisms – Carry out genetic tests to determine occurance of disorders – Gene therapy to treat diseases • Goal: – Remove gene(s) from one organisms & transfer to another organism so gene is expressed in the new organism • Issues: Selective Breeding • Involves whole sets of genes Genetic Engineering • Involves transfer of a single gene from one organism to another Recombinant DNA (rDNA) • DNA that has been altered by genetic engineering • Made by joining DNA from 2 different sources • Contains lengths of nucleotides from different organisms • Organisms containing rDNA are called TRANSGENIC ORGANSIMS – Aka genetically modified organism GMO) – Definition: organism containing recombinant DNA that expresses new genes Transgenic Organisms Steps of Gene Transfer 1. Identify gene – Make desired gene using one of these methods: • • • Gene cut from chromosome Reverse transcription of mRNA Artificial synthesis from nucleotides 2. Make copies of gene – Use Polymerase Chain reaction (PCR) 3. Insert gene into vector – Vectors deliver gene into the cells of organisms Examples: – • • • Plasmids Viruses liposomes 4. Vector carries gene into cells 5. Cells with new gene identified and clones 3 Main Tools for a Gene Technologist: • Enzymes – Restriction nucleases – Ligases – Reverse transcriptase • Vectors – Plasmids – Viruses – liposomes • Genetic Markers – Genes coding for easily identifiable markers that can be used as markers Bacteriophages • Virus • Infects bacteria • “phages” Tools for a Gene Technologist: Restriction Enzymes (RE) • Restriction endonucleases – Class of enzymes from bacteria that breakdown DNA of invading virus (bacteriophage) – Bacteria contain gene that codes for enzymes that cut the phage DNA at sugar-phosphate backbone into smaller pieces at specific spots of the DNA – Purpose: to restrict viral infection of bacteria (protect bacteria) How Restriction Enzymes Work • Bind to specific TARGET SITE of DNA – Called the Restriction site: a specific sequence of bases – Bacterial DNA is NOT cut by enzyme because: • Protective chemical markers OR • Does not have target/restriction site in its DNA – Usually a PALINDROMIC nucleotide sequence • Reads the same in both directions • RACE CAR (same both ways) • GGATCC and complementary strand is CCTAGG How Restriction Enzymes Work • TWO Ways to cut viral DNA: – Staggered patter across sugarphosphate backbone STICKY ENDS • short region of UNPAIRED nucleotides • Unpaired region is called an overhang – sticky because it wants to and will pair with another sticky end that has complementary overhang sequence – Sticky ends are like long-lost twins seeking to hug each other tightly once they meet • EASY for hydrogen bonds to form with complementary bases with another piece of DNA cut with same restriction enzyme – Straight across sugar-phosphate backbone BLUNT ENDS • not sticky because all the nucleotides are already paired between the two strands of DNA • blunt ends of DNA and plasmids are less likely to find each other, and thus ligation of blunt ends requires that more DNA is put into the test tube What happens when you cut LONG pieces of DNA with RE? • DNA pieces of different lengths are created • Need to separate DNA pieces by length in order to locate a specific piece – Use GEL ELCTROPHORESIS and GENE PROBES • Once you have desired DNA segment, you make millions of copies of that piece using POLYMERASE CHAIN REACTION (PCR) Naming Restriction Enzymes • Abbreviation indicates ORIGIN of restriction enzyme – Organism it came from • Roman numerals differentiate between enzymes from the same source – Usually numbered by order which they were found/discovered • EcoRI – From Escherichi coli bacteria (Strain RY) – “I” means it was the first restriction enzyme made from E. Coli Improvements in Gene Technology • Protein sequencing: – technique to determine the amino acid sequence of a protein, as well as which conformation the protein adopts – Genetic code used to determine order of nucleotides & synthesize DNA artificially from nucleotides • Better than cutting gene from chromosome or using reverse transcriptase Improvements in Gene Technology • DNA templates no longer needed to make genes or genome • Steps to create new “novel” genes (such as those in a vaccine) – Choose codons for desired amino acid sequence – Sequence of nucleotides held in computer – This directs synthesis of short fragments of DNA – Fragments of DNA joined to make longer sequences of DNA – Longer sequences of DNA inserted into plasmids for use in genetic engineering