GCD 3022 1st Edition Lecture 33Outline of Last Lecture I. Recombinant DNA technologya. Discoveryb. Importance of recombinant DNA technologyII. Gene cloning a. Cloning experimentsi. cDNA ii. vector DNAiii. preparation of DNAiv. host cellb. restriction enzymesIII. Steps in gene cloning (of human B-globin gene)a. Plasmid: AmpR and lacZb. cDNAc. Restriction enzymesd. Recombinant evente. Testing for the desired genef. Net Result of cloningIV. Polymerase Chain Reactiona. Starting materialsi. Template DNAii. Oligonucleotide primersiii. Deoxynucleoside triphosphatesiv. Taq polymeraseb. Processc. Resultd. Reverse transcriptase PCROutline of Current LectureI. DNA sequencingII. Dideoxy method a. Mechanismb. Dideoxynucleotidesc. Chain terminationThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.d. Automated sequencingIII. Blotting methodsa. DNA librariesb. Southern blottingc. Northern blottingd. Western blotting IV. Biotechnologya. Earlier studiesb. Uses of microorganisms in biotechnologyV. Biological control and bioremediationa. Biological controli. Bacterial species as biological control agentsii. Microorganisms b. BioremediationVI. Geneticall modified animalsa. Purpose of genetically modified animalsi. Livestock ii. Reproductive cloning iii. Stem cellsiv. Cloning of somatic cellsb. Gene additioni. Gene addition in eukaryotesc. Gene replacementi. Mice and gene replacementsii. Gene knockoutiii. Gene knockinVII. Genetically modified plantsa. History b. Transgenic plantsi. A. tumefaciensii. Process VIII. Human gene therapya. Researchb. Transfer methodsCurrent LectureI. DNA sequencing: enables researchers to determine the base sequence of DNAII. Dideoxy method: method of DNA sequencing that uses dideoxyribonucleotides (ddNTPs) and our knowledge of DNA replication.a. Mechanism: DNA polymerase connects adjacent deoxynucleotides by covalently linking the 5’ phosphate of one to the 3’ alcohol of another. The nucleotides missing the 3’ alcohol can then be synthesized. These nucleotides are called dideoxyribonucleotides. b. Chain termination: if a ddNTP is added to a growing DNA strand, the strand can no longer grow. If ddATP is used, termination will always be at an A in the DNAc. Automated sequencing: uses a single tube containing all four ddNTPs, but each type has a different colored fluorescent label attached, which allows them to be differentiated when they are ran through a lane of gel. The process is automated by using a laser and fluorescence detector. III. Blotting methodsa. DNA libraries: collection of thousands of different fragments of DNA, each of which is inserted into a vector. Genomic library when it has chromosomal DNA. Can be a cDNA library when it contains hybrid vectors with cDNA inserts. b. Southern blotting: can detect the presence of a particular gene sequence within a complex genetic background. Developed by E.M. Southern in 1975. Uses include:i. Determining the copy number of a gene in a genomeii. Detecting small gene deletions that cannot be detected by light microscopyiii. Identifying gene familiesiv. Identifying homologous genes among different speciesv. Determining if a transgenic organism is carrying a new or modified genec. Northern blotting: used to identify a specific RNA within a mixture of many RNA molecules. Similar to Southern blotting in that:i. It extracts the RNA of interest from cells and purifies it, then separates its components using electrophoresis. ii. The components are blotted onto nitrocellulose or nylon filters and the filters are placed into a solution containing a radioactive probe. iii. The filters are exposed to x-ray film and the RNAs that are complementary to the radiolabeled probe are detected as dark bands on the x-ray film. Uses include: iv. Determining if a specific gene is transcribed in a particular cell type (ex: nerve vs. muscle cells)v. Determining if a specific gene is transcribed at a particular stage of development (ex: fetal vs. adult cells)vi. Revealing if a pre-mRNA is alternatively splicedd. Western blotting: used to identify a specific protein within a mixture of many protein molecules. Uses include determining if a specific protein is made in a particular cell type and if a specific protein is made at a particular stage of development. The process of Western blotting involves:i. Extraction of proteins from the cellsii. Separation of protein components on a geliii. Blotting of components onto filters and placement of the filters into a solution containing a primary antibody (which recognizes the protein of interest)iv. The secondary antibody (which recognizes the constant region of the primary antibody) is added to the mixture.v. A colorless dye XP is added, where it is converted to a black compound bythe secondary antibodyvi. The proteins of interest are indicated by dark bandsIV. Biotechnology: technologies that involve the use of living organisms, or their products, to benefit humans.a. Earlier studies: study of biotechnology began about 12,000 years ago when humans began to domesticate animals and plants for the production of food. Since the 1970s there have been new and improved ways to make use of organisms to benefit humans. b. Uses of microorganisms in biotechnology: relatively new area of research in biotechnology, problems with safety concerns and negative public view.V. Biological control and bioremediation: two methods of using bacterial species to clean up pollutants or keep plants healthy. a. Biological control: the use of microorganisms or their products to alleviate plant problems (disease or environmental damage). i. Bacterial species as biological control agents: Bacillus thuringiensis toxins (Bt toxins) are lethal to many caterpillars and beetles but harmless to plants and humans. Thus they are used in powder form to protect plants from pests. b. Bioremediation: the use of microorganisms to reduce environmental pollutants. Enzymes produced by a microorganism transform the structure of the toxic pollutant (called biotransformation). Biotransformation usually results in biodegradation (toxic converted to nontoxic metabolites). Biotransformation without biodegradation can also occur (pollutant becomes less toxic).VI. Geneticall modified animals: the production of transgenic animals to help humans.a. Purpose of genetically modified animals: animal systems help scientists understand