GCD 3022 1st Edition Lecture 34Outline of Last Lecture I. DNA sequencingII. Dideoxy method a. Mechanismb. Dideoxynucleotidesc. Chain terminationd. 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 plantsThese 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.a. History b. Transgenic plantsi. A. tumefaciensii. Process VIII. Human gene therapya. Researchb. Transfer methodsOutline of Current LectureI. Cloninga. Selectable marker b. Restriction endonucleases c. Stem celld. Dolly e. cDNA II. Sequencing a. PCRi. Taq polymeraseb. Dideoxy methodi. ddNTPii. Purpose of dideoxynucleotidesc. Blottingi. Western blottingIII. Gene manipulationa. Gene knockout b. Mouse model c. Gene replacementd. Gene addition Current LectureIV. Cloninga. Selectable marker: when using a vector with a B-galactosidasescreenable marker,a lacZ strain of bacteria would be used as a host cell for cloning. b. Restriction endonucleases: recognize specific sequences in DNA, produced by bacterial cells as a primitive immune system, and often generate short single stranded sequences. c. Stem cell: a cell that can divide and remain undifferentiated.d. Dolly: was created when a mammary cell was fused with an egg cell that had its nucleus removed.e. cDNA: complementary DNA (DNA copy of RNA). Synthesized by starting with RNAthat has a polyA tail, adding an oligodT primer, reverse transcriptase, and dNTPs (for single stranded cDNA). For double stranded, use RNaseH to digest some of the RNA and generate RNA primers that polymerase or reverse transcriptase will use to make the second strand of DNA. DNA ligase then seals the nicks. V. Sequencing a. PCRi. Taq polymerase: used in PCR because it is not damaged by exposure to high temperatures. b. Dideoxy methodi. ddNTP: the functional groups of ddNTP are 2’ H and 3’ H, while dNTPs have 2’ H and 3’ OH. ii. Purpose of dideoxynucleotides: used in dideoxy method of DNA sequencing and are incorporated into DNA strands but prevent further strand growth. c. Blotting: used to sequence DNA or RNA or determine the identity of proteinsi. Western blotting: detects proteins using a antibodies as a probe.VI. Gene manipulationa. Gene knockout: when a functional gene is replaced with a mutant copy. b. Mouse model: a strain of mice engineered to carry a mutation analogous to a disease-causing mutation in a human gene. c. Gene replacement: the gene that is introduced into the cell is swapped with a homologous gene that is already present in the genome. Used to correct a mutant gene or insert a mutant gene to determine effects. d. Gene addition: occurs when a gene is introduced into a cell and it integrates into the chromosomal DNA by nonhomologous recombination. This method would beused when you wish to introduce an additional copy of a gene or add a gene to a genome that doesn’t normally contain
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