GCD 3022 1st Edition Lecture 34 Outline of Last Lecture I DNA sequencing II Dideoxy method a Mechanism b Dideoxynucleotides c Chain termination d Automated sequencing III Blotting methods a DNA libraries b Southern blotting c Northern blotting d Western blotting IV Biotechnology a Earlier studies b Uses of microorganisms in biotechnology V Biological control and bioremediation a Biological control i Bacterial species as biological control agents ii Microorganisms b Bioremediation VI Geneticall modified animals a Purpose of genetically modified animals i Livestock ii Reproductive cloning iii Stem cells iv Cloning of somatic cells b Gene addition i Gene addition in eukaryotes c Gene replacement i Mice and gene replacements ii Gene knockout iii Gene knockin VII Genetically modified plants These 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 plants i A tumefaciens ii Process VIII Human gene therapy a Research b Transfer methods Outline of Current Lecture I Cloning a Selectable marker b Restriction endonucleases c Stem cell d Dolly e cDNA II Sequencing a PCR i Taq polymerase b Dideoxy method i ddNTP ii Purpose of dideoxynucleotides c Blotting i Western blotting III Gene manipulation a Gene knockout b Mouse model c Gene replacement d Gene addition Current Lecture IV Cloning a 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 V VI e cDNA complementary DNA DNA copy of RNA Synthesized by starting with RNA that 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 Sequencing a PCR i Taq polymerase used in PCR because it is not damaged by exposure to high temperatures b Dideoxy method i 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 proteins i Western blotting detects proteins using a antibodies as a probe Gene manipulation a 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 be used when you wish to introduce an additional copy of a gene or add a gene to a genome that doesn t normally contain it
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