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U of M GCD 3022 - Chapter 21: DNA technologies and gene cloning
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GCD 3022 1st Edition Lecture 33 Outline of Last Lecture I Recombinant DNA technology a Discovery b Importance of recombinant DNA technology II Gene cloning a Cloning experiments i cDNA ii vector DNA iii preparation of DNA iv host cell b restriction enzymes III Steps in gene cloning of human B globin gene a Plasmid AmpR and lacZ b cDNA c Restriction enzymes d Recombinant event e Testing for the desired gene f Net Result of cloning IV Polymerase Chain Reaction a Starting materials i Template DNA ii Oligonucleotide primers iii Deoxynucleoside triphosphates iv Taq polymerase b Process c Result d Reverse transcriptase PCR Outline of Current Lecture I DNA sequencing II Dideoxy method a Mechanism b Dideoxynucleotides c Chain termination 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 III IV V VI VII VIII d Automated sequencing Blotting methods a DNA libraries b Southern blotting c Northern blotting d Western blotting Biotechnology a Earlier studies b Uses of microorganisms in biotechnology Biological control and bioremediation a Biological control i Bacterial species as biological control agents ii Microorganisms b Bioremediation 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 Genetically modified plants a History b Transgenic plants i A tumefaciens ii Process Human gene therapy a Research b Transfer methods Current Lecture I DNA sequencing enables researchers to determine the base sequence of DNA II III 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 DNA c 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 Blotting methods a 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 genome ii Detecting small gene deletions that cannot be detected by light microscopy iii Identifying gene families iv Identifying homologous genes among different species v Determining if a transgenic organism is carrying a new or modified gene c 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 IV V 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 spliced d 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 cells ii Separation of protein components on a gel iii 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 by the secondary antibody vi The proteins of interest are indicated by dark bands 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 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 and treat human diseases i Livestock in the process called molecular pharming cows are genetically modified to produce milk with a specific


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U of M GCD 3022 - Chapter 21: DNA technologies and gene cloning

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