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Download Introduction Biotechnology Recombinant DNA Genetic Engineering
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Introduction Biotechnology o Recombinant DNA o Genetic Engineering Limitations to DNA Research o Molecule Size o Gene to Chromosome Size o Noncoding Sequences o Gene to Non-Gene Distinctions DNA Extraction Membrane Dissolution Centrifuge & Cell Fractionalization DNA Cloning & Expression Restriction Enzymes & DNA Fragmentation o Restriction Site (4-8 bases) o Restriction Enzyme Specificity & Natural Short Sequence Repetition Restriction Fragments o Sticky End Restrictors o DNA Ligase DNA Cloning o Plasmids o Plasmid Modification & Bacterial Transformation by Foreign DNA o Recombinant DNA & Genetic Engineered Organism o Cell Cycle & Gene Cloning o Cloning Vector o Using Restriction Enzymes to to Mix/Alter Plasmids o Plate Identification DNA Libraries o Genomic Basics Specific Bacterial Plasmids – Plasmid Cone Genomic Library Bacteriophage Bacterial Artificial Chromosomes (BAC) Multiwell storage Advantages Best for unknown genes & unmapped genes Regulatory & Intron sequences included cDNA o Steps Mature mRNA Primers + Bases + Enzymes Reverse Transcription mRNA Degradation DNA Polymerase + Primer cDNA o Advantages Specific protein / phenotype Studying specific cells Best for known gene / mapped gene Study gene expression patterns by repeated observation of expressing cell DNA Library Screening (DNA Hybridization) o DNA Hybridization o Nucleic Acid Probe (Radioactively tagged / Dyed) o Multiwell Probe coated nylon membrane Identified strand Cloned DNA Expression o Expression Vector o Yeast vs. Bacteria Cells RNA processing Post-Translational Modification o DNA Insertion Techniques Phage Electroporation Heat Shock Bacteria (e.g. Agrobacterium in plants) o Cross Gene Expression & Evolutionary Ancestry DNA Amplification Polymerase Chain Reaction Advantages o Skips Library Formation & Screening Step o Faster o No cell culturing o No plate identification Parts o Heat stable DNA Polymerase (Taq DNA Polymerase from hot spring bacteria) o Heat induced primed DNA molecule o Repeated cycles (2n correct sequence, n = cycle number) Disadvantages o Errors o Sequence length limitation o Need to sequence array to identify error free fragments Applications o DNA Fingerprinting o Species genomic analysis & sequencing o Prenatal diagnosis o Cancer genomic analysis o Viral genomic analaysis DNA Sequencing & Analysis DNA Sequencing o Applications of DNA Sequencing Comparative genome analysis Origin of phonotypical differences (epigenetics) Phylogenetic & Evolutionary Timeline Central Dogma Analysis Temporal and local gene expression analysis Gene function o Electrophoresis (Separating DNA) Agorose gel Electrostatic potential / magnetic flow (Phosphate Gel attraction / Density & Fragment travel rate Resolution power DNA Cloning + Fragmentation DNA fragment separation Restriction fragment length polymorphism o Southern Blotting Eletrophoresis + DNA Hybridization with Radioactive probe o Dideoxy Chain Termination Sequencing (Next-Generation Sequencing) - Sanger o Synthesis Sequencing (Third Generation Sequencing) Gene Expression Analysis o Northern Blotting Screening for mRNA coding for protein of interest at different parts/timings of the metabolic pathway / developmental stages Southern-blot similarity (Electrophoresis + Hybridization with Radioactive probe) o Western Blot Screening for protein of interest at different parts/times of metabolic pathway / developmental stages Amino-acid sequence detection via hybridization with probes o Reverse transcriptase-polymerase chain reaction cDNA synthesis from mRNA present at time of interest during metabolic pathway / developmental stages PRC amplification using gene specific primers Gel electrophoresis indicates presence of mRNA coming from gene only when protein is active at the time of sampling o In situ hybridization Probes dyed will show active mRNA elements in cells that express gene. o System Approach Developmental tracking of gene expression Gene to gene relationships (epistasis, polygenic traits, pleitropy) Gene to environment interactions (epistasis, signal transduction)) cDNA vs. DNA comparison for identification of active genes Above vs. gene mapping (Linkage; Physical; Cytogenic; Sequencing; Coiling) o DNA Microarrays mRNA isolation cDNA synthesis with fluorescent/dyed nucleotides Hybridization with fragments from organisms genes Different gene for each spot Active genes will be died Can test multiple genes at once Gene Function Analysis o In vitro mutagenesis o RNAi via siRNA o Genome-wide studies o Single Nucleotide Polymorphism o Restriction fragment length polymorphism Cloaning & Stem Cell Research Basics o DNA vs. Cell vs. Organism Cloning o Cloning & Genomic Equivalence After Differentiation o Totipotent / Stem Cells Plant Cloning o Seeds o Cutting Animal Cloning o In Vitro Fertilization o Dnucleation / UV Nuclear Sterilization o Nuclear Transplantation o Embryonic Implantation o Differentiation & Limits to Nuclear Transplantation o Cell cycle arrest & dedifferentiation o Clone Phenotype Differences (Epigenetics & Chromosomal/Gene Activation/Deactivation) o Stem Cells Embryonic: Undifferentiated (Totipotent) Adult: Semi differentiated (Muscle Base; Bone Marrow; Umbilical cord) (Pluripotent) Reversed: Dedifferentiated (Induced Pluripotent) o Cloning by Blastula Separation Applications o Trait proliferation o Specimen Preservation/Conservation o Species Recovery o Therapeutic Cloning o Cell Reversal as Disease Treatment and Understanding Limitations o Ethics o Underdevelopment (Nuclear Deactivation) o Biodiversity Biotechnology Applications Medical o Identification of Genetic Disorders by Gene Sequencing & Analysis o Embryonic defect detection (amniocentesis) o Study of Defective Genes o Genome Studies & Endemics o Disease Profiling o Personalized Medicine (Based on Gene/Expression Patterns) o Cell Targeting o Gene Therapy Vector (Retroviral / Manual Cell Implantation) modification of genetic code Present: Stem Cell Application (Bone Marrow) vs. Future: System wide gene therapy Embryonic genetic engineering Limitations: Specificity of vectors Gene expression control Pleitropy / Epistasis Eugenics Ethics Pharmaceutical Products o Specific cell delivery o Small molecular synthesis o Protein Production o Transgenic Animals & “Pharm”ing Type / need matching Purity testing Forensics o Genetic Profiling Extraction Cleaving Cloning (usually by PCR) Eletrophoresis Short tandem repeats Crime Scene Investigation & Paternity Environmental Cleanup Agricultural o Organism Hybridization o Artificial Selection o Cloning good crops (Easier to dedifferentiate plants o Genetic engineering Transgenic organisms for protection / yield Gene modification / therapy Common Vector: Ti plasmid in Agrobacterium tumefaciens o Food modifications & Human Interactions