Download Introduction Biotechnology Recombinant DNA Genetic Engineering

Introduction
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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
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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
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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
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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
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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)
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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
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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
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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
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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