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Biotechnology **You must be fluent in the language of biotechnology to understand it** Terms to Know:1. genetic engineering2. biotechnology3. recombinant DNA4. gene cloning5. restriction enzymes6. sticky ends7. DNA ligase8. cloning vector9. nucleic acid hybridization10. genomic library11. cDNA library12. PCR13. gel electrophoresis14. southern blotting15. DNA microarray assay16. SNPs17. RFLPs18. stem cells19. gene therapy20. GMO (genetically modified organism) GeneralGenetic engineering = process of manipulating genes and genomes Biotechnology = process of manipulating organisms or their components for the purpose of making useful products Recombinant DNA = DNA that has been artificially made, using DNA from different sources. ex: human gene inserted into E.coli Gene cloning = process by which scientists can produce multiple copies of specific segments of DNA that they can then work with in the lab. Tools of Genetic Engineering:Restriction enzymes (restriction endonucleases) = used to cut strands of DNA at specific locations (restriction sites) Restriction fragments = have at least 1 sticky end (single-stranded end) DNA ligase = joins DNA fragments Cloning vector = carries the DNA sequence to be cloned (eg. bacterial plasmid) We use restriction enzymes (RE) and DNA ligase to make recombinant DNA (cloning vectors) Steps of gene cloning:1. gene of interest inserted into isolated bacterial plasmid using RE and ligase2. plasmid put into bacterial cell3. host cell grown in culture to form a clone of cells containing the cloned gene of interest4. basic research and various applicationsex: gene for pest resistance inserted into plants, gene used to alter bacteria for cleaning up toxic waste, protein dissolves blood clots in heart attack therapy, human growth hormone treats stunted growthTechniques of Genetic EngineeringTransformation = bacteria takes up plasmid (w/gene of interest) Nucleic acid hybridization = used to track gene of interestUse multi-well plates holding library clones, apply radioactively labeled probe molecules, will bind to gene of interest PCR (polymerase chain reaction) = amplify (copy) piece of DNA w/o the use of cells. Denature, anneal, extend cycle over and over Gel electrophoresis = used to separate DNA molecules on basis of size and charge using an electric current (negatively charged DNA moves toward the + pole)Put a mix of DNA molecules at the – end. Longer molecules et stuck in the gel matrix closer to the starting end as the DNA mixture moves to the + end. Southern blotting = used to find a specific human gene1. Prep restriction fragments from genes of interest.2. Run electrophoresis separate fragments based on size.3. Transfer fragments to nitrocellulose membrane (referred to as blotting).4. Hybridize w radiolabeled probe to determine gene presence. DNA microarray assays = study many genes at the same time1. isolate mRNA2. make cDNA by reverse transcription, using fluorescently labeled nucleotides3. apply the cDNA mixture to a microarray, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray.4. Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot represents a gene expressed in the tissue sample. Cloning organismsNuclear transplantation = nucleus of egg is removed and replaced with nucleus of body cell1. body cell donor2. egg cell donor3. fuse cells4. grow cells in culture to early embryo stage5. inplant in uterus of 3rd animal, surrogate6. embryonic development results à dolly the sheepProblems: cloned embryos exhibit various defects. DNA of fully differentiated cell have epigenetic changes. Stem CellsStem cells = can reproduce itself indefinitely and produce other specialized cells-zygote – totipotent (any type of cell)-embryonic stem cells – pluripotent (many cell types)-adult stem cells – multipotent (a few cell types) or induced pluripotent, iPS (forced to be pluripotent) Can use stem cells for disease treatment-ex: stem cells to generate cell types for tissue repair Applications of DNA Technology1. diagnosis of disease – identify alleles, viral DNA2. gene therapy – alter afflicted genes3. production of pharmaceuticals – ex ‘pharm’ animas produce human protein secreted in milk for medical use4. forensic applications – DNA profiling, fingerprinting5. environmental cleanup – use microorganisms RFLPs – restriction fragment length polymorphisms-cut DNA with different restriction enzymes-each person has different #s of DNA fragments created-analyze DNA samples on a gel for disease diagnosis-outdated method of DNA profiling (required a quarter-sized sample of blood)-can be used in disease diagnosis STR Analysis-STR = short tandem repeats-noncoding DNA has regions with sequences (2-5 bases length) that are repeated-each person has different # of repeats at different locations (loci)-current method of DNA fingerprinting used (only need 20 cells for analysis)
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