2Biotechnology Terms Biotechnology Genetic Engineering Recombinant DNA Process of manipulating organisms or their components to make useful productsGenetic engineering + tissue/cell culturing technologiesGenetic EngineeringManipulation of individual genes or entire genomesInsulin (insulin E. coli bacteria OR yeast) & GMO (Genetically Modified Organism)Recombinant DNAArtificially created DNATypically, DNA is integrated from another species
3Biotechnology Terms (Page 2) Gene CloningLaboratory production of multiple copies of DNA segmentTherapeutic cloning – embryonic stem cellsSpinal cord injuriesReproductive (organismal) cloning – Dolly the sheepRestriction EnzymesEnzymes that cut DNA at specific locationsUsually, derived from bacteriaCut sites of DNA = restriction fragmentsSticky ends – restriction fragments usually have one end longer than the other
6Quick Assignment Relate the 6 terms just discussed in a concept map. Be prepared to defend your arrangement
7Cloning Process 5 steps (first 2) 1. Identify & isolate the gene of interestInvolves finding a cloning vector – plasmid or organism used to carry the DNA sequence to be cloned2. Cut gene of interest from original site & open up vector’s DNA using a ________ ________This ensures matching sticky ends on gene of interest & vector DNA
8Cloning Process (Page 2) 5 steps (3-4)3. Combine the 2 DNA pieces (into a recombinant plasmid?)Recombinant plasmid – plasmid + DNA fragmentsSealed together using DNA LigaseRemember: we used ________ ________ to cut gene of interest from original site & cut vector’s DNAThis ensures matching sticky ends on gene of interest & vector DNA4. Transfer the vector (recombinant plasmid) into a host cellUsually involves bacterial transformation
9Bacteria & Genetic Recombination ConjugationBacterial SexGenetic material is exchanged by direct contactTransductionPhage transfer of DNAInvolves a phage vectorPhage moves the DNA from bacterium to other bacterium
10Bacteria & Genetic Recombination TransformationUptake of exogenous DNAGriffith’s experiment - pathogenic DNA was transferred to benign bacteriaMost common method for genetic engineering
11Step 5 Select for transformed cells Link the gene of interest with a reporter geneSuch as pBLU or pGLOpBLU = Blue colorationpGLO = fluorescent green under UV lightIn Lab 6, we will insert the coloration gene and an ampicillin resistance gene to select for transformed cells
13At this point… You know which cells have the gene of interest You can identify the cells that have the gene of interestNow what?You need to extract the gene of interestHow would you do that?
14Nucleic Acid Hybridization Detects the gene of interestUses a short, single stranded DNA or RNA called a nucleic acid probeThe nucleic acid probe is complementary to a known sequence in the gene of interestUsually attach a radioactive isotope or fluorescent tag protein so that it is detectable
15Genomic LibrariesNucleic Acid Hybridization repeated many times produces a genomic libraryThousands of recombinant clonesEach has a piece of the original genome being studied
16cDNA Library cDNA = complementary DNA mRNA is extracted from cells Use what enzyme to make DNA from this mRNA?Then make another strand of DNA using what enzyme?cDNA library is only a portion of the genomePortion that codes for mRNAExons? Introns? tRNA? rRNA?
17Microarray Assay Genome-wide study of gene expression Different genes are in each wellIdentifies gene interactions + provides clues to gene functionsTake samples throughout development + assay to determine which genes are expressed and at what stagesDetect patterns of expression throughout developmentDetect likely response to a pathogenic agent
24DNA Technology affects us… Disease DiagnosisPCR used to detect traces of viral DNA or RNA in sampleRFLP (Restriction Fragment Length Polymorphisms)Different alleles have different RFLPsGene Therapy – alter afflicted genesPharmaceutical Production – Insulin productionForensic Application – DNA fingerprints
25Page 2 Environmental cleanup Agricultural applications Genetically engineered microbesDetoxification of specific wastesAgricultural applicationsInsert pest-resistant or drought-resistant genesGMO (Genetically Modified Organisms)You eat GMO corn, soybeans, canola and cottonseed oilProbably at least weekly46% of GMOs are grown in USEurope had 12 year moratorium on growing GE foods