Presentation on theme: "Ch. 20 Biotechnology Objective:"— Presentation transcript:
1Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly used technologies.
2Understanding and Manipulating Genomes Sequenced the human genome in 2003 through:Biotechnology: manipulation of organismsGenetic engineering: manipulation of genesRecombinant DNA: 2 DNAs combined
3Using Bacteria as Tools Circular DNAPlasmidExtra genetic materialSmall, circular DNANot necessary, but usually beneficial
4Using Bacteria as Tools Bacterial TransformationUptake of DNA from the fluid surrounding the cellCauses genetic recombinationAllow insertion of gene of interest
520.1: DNA (Gene) CloningUses: make many copies (amplify) quickly and produces proteinsBasic Method:Use bacterial plasmids (cloning vector).Insert desired gene (recombinant DNA).Return plasmid to bacteria.Bacteria reproduce.Various applications.
6Making Recombinant DNA Restriction enzymes (nucleases) cut DNA in specific places (restriction site) to form restriction fragments.Must use same enzyme on plasmid and desired geneForms sticky ends: unbonded nucleotidesAdd DNA ligase to rebond recombinant DNA.
7Cloning a Eukaryotic Gene in a Bacterial Plasmid In gene cloning, the original plasmid is called a cloning vectorA cloning vector is a DNA molecule that can carry foreign DNA into a cell and replicate there
8Cloning a Eukaryotic Gene in a Bacterial Plasmid Only a cell that took up a plasmid, which has the ampR gene, will reproduce and form a colony.Colonies with nonrecombinant plasmids will be blue, because they can hydrolyze X-gal.Colonies with recombinant plasmids, in which lacZ is disrupted, will be white, because they cannot hydrolyze X-gal.By screening the white colonies with a nucleic acid probe (see Figure 20.5), researchers can identify clones of bacterial cells carrying the gene of interest.
9Storing Cloned GenesGenomic Library: complete set of plasmid clones saved.Phages are also used so they are saved as phage library.A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insertComplementary DNA (cDNA) can be made by reverse transcription of mRNA to make a cDNA library.
10ID Clone Carrying Gene of Interest Nucleic acid probe (RNA or DNA) radioactively labeled which hybridizes to gene.
11Eukaryotic Genes in Bacterial Expression Systems To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active prokaryotic promoterTo overcome inability to remove introns, use cDNA form of the gene
12Eukaryotic Cloning and Expression Systems The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problemsYACs behave normally in mitosis and can carry more DNA than a plasmidEukaryotic hosts can provide the posttranslational modifications that many proteins require
13Amplifying DNA: Polymerase Chain Reaction 1 DNA strand → billions in hours.Denature: Heat DNA to break H-bondsAnnealing: Add primers and coolExtension: Add heat resistant DNA polymerase and nucleotidesRepeat using thermocycler
1420.2 Restriction Fragment Analysis Gel ElectrophoresisDNA is – charge; attracted to +Gel that separates DNA by length; smaller pieces can travel faster/further.Make fragments by restriction enzymes and separate them.Alleles have different sequences of DNA so are cut differently.
15Southern BlottingA technique called Southern blotting combines gel electrophoresis with nucleic acid hybridizationSpecific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel
16Restriction Fragment Length Polymorphisms (RFLPs) Restriction fragments made using the same enzyme on homologues.Used as a marker (fingerprint) for individuals.Paternity Test
17DNA SequencingRelatively short DNA fragments can be sequenced by the dideoxy chain-termination methodInclusion of special dideoxyribonucleotides in the reaction mix ensures that fragments of various lengths will be synthesized
20How to ID Unknown Genes Compare to known genes of other organisms. Disable the gene and observe the consequence.In vitro interference: use copies DNA gene, introduce mutagen, reinsert into cell, observe consequence.
21Studying Expression of Interacting Groups of Genes DNA Microarray AssaysTake mRNAMake cDNA (single strand)Fluorescently labelApply to array chip (contains known DNA fragments the cDNA will bond to)Look for fluorescence.
22Determining Gene Function One way to determine function is to disable the gene and observe the consequences (knock-outs)Using in vitro mutagenesis, mutations are introduced into a cloned gene, altering or destroying its functionWhen the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotypeA transgenic mouse with an active rat growth hormone gene (left). This transgenic mouse is twice the size of a normal mouse (right).
23Comparing Genomes of Different Species Allows us to look for evolutionary relationships.Comparative data on simple organisms helps us understand more complex ones.Closely related species: figure out one and use as a template for the others.
24Future Directions Proteomics: study proteins encoded by genomes. Single Nucleotide Polymorphisms (SNPs): single base-pair differences from one human to another.People are 99.99% identical on genetic level.
2520.3 Cloning In Plants: Totipotent: cells can dedifferentiate. Tranplanting a clipping or root causing a clone to be made.
26Cloning In Animals Remove nucleus from egg Add nucleus from somatic cell of donorGrow in cultureImplant in uterusClone is born!
27CC, the first cloned catAlthough CC is a clone of her mother, they are not identical due to the X-inactivation mechanism and different environmental influencesFigure CC, the first cloned cat, and her single parent.Figure 20.20
28Stem Cells of AnimalsGoal of cloning human embryos → stem cell productionStem cell = undifferentiated cellEmbryonic stem cells have the potential to become anything (pluripotent).Adult stem cells can’t.
29Regenerative Medicine? Human ear grown in a lab from stem cells.Human pluripotent stem cells crucial for the development of regenerative medicineCan allow for growing a whole new heart or liver, since they can be converted into any cell type in the body
3020.4 Applications of Genetic Engineering Medical Applications:Identifying genes that cause disease/disordersGene therapy: changing disease causing genes in humans.
3120.4 Applications of Genetic Engineering Pharmaceutical ProductsInsulinHuman growth hormoneTissue plasminogen activator to dissolve blood clotsHIV blockersVaccines
32Environmental Cleanup Forensic EvidenceDNA fingerprinting using gel electrophoresisEnvironmental CleanupMining bacteria (copper, lead, nickel, etc)Cleaning toxic wasterClean oil spills
33Agricultural Applications Animal Husbandry and “Pharm” animalsTransgenic animals (has recombinant DNA) to make better wool, leaner meat, shorter maturation time, pharmaceutical factories for blood clotting factors.Genetic Engineering in PlantsDelayed ripening, resistance to spoilage/disease, increase nutritional value.Uses Ti plasmid recombined with desired genes.
34Transgenic AnimalsHuman gene for antithrombin inserted into a goat’s genome and the protein is produced in the milk
35Genetic Engineering in Plants Agricultural scientists have endowed a number of crop plants with genes for desirable traitsThe Ti plasmid is the most commonly used vector for introducing new genes into plant cells
36Transgenic PlantsBt transgenic corn is normal corn that contains a gene from the soil bacterium Bacillus thuringiensis. Gene allows production of a toxic protein that can kill many types of caterpillars(http://www.ces.ncsu.edu/plymouth/pubs/btcorn99.html)Flavr Savr Tomato. 1st engineered food in stores. Engineered to remain firm even as it turns red and ripe.
37Safety and EthicsPotential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or proceduresMost public concern about possible hazards centers on genetically modified (GM) organisms used as food