Presentation on theme: "Ch. 20 Biotechnology Objective: LO 3.5 The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly."— Presentation transcript:
Ch. 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.
Understanding and Manipulating Genomes Sequenced the human genome in 2003 through: – Biotechnology: manipulation of organisms Genetic engineering: manipulation of genes – Recombinant DNA: 2 DNAs combined
Using Bacteria as Tools Bacteria – Circular DNA – Plasmid Extra genetic material Small, circular DNA Not necessary, but usually beneficial ogyPages/R/RecombinantPlasmid.gif
Using Bacteria as Tools Bacterial Transformation – Uptake of DNA from the fluid surrounding the cell – Causes genetic recombination – Allow insertion of gene of interest ures/lecture%203/transformation.jpg
20.1: DNA (Gene) Cloning Uses: make many copies (amplify) quickly and produces proteins Basic Method: 1.Use bacterial plasmids (cloning vector). 2.Insert desired gene (recombinant DNA). 3.Return plasmid to bacteria. 4.Bacteria reproduce. 5.Various applications.
Making Recombinant DNA Restriction enzymes (nucleases) cut DNA in specific places (restriction site) to form restriction fragments. – Must use same enzyme on plasmid and desired gene – Forms sticky ends: unbonded nucleotides – Add DNA ligase to rebond recombinant DNA.
Cloning a Eukaryotic Gene in a Bacterial Plasmid In gene cloning, the original plasmid is called a cloning vector A cloning vector is a DNA molecule that can carry foreign DNA into a cell and replicate there
Cloning a Eukaryotic Gene in a Bacterial Plasmid Only a cell that took up a plasmid, which has the amp R 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.
Storing Cloned Genes Genomic 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 insert Complementary DNA (cDNA) can be made by reverse transcription of mRNA to make a cDNA library.
ID Clone Carrying Gene of Interest Nucleic acid probe (RNA or DNA) radioactively labeled which hybridizes to gene.
Eukaryotic 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 promoter To overcome inability to remove introns, use cDNA form of the gene
Eukaryotic Cloning and Expression Systems The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems YACs behave normally in mitosis and can carry more DNA than a plasmid Eukaryotic hosts can provide the posttranslational modifications that many proteins require
Amplifying DNA: Polymerase Chain Reaction 1 DNA strand → billions in hours. 1.Denature: Heat DNA to break H-bonds 2.Annealing: Add primers and cool 3.Extension: Add heat resistant DNA polymerase and nucleotides 4.Repeat using thermocycler
20.2 Restriction Fragment Analysis Gel Electrophoresis DNA 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.
Southern Blotting A technique called Southern blotting combines gel electrophoresis with nucleic acid hybridization Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel
Restriction Fragment Length Polymorphisms (RFLPs) Restriction fragments made using the same enzyme on homologues. Used as a marker (fingerprint) for individuals. Paternity Test
DNA Sequencing Relatively short DNA fragments can be sequenced by the dideoxy chain-termination method Inclusion of special dideoxyribonucleotides in the reaction mix ensures that fragments of various lengths will be synthesized
How 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.
Studying Expression of Interacting Groups of Genes DNA Microarray Assays – Take mRNA – Make cDNA (single strand) – Fluorescently label – Apply to array chip (contains known DNA fragments the cDNA will bond to) – Look for fluorescence.
Determining 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 function When the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotype A transgenic mouse with an active rat growth hormone gene (left). This transgenic mouse is twice the size of a normal mouse (right).
Comparing 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.
Future 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.
20.3 Cloning In Plants: – Totipotent: cells can dedifferentiate. – Tranplanting a clipping or root causing a clone to be made.
Cloning In Animals – Remove nucleus from egg – Add nucleus from somatic cell of donor – Grow in culture – Implant in uterus – Clone is born!
CC, the first cloned cat Although CC is a clone of her mother, they are not identical due to the X-inactivation mechanism and different environmental influences Figure 20.20
Stem Cells of Animals Goal of cloning human embryos → stem cell production Stem cell = undifferentiated cell Embryonic stem cells have the potential to become anything (pluripotent). Adult stem cells can’t.
Regenerative Medicine? Human pluripotent stem cells crucial for the development of regenerative medicine Can allow for growing a whole new heart or liver, since they can be converted into any cell type in the body Human ear grown in a lab from stem cells. mutate-in-time/
20.4 Applications of Genetic Engineering Medical Applications: – Identifying genes that cause disease/disorders – Gene therapy: changing disease causing genes in humans.
20.4 Applications of Genetic Engineering Pharmaceutical Products – Insulin – Human growth hormone – Tissue plasminogen activator to dissolve blood clots – HIV blockers – Vaccines
Forensic Evidence – DNA fingerprinting using gel electrophoresis Environmental Cleanup – Mining bacteria (copper, lead, nickel, etc) – Cleaning toxic waster – Clean oil spills
Agricultural Applications – Animal Husbandry and “Pharm” animals Transgenic animals (has recombinant DNA) to make better wool, leaner meat, shorter maturation time, pharmaceutical factories for blood clotting factors. – Genetic Engineering in Plants Delayed ripening, resistance to spoilage/disease, increase nutritional value. Uses Ti plasmid recombined with desired genes.
Transgenic Animals Human gene for antithrombin inserted into a goat’s genome and the protein is produced in the milk 12.html
Genetic Engineering in Plants Agricultural scientists have endowed a number of crop plants with genes for desirable traits The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
Transgenic Plants Flavr Savr Tomato. 1 st engineered food in stores. Engineered to remain firm even as it turns red and ripe. Bt 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)
Safety and Ethics – Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures – Most public concern about possible hazards centers on genetically modified (GM) organisms used as food