Presentation on theme: "DNA Technology and Genomics"— Presentation transcript:
1 DNA Technology and Genomics Chapter 20DNA Technology and Genomics
2 DNA TechnologyGenetic Engineering – direct manipulation of genes for practical purposesScientists can make recombinant DNA and then introduce it into cultured cells that replicate the DNA and may express its genes, yielding a desired protein.Often, E. coli is used as the “host”Biotechnology – manipulations of organisms to make useful products.Recombinant DNA – genes from two different sources are combined in vitro (outside the living body in a lab).
4 Techniques for Gene Cloning Plasmid – circular DNA that replicates within bacterial cells (separate from bacterial chromosomes)Isolation of plasmidGene insertion into plasmidPlasmid put into bacterial cellCell clonedIdentification of desired cloneCopies of the gene/Copies of the proteinUsed in gene therapy (HGH) and agriculture (resistance to pests)
5 Restriction EnzymesRestriction enzymes cut up foreign DNA – recognize short nucleotide sequencesRestriction site – area recognized by restriction enzyme (5’ – 3’)Restriction fragments – nucleotide sequence left after DNA has been cut.Sticky end – single-stranded DNA fragment; DNA ligase can permanently fuse fragments together
10 DNA Cloning Cloning Vector – the original bacterial plasmid Procedure: Cloning a Eukaryotic Gene in a Bacterial PlasmidThe E. coli plasmid has two useful genes: ampR (resistant to ampicillian) and lacZ (catalyses hydrolysis of lactose)Isolation of vector (bacterial plasmid) and gene-source DNA (human tissue)
11 DNA Cloning Insertion of DNA into the vector Restriction enzyme cuts plasmid DNA at single restriction siteCuts human DNA making thousands of fragmentsOne of these fragments carries the gene we wantThe restriction enzyme creates compatible sticky ends on both the human DNA fragments and the plasmid DNAThe DNA and plasmids are combined – DNA ligase “glues” the pieces together
12 DNA Cloning Introduction of cloning vector into cells Cloning of cells Transformation – uptake of naked DNA from surrounding solutionSome acquire the desired DNA; others take up other DNACloning of cellsPut bacterial cells on a plate containing medium with ampicillin and X-galBacteria with recombinant plasmids carrying foreign DNA with form white colonies on medium containing ampicillin and X-gal
13 DNA CloningIdentification of cell colonies carrying the gene of interestNucleic acid hybridization – base pairing between the gene and a complementary sequence using a nucleic acid probeThe probe can be labeled with a radioactive isotope or a fluorescent tagThen the strand of DNA is separated by denaturing it with heat or chemicalsThe desired DNA can then be isolated an grown in large amounts
14 Cloned Genes Are Stored in DNA Libraries A complete set of thousands of recombinant plasmid clonesCan be stored as a plasmid library (bacterial cells) or phage libraries (viruses)
15 Polymerase Chain Reaction (PCR) Clones DNA In Vitro Any piece of DNA can be quickly copied many times without using a host cellDNA is placed in a test tube with:Special DNA polymerases (first isolated from bacteria growing in hot springs – the enzyme does not denature with heat)Supply of nucleotidesSynthetic single-stranded DNA that serves as primersCan be used to amplify a specific gene prior to cloning
16 PCR can be used to quickly amplify DNA from a 40,000 year old woolly mammoth; tiny amounts of blood, tissue, or semen from a crime scene; and DNA of viral genes from HIV-infected cells
17 DNA Analysis and Genomics Suppose we have cloned a DNA segment carrying a human gene of interest – now we begin to ask some far-reaching questions:Do genes differ in different people, and are certain alleles associated with a hereditary disorder?Where and when is it expressed?What is its location within the genome?Evolutionary questions - Species to species differentiation?
18 DNA Analysis and Genomics Genomics – the study of whole sets of genes and their interactionsGel Electrophoresis – method of sorting DNA molecules into bands, each consisting of molecules of the same lengthSeparation on the basis of size, electrical charge, and other physical propertiesRestriction fragments – detect DNA differences that affect restriction sitesGel Electrophoresis used to sort DNA fragments by size after being treated with restriction enzymes
19 Gel ElectrophoresisDigest two different DNA samples with same restriction enzyme (the samples should differ in one or more restriction site)Use nucleic acid hybridization with a specific probe to label discrete bands that derive from our gene of interest (usually used in combination with Southern blotting)
22 Gel ElectrophoresisThe DNA fingerprints below represent four different individuals.Which of the following statements is consistent with the results?B is the child of A and C.C is the child of A and BWhich of the following are probably siblings?A and DC and D
23 Entire Genomes Can Be Mapped at the DNA Level Human Genome Project – begun in 1990 to map all the human genesSurprisingly, there are few genes in the Human Genome
24 Practical Application of DNA Technology Diagnosis of DiseasesPCR can be used to amplify and detect HIV DNA in blood or tissue samplesScientists can diagnose hundreds of human genetic disorders before the onset of symptoms, even before birthWe can identify symptomless carriers of potentially harmful recessive allelesGenes have been cloned for many human diseases, including hemophilia, cystic fibrosis, and Duchenne muscular dystrophy
25 Practical Application of DNA Technology Human Gene Therapy – the alteration of an afflicted individual’s genesTheoretically, it is possible to replace or supplement the defective gene with a normal alleleThe new allele could be inserted into the somatic cells of the tissue affected by the disorderThe cells that receive the normal allele must be ones that will continue to reproduce during the patient’s life – one example is stem cells from bone marrow that give rise to all blood and immune cells
26 Practical Application of DNA Technology Use of stem cells to promote healthy bone marrow
27 Practical Application of DNA Technology Pharmaceutical ProductsMostly proteinsHuman insulinHuman growth hormone (HGH)Tissue plasminogen activator (TPA) – protein that helps dissolve blood clots (very expensive)Drugs that mimic a receptor protein that HIV binds to in entering white blood cells – the HIV binds to the drugs and fails to enter the blood cellVaccines
28 Practical Application of DNA Technology Reproductive CloningProducing complete, genetically identical animalsUsually talked about in the pressDOLLY the SHEEP.
29 Practical Application of DNA Technology Forensic UsesDNA fingerprintingEnvironment UsesSewage treatment plants rely on the ability of microbes (bacteria and protists, mainly) to degrade many organic compounds into nontoxic formsAgricultural UsesAnimal Husbandry and “Pharm” AnimalsTransgenic organisms – their genomes carry genes from another specie
30 Practical Application of DNA Technology Agricultural Uses (continued)Genetic engineering in plants (editable cotton seed – 2006)Delayed ripeningResistance to spoilage and diseaseIncrease nutritional value (“golden rice”)
31 WITH EVOLUTION IN THIS WAY? SO THE BIG QUESTION IS:SHOULD WE INTERFEREWITH EVOLUTION IN THIS WAY?