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Chapter 20 DNA Technology and Genomics PowerPoint Lectures for Biology, Seventh Edition Neil Campbell and Jane Reece Lectures by Chris Romero Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Using Restriction Enzymes to Make Recombinant DNA Restriction site • Bacterial restriction enzymes DNA 53 cut DNA molecules at DNA Restriction enzyme cuts sugar-phosphate sequences called restriction the backbones at each arrow. sites – fragments with “sticky ends” 3 5 Sticky end DNA fragment from another source is added. Base pairing Fragment from different of sticky ends produces DNA molecule cut by the various combinations. same restriction enzyme One possible combination DNA ligase seals the strands. Animation: Restriction Enzymes Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Recombinant DNA molecule 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 be replicated. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 20-4_1 Bacterial cell Isolate plasmid DNA and human DNA. lacZ gene (lactose breakdown) Human cell Restriction site ampR gene (ampicillin resistance) Cut both DNA samples with the same restriction enzyme. Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids LE 20-4_2 Bacterial cell Isolate plasmid DNA and human DNA. lacZ gene (lactose breakdown) Human cell Restriction site ampR gene (ampicillin resistance) Cut both DNA samples with the same restriction enzyme. Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria LE 20-4_3 Bacterial cell Isolate plasmid DNA and human DNA. lacZ gene (lactose breakdown) Human cell Restriction site ampR gene (ampicillin resistance) Cut both DNA samples with the same restriction enzyme. Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria Plate the bacteria on agar containing ampicillin and X-gal. Incubate until colonies grow. Colony carrying nonrecombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene Bacterial clone Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) 5 3 Target sequence • The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA Genomic DNA Denaturation: Heat briefly to separate DNA strands Cycle 1 yields 2 molecules Extension: DNA polymerase adds nucleotides to the 3 end of each primer Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence 3 5 5 3 3 5 Primers New nucleotides Gel Electrophoresis and Southern Blotting • Gel electrophoresis - technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size Cathode Power source Mixture of DNA molecules of different sizes Shorter molecules Gel Glass plates Anode Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Video: Biotechnology Lab Longer molecules Normal b-globin allele • Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene 175 bp Ddel 201 bp Ddel Large fragment Ddel Ddel Sickle-cell mutant b-globin allele 376 bp Ddel Large fragment Ddel Ddel Ddel restriction sites in normal and sickle-cell alleles of b-globin gene Normal allele Sickle-cell allele Large fragment 376 bp 201 bp 175 bp Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Electrophoresis of restriction fragments from normal and sickle-cell alleles Forensic Evidence • DNA “fingerprints” obtained by analysis of tissue or body fluids can provide evidence in criminal and paternity cases Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Defendant’s blood (D) Blood from defendant’s clothes Victim’s blood (V) Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Concept 20.5: The practical applications of DNA technology affect our lives in many ways • Many fields benefit from DNA technology and genetic engineering Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Human Gene Therapy • Gene therapy is the alteration of an afflicted individual’s genes • Gene therapy holds great potential for treating disorders traceable to a single defective gene • Vectors are used for delivery of genes into cells Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 20-16 Cloned gene Insert RNA version of normal allele into retrovirus. Viral RNA Retrovirus capsid Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient Inject engineered cells into patient. Bone marrow Pharmaceutical Products • Some pharmaceutical applications of DNA technology: – Large-scale production of human hormones and other proteins with therapeutic uses – Production of safer vaccines Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Environmental Cleanup • Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Genetic Engineering in Plants Agrobacterium tumefaciens • Agricultural scientists have endowed a number of crop plants with genes for desirable traits Ti plasmid Site where restriction enzyme cuts T DNA DNA with the gene of interest Recombinant Ti plasmid Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Plant with new trait