Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings PowerPoint Lectures for Biology, Seventh Edition Neil Campbell and Jane Reece Lectures by Chris Romero Chapter 20 DNA Technology and Genomics

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Restriction site DNA Restriction enzyme cuts the sugar-phosphate backbones at each arrow. One possible combination DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. Fragment from different DNA molecule cut by the same restriction enzyme DNA ligase seals the strands. Recombinant DNA molecule Sticky end Using Restriction Enzymes to Make Recombinant DNA Bacterial restriction enzymes cut DNA molecules at DNA sequences called restriction sites – fragments with “sticky ends” Animation: Restriction Enzymes Animation: Restriction Enzymes

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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.

LE 20-4_1 Isolate plasmid DNA and human DNA. Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Bacterial cell lacZ gene (lactose breakdown) Human cell Restriction site amp R gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Recombinant DNA plasmids

LE 20-4_2 Isolate plasmid DNA and human DNA. Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Bacterial cell lacZ gene (lactose breakdown) Human cell Restriction site amp R gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria

LE 20-4_3 Isolate plasmid DNA and human DNA. Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Bacterial cell lacZ gene (lactose breakdown) Human cell Restriction site amp R gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments 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 non- recombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene Bacterial clone

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA Genomic DNA Target sequence Primers Denaturation: Heat briefly to separate DNA strands Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence Extension: DNA polymerase adds nucleotides to the 3 end of each primer Cycle 1 yields 2 molecules New nucleo- tides 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 Cathode Power source Anode Mixture of DNA molecules of differ- ent sizes Gel Glass plates Longer molecules Shorter molecules Gel Electrophoresis and Southern Blotting Gel electrophoresis - technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size Video: Biotechnology Lab Video: Biotechnology Lab

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene Normal  -globin allele 175 bp201 bpLarge fragment Sickle-cell mutant  -globin allele 376 bpLarge fragment Ddel Ddel restriction sites in normal and sickle-cell alleles of  -globin gene Normal allele Sickle-cell allele Large fragment 376 bp 201 bp 175 bp Electrophoresis of restriction fragments from normal and sickle-cell alleles

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Forensic Evidence DNA “fingerprints” obtained by analysis of tissue or body fluids can provide evidence in criminal and paternity cases 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

LE Cloned gene Retrovirus capsid Bone marrow cell from patient Inject engineered cells into patient. Insert RNA version of normal allele into retrovirus. Viral RNA 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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 Agricultural scientists have endowed a number of crop plants with genes for desirable traits Agrobacterium tumefaciens Ti plasmid Site where restriction enzyme cuts DNA with the gene of interest T DNA Recombinant Ti plasmid Plant with new trait