Chapter 20

1. Chapter 20 DNA Technology and Genomics

2. Using Restriction Enzymes to Make Recombinant DNA Restriction site 5¢ 3¢ DNA 3¢ 5¢ • Bacterial restriction enzymes cut DNA molecules at DNA sequences called restriction sites • fragments with “sticky ends” Restriction enzyme cuts the sugar-phosphate backbones at each arrow. Sticky end 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 One possible combination DNA ligase seals the strands. Animation: Restriction Enzymes Recombinant DNA molecule

3. 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.

4. LE 20-4_1 Bacterial cell lacZ gene (lactose breakdown) Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments 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. Recombinant DNA plasmids

5. LE 20-4_2 Bacterial cell lacZ gene (lactose breakdown) Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments 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. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria

6. LE 20-4_3 lacZ gene (lactose breakdown) Bacterial cell Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments 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. 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 recombinant plasmid with disrupted lacZ gene Colony carrying non- recombinant plasmid with intact lacZ gene Bacterial clone

7. Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) 5¢ 3¢ Target sequence Genomic DNA 3¢ 5¢ • The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA 5¢ 3¢ Denaturation: Heat briefly to separate DNA strands 3¢ 5¢ Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence Cycle 1 yields 2 molecules Primers Extension: DNA polymerase adds nucleotides to the 3¢ end of each primer New nucleo- tides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

8. Gel Electrophoresis and Southern Blotting Mixture of DNA molecules of differ- ent sizes Longer molecules • Gel electrophoresis - technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size Cathode Shorter molecules Power source Gel Glass plates Anode Video: Biotechnology Lab

9. Normal b-globin allele 175 bp 201 bp Large fragment • Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene Ddel Ddel Ddel Ddel Sickle-cell mutant b-globin allele 376 bp Large fragment Ddel Ddel 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

10. Forensic Evidence Defendant’s blood (D) Blood from defendant’s clothes Victim’s blood (V) • DNA “fingerprints” obtained by analysis of tissue or body fluids can provide evidence in criminal and paternity cases

12. Concept 20.5: The practical applications of DNA technology affect our lives in many ways • Many fields benefit from DNA technology and genetic engineering

13. 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

14. LE 20-16 Cloned gene 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. Retrovirus capsid Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient Bone marrow Inject engineered cells into patient.

15. Pharmaceutical Products • Some pharmaceutical applications of DNA technology: • Large-scale production of human hormones and other proteins with therapeutic uses • Production of safer vaccines

16. Environmental Cleanup • Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials

17. Genetic Engineering in Plants Agrobacterium tumefaciens Ti plasmid • Agricultural scientists have endowed a number of crop plants with genes for desirable traits Site where restriction enzyme cuts T DNA DNA with the gene of interest Recombinant Ti plasmid Plant with new trait