CHAPTER 12 DNA Technology and the Human Genome

1. CHAPTER 12DNA Technology and the Human Genome Modules 12.1 – 12.6

2. From E.Coli to a Map of Our Genes • Research on E. coli revealed that these bacteria have a sexual mechanism that can bring about the combining of genes from two different cells • This discovery led to the development of recombinant DNA technology • a set of techniques for combining genes from different sources

3. The Human Genome Project • The production of vaccines, cancer drugs, and pesticides • Engineered bacteria that can clean up toxic wastes • DNA technology has many useful applications

4. BACTERIA AS TOOLS FOR MANIPULATING DNA 12.1 In nature, bacteria can transfer DNA in three ways • Transformation, the taking up of DNA from the fluid surrounding the cell DNA enterscell Fragment ofDNA from anotherbacterial cell Bacterial chromosome(DNA) Figure 12.1A

5. Conjugation, the union of cells and the DNA transfer between them • Transduction, the transfer of bacterial genes by a phage Mating bridge Phage Fragment ofDNA from anotherbacterial cell(former phagehost) Sex pili Donor cell(“male”) Recipient cell(“female”) Figure 12.1B Figure 12.1C

6. The transferred DNA is then integrated into the recipient cell’s chromosome Donated DNA Degraded DNA Crossovers Recipient cell’schromosome Recombinantchromosome Figure 12.1D

7. 12.2 Bacterial plasmids can serve as carriers for gene transfer F factor (integrated) Male (donor) cell Origin of F replication • An F factor is a DNA segment in bacteria that enables conjugation and contains an origin of replication Bacterial chromosome F factor startsreplication andtransfer of chromosome Recipient cell Only part of thechromosome transfers Figure 12.2A Recombination can occur

8. F factor (plasmid) Male (donor) cell Bacterial chromosome • An F factor can exist as a plasmid, a small circular DNA molecule separate from the bacterial chromosome F factor startsreplication andtransfer Plasmids Plasmid completestransfer andcircularizes Cell now male Figure 12.2B, C

9. 12.3 Plasmids are used to customize bacteria: An overview • Plasmids are key tools for DNA technology • Researchers use plasmids to insert genes into bacteria

10. Cell containing geneof interest 1 Bacterium Plasmidisolated 2 DNA isolated 3 Gene inserted into plasmid Bacterialchromosome Plasmid Gene ofinterest Recombinant DNA(plasmid) DNA 4 Plasmid put intobacterial cell Recombinantbacterium 5 Cell multiplies withgene of interest Copies of gene Copies of protein Gene for pestresistanceinserted intoplants Clones of cell Protein used to make snow format highertemperature Gene used to alter bacteriafor cleaning up toxic waste Protein used to dissolve bloodclots in heart attack therapy • Figure 12.3

11. 12.4 Enzymes are used to “cut and paste” DNA Restriction enzymerecognition sequence 1 DNA Restriction enzymecuts the DNA intofragments Restriction enzymecuts the DNA intofragments • Restriction enzymes cut DNA at specific points • DNA ligase “pastes” the DNA fragments together • The result is recombinant DNA 2 Sticky end Addition of a DNAfragment fromanother source 3 Two (or more)fragments sticktogether bybase-pairing 4 DNA ligasepastes the strand 5 Figure 12.4 Recombinant DNA molecule

12. 12.5 Genes can be cloned in recombinant plasmids: A closer look • Bacteria take the recombinant plasmids and reproduce • This clones the plasmids and the genes they carry • Products of the gene can then be harvested

13. 1 Isolate DNAfrom two sources Human cell E. coli 2 Cut both DNAs with the same restriction enzyme Plasmid DNA Gene V Sticky ends 3 Mix the DNAs; they joinby base-pairing 4 Add DNA ligaseto bond the DNA covalently Recombinant DNAplasmid Gene V 5 Put plasmid into bacteriumby transformation 6 Clone the bacterium Bacterial clone carrying manycopies of the human gene Figure 12.5

14. 12.6 Cloned genes can be stored in genomic libraries Genome cut up with restriction enzyme Recombinantplasmid • Recombinant DNA technology allows the construction of genomic libraries • Genomic libraries are sets of DNA fragments containing all of an organism’s genes • Copies of DNA fragments can be stored in a cloned bacterial plasmid or phage Recombinantphage DNA OR Phage clone Bacterialclone Plasmid library Phage library Figure 12.6