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

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

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

12.1 In nature, bacteria can transfer DNA in three ways 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 enters cell Fragment of DNA from another bacterial cell Bacterial chromosome (DNA) Figure 12.1A

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

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

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

F factor (plasmid) An F factor can exist as a plasmid, a small circular DNA molecule separate from the bacterial chromosome Male (donor) cell Bacterial chromosome F factor starts replication and transfer Plasmids Plasmid completes transfer and circularizes Cell now male Figure 12.2B, C

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

1 2 3 4 5 Cell containing gene of interest Bacterium Plasmid isolated DNA isolated 3 Gene inserted into plasmid Bacterial chromosome Plasmid Gene of interest Recombinant DNA (plasmid) DNA 4 Plasmid put into bacterial cell Recombinant bacterium 5 Cell multiplies with gene of interest Copies of gene Copies of protein Gene for pest resistance inserted into plants Clones of cell Protein used to make snow form at higher temperature Gene used to alter bacteria for cleaning up toxic waste Protein used to dissolve blood clots in heart attack therapy Figure 12.3

12.4 Enzymes are used to “cut and paste” DNA Restriction enzyme recognition sequence Restriction enzymes cut DNA at specific points DNA ligase “pastes” the DNA fragments together The result is recombinant DNA 1 DNA Restriction enzyme cuts the DNA into fragments Restriction enzyme cuts the DNA into fragments 2 Sticky end Addition of a DNA fragment from another source 3 Two (or more) fragments stick together by base-pairing 4 DNA ligase pastes the strand 5 Figure 12.4 Recombinant DNA molecule

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

Bacterial clone carrying many copies of the human gene 1 Isolate DNA from 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 join by base-pairing 4 Add DNA ligase to bond the DNA covalently Recombinant DNA plasmid Gene V 5 Put plasmid into bacterium by transformation 6 Clone the bacterium Bacterial clone carrying many copies of the human gene Figure 12.5

12.6 Cloned genes can be stored in genomic libraries 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 Genome cut up with restriction enzyme Recombinant plasmid Recombinant phage DNA OR Phage clone Bacterial clone Plasmid library Phage library Figure 12.6