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BIOLOGY CONCEPTS & CONNECTIONS Fourth Edition Neil A. Campbell • Jane B. Reece • Lawrence G. Mitchell • Martha R. Taylor CHAPTER 12 DNA Technology and the Human Genome Modules 12.1 – 12.6 From PowerPoint® Lectures for Biology: Concepts & Connections Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 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 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings • 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 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 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 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings • 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”) Figure 12.1B Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Figure 12.1C Recipient cell (“female”) • The transferred DNA is then integrated into the recipient cell’s chromosome Donated DNA Degraded DNA Crossovers Recipient cell’s chromosome Figure 12.1D Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Recombinant chromosome 12.2 Bacterial plasmids can serve as carriers for gene transfer • An F factor is a DNA segment in bacteria that enables conjugation and contains an origin of replication F factor (integrated) 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 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Recombination can occur F factor (plasmid) Male (donor) cell Bacterial chromosome F factor starts replication and transfer • An F factor can exist as a plasmid, a small circular DNA molecule separate from the bacterial chromosome Plasmids Plasmid completes transfer and circularizes Cell now male Figure 12.2B, C Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 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 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 1 Bacterium Plasmid isolated 2 DNA isolated 3 Gene Bacterial chromosome Cell containing gene of interest inserted into plasmid Plasmid Gene of interest Recombinant DNA (plasmid) 4 DNA Plasmid put into bacterial cell Recombinant bacterium 5 Cell multiplies with gene of interest Copies of gene Gene for pest resistance inserted into plants Copies of protein Clones of cell Gene used to alter bacteria for cleaning up toxic waste Protein used to make snow form at higher temperature Protein used to dissolve blood clots in heart attack therapy Figure 12.3 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 12.4 Enzymes are used to “cut and paste” DNA • Restriction enzymes cut DNA at specific points • DNA ligase “pastes” the DNA fragments together Restriction enzyme recognition sequence 1 DNA 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 • The result is recombinant DNA 4 DNA ligase pastes the strand 5 Figure 12.4 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 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 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings E. coli 1 Isolate DNA Human cell from two sources 2 Cut both Plasmid DNAs with the same restriction enzyme 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 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Figure 12.5 12.6 Cloned genes can be stored in genomic libraries • Recombinant DNA technology allows the construction of genomic libraries Genome cut up with restriction enzyme Recombinant plasmid OR – 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 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Bacterial clone Plasmid library Recombinant phage DNA Phage clone Phage library Figure 12.6 OTHER TOOLS OF DNA TECHNOLOGY 12.7 Reverse transcriptase helps make genes for cloning • Reverse transcriptase can be used to make smaller cDNA libraries – These contain only the genes that are transcribed by a particular type of cell Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings CELL NUCLEUS DNA of eukaryotic gene Exon Intron Exon Intron Exon 1 Transcription RNA transcript 2 RNA splicing (removes introns) mRNA 3 Isolation of mRNA TEST TUBE Reverse transcriptase from cell and addition of reverse transcriptase; synthesis of DNA strand 4 Breakdown of RNA cDNA strand 5 Synthesis of second DNA strand cDNA of gene (no introns) Figure 12.7 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 12.8 Nucleic acid probes identify clones carrying specific genes • A nucleic acid probe can tag a desired gene in a library Radioactive probe (DNA) Mix with singlestranded DNA from various bacterial (or phage) clones Single-stranded DNA Base pairing indicates the gene of interest Figure 12.8A Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings • DNA probes can identify a bacterial clone carrying a specific gene Bacterial colonies containing cloned segments of foreign DNA Radioactive DNA 1 Transfer Solution containing probe cells to filter Filter paper 2 Treat cells on filter to separate DNA strands 3 Add probe to filter Probe DNA Gene of interest Single-stranded DNA from cell Hydrogen-bonding 4 Autoradiography Colonies of living cells containing gene of interest Developed film 5 Compare autoradiograph with master plate Master plate Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Figure 12.8B 12.9 Connection: DNA microarrays test for the expression of many genes at once • A labeled probe can reveal patterns of gene expression in different kinds of cells • This technique may revolutionize the diagnosis and treatment of cancer cDNA DNA of gene DNA microarray, actual size (6,400 genes) Figure 12.9 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 12.10 Gel electrophoresis sorts DNA molecules by size • Restriction fragments of DNA can be sorted by size Mixture of DNA molecules of different sizes Longer molecules Power source Gel Shorter molecules Glass plates Completed gel Figure 12.10 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 12.11 Restriction fragment analysis is a powerful method that detects differences in DNA sequences • Scientists can compare DNA sequences of different individuals based on the size of the fragments Allele 1 Allele 2 w Cut z x Cut y Figure 12.11A Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Cut y DNA from chromosomes 1 2 Longer fragments Shorter fragments Figure 12.11B Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings • Radioactive probes are also used to make comparisons 1 Restriction fragment preparation Restriction fragments 2 Gel electrophoresis 3 Blotting 4 Radioactive probe Filter paper Radioactive, singlestranded DNA (probe) Probe 5 Detection of radioactivity (autoradiography) Film Figure 12.11C Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 12.12 The PCR method is used to amplify DNA sequences • The polymerase chain reaction (PCR) can quickly clone a small sample of DNA in a test tube Initial DNA segment 1 2 4 Number of DNA molecules Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 8 Figure 12.12 THE CHALLENGE OF THE HUMAN GENOME 12.13 Most of the human genome does not consist of genes • The 23 chromosomes in the haploid human genome contain about 3 billion nucleotide pairs – This DNA is believed to include about 35,000 genes and a huge amount of noncoding DNA Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings • Much of the noncoding DNA consists of repetitive nucleotide sequences – One example includes telomeres at the end of the chromosomes Repeated unit End of DNA molecule NUCLEOTIDE SEQUENCE OF A HUMAN TELOMERE Figure 12.13A Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings • Barbara McClintock discovered that segments of DNA called transposons can move about within a cell’s genome Figure 12.13B, C Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 12.14 Connection: The Human Genome Project is unlocking the secrets of our genes • The Human Genome Project involves: – genetic and physical mapping of chromosomes – DNA sequencing – comparison of human genes with those of other species Figure 12.14 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings OTHER APPLICATIONS OF DNA TECHNOLOGY 12.15 Connection: DNA technology is used in courts of law • DNA fingerprinting can help solve crimes Defendant’s blood Blood from defendant’s clothes Victim’s blood Figure 12.15A, B Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 12.16 Connection: Recombinant cells and organisms can mass-produce gene products • Recombinant cells and organisms are used to manufacture useful proteins Table 12.16 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings • These sheep carry a gene for a human blood protein that is a potential treatment for cystic fibrosis Figure 12.16 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 12.17 Connection: DNA technology is changing the pharmaceutical industry and medicine • Hormones, cancer-fighting drugs, and new vaccines are being produced using DNA technology – This lab equipment is used to produce a vaccine against hepatitis B Figure 12.17 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 12.18 Connection: Genetically modified organisms are transforming agriculture • New genetic varieties of animals and plants are being produced – A plant with a new trait can be created using the Ti plasmid Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Agrobacterium tumefaciens DNA containing gene for desired trait 1 Ti plasmid T DNA Insertion of gene into plasmid using restriction enzyme and DNA ligase Plant cell 2 Recombinant Ti plasmid Restriction site Introduction into plant cells in culture 3 Regeneration of plant T DNA carrying new gene within plant chromosome Plant with new trait Figure 12.18A Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings • “Golden rice” has been genetically modified to contain beta-carotene – This rice could help prevent vitamin A deficiency Figure 12.18B Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 12.19 Connection: Gene therapy may someday help treat a variety of diseases • Techniques for manipulating DNA have potential for treating disease by altering an afflicted individual’s genes Cloned gene (normal allele) 1 Insert normal gene into virus Viral nucleic acid Retrovirus 2 Infect bone marrow cell with virus – Progress is slow, however 3 Viral DNA inserts into chromosome – There are also ethical questions related to gene therapy Bone marrow cell from patient Bone marrow 4 Inject cells Figure 12.19 Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings into patient RISKS AND ETHICAL QUESTIONS 12.20 Connection: Could GM organisms harm human health or the environment? • Genetic engineering involves some risks – Possible ecological damage from pollen transfer between GM and wild crops – Pollen from a transgenic variety of corn that contains a pesticide may stunt or kill monarch caterpillars Figure 12.20A, B Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings 12.21 Connection: DNA technology raises important ethical questions • Our new genetic knowledge will affect our lives in many ways • The deciphering of the human genome, in particular, raises profound ethical issues – Many scientists have counseled that we must use the information wisely Figure 12.21A-C Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings