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Chapter 12 DNA Technology and Genomics PowerPoint Lectures for Biology: Concepts and Connections, Fifth Edition – Campbell, Reece, Taylor, and Simon Lectures by Chris Romero Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings DNA and Crime Scene Investigations • Many violent crimes go unsolved – lack of enough evidence • If biological fluids are left at a crime scene – DNA can be isolated from them Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • DNA fingerprinting: set of laboratory procedures – determines w/near certainty whether 2 samples of DNA are from same individual – powerful tool for CSIs Investigator at one of the crime scenes (above), Narborough, England (left) Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings BACTERIAL PLASMIDS AND GENE CLONING 12.1 Plasmids are used to customize bacteria: An overview • Gene cloning is 1 application • DNA technology – Methods for studying & manipulating genetic material Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Researchers can insert desired genes into plasmids, creating recombinant DNA – And insert those plasmids into bacteria Figure 12.1 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Bacterium 1 Plasmid isolated Cell containing gene of interest 2 DNA isolated 3 Bacterial Plasmid chromosome Gene inserted into plasmid DNA Recombinant DNA (plasmid) Gene of interest 4 Plasmid put into bacterial cell Recombinant bacterium Gene for pest Resistance inserted into plants 5 Cell multiplies with gene of interest Copies of gene Copies of protein Clone of cells Protein used to make snow form at higher temperature Figure 12.1 Gene used to alter bacteria for cleaning up toxic waste Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Protein used to dissolve blood clots in heart attack therapy • If the recombinant bacteria multiply into a clone – The foreign genes are also copied Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.2 Enzymes are used to “cut and paste” DNA • The tools used to make recombinant DNA are – Restriction enzymes, which cut DNA at specific sequences – DNA ligase, which “pastes” DNA fragments together Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Creating recombinant DNA using REs & DNA ligase 1 GAATTC CTTAAG DNA Restriction enzyme recognition sequence 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 G A AT T C C T TA A G G A AT T C C T TA A G 4 DNA ligase pastes the strand 5 Figure 12.2 Recombinant DNA molecule Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.3 Genes can be cloned in recombinant plasmids: A closer look • Bacteria take the recombinant plasmids from their surroundings – reproduce – cloning the plasmids and the genes they carry Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Cloning a gene in a bacterial plasmid 1 Isolate DNA from two sources E.coli Human cell DNA Plasmid Gene V Sticky ends 3 Mix the DNAs; they join by base-pairing 2 Cut both DNAs with the same restriction enzyme 4 Add DNA ligase to bond the DNA covalently Gene V Recombinant DNA plasmid 5 Put plasmid into bacterium by transformation Recombinant bacterium 6 Clone the bacterium Figure 12.3 Bacterial clone carrying many copies of the human gene Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings CONNECTION 12.6 Recombinant cells and organisms can massproduce gene products • Applications of gene cloning include – medical and other uses Table 12.6 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Different organisms, including bacteria, yeast, and mammals – Can be used for this purpose Figure 12.6 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings CONNECTION 12.7 DNA technology is changing the pharmaceutical industry • widely used to produce medicines and to diagnose diseases Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Therapeutic hormones • In 1982, humulin, human insulin produced by bacteria – 1st recombinant drug approved by FDA Figure 12.7A Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Diagnosis and Treatment of Disease • DNA technology – Is being used increasingly in disease diagnosis Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Vaccines • DNA technology – Is also helping medical researchers develop vaccines Figure 12.7B Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings pGLO LAB Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings RESTRICTION FRAGMENT ANALYSIS AND DNA FINGERPRINTING 12.10 Gel electrophoresis sorts DNA molecules by size Mixture of DNA molecules of different sizes – – Longer molecules Power source Gel + Shorter molecules + Figure 12.10 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Completed gel 12.11 Restriction fragment length polymorphisms can be used to detect differences in DNA sequences Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings How Restriction Fragments Reflect DNA Sequence • Restriction fragment length polymorphisms (RFLPs) – Reflect differences in the sequences of DNA samples Crime scene Suspect w Cut C C G G G G C C z A C G G T G C C C C G G G G C C x Cut y Figure 12.11A Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings C C G G G G C C Cut y DNA from chromosomes • After digestion by restriction enzymes – fragments are run through a gel 1 – 2 Longer fragments z x w Shorter fragments Figure 12.11B y + Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings y Using DNA Probes to Detect Harmful Alleles • Radioactive probes – Can reveal DNA bands of interest on a gel Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Detecting a harmful allele using restriction fragment analysis 1 Restriction fragment preparation I II III Restriction fragments 2 Gel electrophoresis I II III 3 Blotting Filter paper 4 Radioactive probe Radioactive, singlestranded DNA (probe) Probe 5 Detection of radioactivity (autoradiography) I II III Film Figure 12.11C Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings I II III CONNECTION 12.12 DNA technology is used in courts of law Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • DNA fingerprinting can help solve crimes Defendant’s blood Blood from defendant’s clothes Victim’s blood Figure 12.12A Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Figure 12.12B CONNECTION 12.13 Gene therapy may someday help treat a variety of diseases • Gene therapy – Is the alteration of an afflicted individual’s genes Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Gene Therapy Cloned gene 1 Insert normal gene (normal allele) into virus Retrovirus Viral nucleic acid 2 Infect bone marrow cell with virus 3 Viral DNA inserts into chromosome Bone marrow cell from patient Figure 12.13 4 Inject cells into patient Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Bone marrow • Gene therapy – May one day be used to treat both genetic diseases and nongenetic disorders • Unfortunately, progress is slow Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.14 The PCR method is used to amplify DNA sequences • The polymerase chain reaction (PCR) – Can be used to clone a small sample of DNA quickly, producing enough copies for analysis Initial DNA segment 1 Figure 12.14 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 2 4 8 Number of DNA molecules GENOMICS CONNECTION 12.15 The Human Genome Project is an ambitious application of DNA technology • The Human Genome Project, begun in 1990 and now largely completed – Genetic and physical mapping of chromosomes, followed by DNA sequencing Figure 12.15 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • The data are providing insight into – Development, evolution, and many diseases Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.16 Most of the human genome does not consist of genes • The haploid human genome contains about 25,000 genes – And a huge amount of noncoding DNA Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Much of the noncoding DNA consists of repetitive nucleotide sequences – And transposons that can move about within the genome Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings CONNECTION 12.17 The science of genomics compares whole genomes • The sequencing of many prokaryotic and eukaryotic genomes Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Besides being interesting themselves... – Nonhuman genomes can be compared with the human genome Table 12.17 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Proteomics – Is the study of the full sets of proteins produced by organisms Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings GENETICALLY MODIFIED ORGANISMS: CONNECTION 12.18 Genetically modified organisms are transforming agriculture Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Recombinant DNA technology – Can be used to produce new genetic varieties of plants and animals, genetically modified (GM) organisms Agrobacterium tumefaciens DNA containing gene for desired trait 1 Ti plasmid T DNA Plant cell Recombinant Ti plasmid Insertion of gene into plasmid using restriction enzyme and DNA ligase Restriction site 2 Introduction Regeneration into plant of plant cells in culture T DNA carrying new Plant with new trait gene within plant chromosome Figure 12.18A Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 3 • Transgenic organisms – have had genes from other organisms inserted into their genomes Figure 12.18B Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • A number of important crops and plants – Are genetically modified Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings CONNECTION 12.19 Could GM organisms harm human health or the environment? • Development of GM organisms – Requires significant safety measures Figure 12.19A Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • Genetic engineering involves risks – Such as ecological damage from GM crops Figure 12.19B Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings CONNECTION 12.20 Genomics researcher Eric Lander discusses the Human Genome Project • Genomics pioneer Eric Lander – Points out that much remains to be learned from the Human Genome Project Figure 12.20 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.17 The science of genomics compares whole genomes • The sequencing of many prokaryotic and eukaryotic genomes – Has produced data for genomics, the study of whole genomes Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.4 Cloned genes can be stored in genomic libraries • Genomic libraries, sets of DNA fragments containing all of an organism’s genes – Can be constructed and stored in cloned bacterial plasmids or phages Genome cut up with restriction enzyme Recombinant plasmid Recombinant phage DNA or Bacterial clone Figure 12.4 Plasmid library Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Phage clone Phage library 12.5 Reverse transcriptase helps make genes for cloning • Reverse transcriptase can be used to make smaller, complementary DNA (cDNA) libraries – Containing only the genes that are transcribed by a particular type of cell Cell nucleus Exon Intron DNA of eukaryotic gene Exon Intron Exon 1 Transcription RNA transcript 2 RNA splicing (removes introns) mRNA Test tube Reverse transcriptase 3 Isolation of mRNA from cell and addition of reverse transcriptase; synthesis of DNA strand cDNA strand 4 Breakdown of RNA 5 Synthesis of second DNA strand Figure 12.5 cDNA of gene (no introns) Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings RESTRICTION FRAGMENT ANALYSIS AND DNA FINGERPRINTING 12.8 Nucleic acid probes identify clones carrying specific genes • DNA technology methods – Can be used to identify specific pieces of DNA Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • A nucleic acid probe – Is a short, single-stranded molecule of radioactively labeled or fluorescently labeled DNA or RNA – Can tag a desired gene in a library Radioactive probe (DNA) Mix with singlestranded DNA from various bacterial (or phage) clones Single-stranded DNA Figure 12.8 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Base pairing indicates the gene of interest CONNECTION 12.9 DNA microarrays test for the expression of many genes at once • DNA microarray assays – Can reveal patterns of gene expression in different kinds of cells Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings • DNA microarray DNA microarray Each well contains DNA from a particular gene 1 mRNA isolated Reverse transcriptase and fluorescent DNA nucleotides 2 cDNA made from mRNA Actual size (6,400 genes) 4 Unbound cDNA rinsed away Fluorescent spot 3 cDNA applied to wells Nonfluorescent spot cDNA DNA of an expressed gene Figure 12.9 Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings DNA of an unexpressed gene