Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings PowerPoint Lectures for Biology: Concepts and Connections, Fifth Edition – Campbell, Reece, Taylor, and Simon Lectures by Chris Romero Chapter 12 DNA Technology and Genomics

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 one application of DNA technology –Methods for studying and 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 Bacterium Bacterial chromosome Plasmid 1 isolated 3 Gene inserted into plasmid 2 DNA isolated Cell containing gene of interest DNA Gene of interest Recombinant DNA (plasmid) 4 Plasmid put into bacterial cell Recombinant bacterium 5 Cell multiplies with gene of interest Copies of protein Copies of gene Clone of cells Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein used to dissolve blood clots in heart attack therapy Protein used to make snow form at higher temperature Figure 12.1

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 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 restriction enzymes and DNA ligase Restriction enzyme recognition sequence G A A T T C C T T A A G DNA C T T A A A AT TC G C T T A A Addition of a DNA fragment from another source 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 5 DNA ligase pastes the strand Restriction enzyme cuts the DNA into fragments Recombinant DNA molecule G G Sticky end G Figure 12.2

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 –And reproduce, thereby 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 2 Cut both DNAs with the same restriction enzyme E.coli Plasmid DNA Gene V Sticky ends 3Mix the DNAs; they join by base-pairing 4 Add DNA ligase to bond the DNA covalently 5 Put plasmid into bacterium by transformation Gene V Recombinant DNA plasmid Recombinant bacterium 6 Clone the bacterium Bacterial clone carrying many copies of the human gene Human cell Figure 12.3

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings CONNECTION 12.6 Recombinant cells and organisms can mass- produce gene products Applications of gene cloning include –The mass production of gene products for 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 12.7 DNA technology is changing the pharmaceutical industry DNA technology –Is widely used to produce medicines and to diagnose diseases CONNECTION

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Therapeutic hormones In 1982, humulin, human insulin produced by bacteria –Became the first recombinant drug approved by the Food and Drug Administration 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 12.8 Nucleic acid probes identify clones carrying specific genes DNA technology methods –Can be used to identify specific pieces of DNA RESTRICTION FRAGMENT ANALYSIS AND DNA FINGERPRINTING

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) Single-stranded DNA Mix with single- stranded DNA from various bacterial (or phage) clones Base pairing indicates the gene of interest A T C C G A A T G C G C T T A T C G A G C C T T A T G C A T A T C C G A A G G T A G G C T A A Figure 12.8

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 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 CONNECTION

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings DNA microarray 1 mRNA isolated Reverse transcriptase and fluorescent DNA nucleotides 2 cDNA made from mRNA 4Unbound cDNA rinsed away 3 cDNA applied to wells DNA microarray Each well contains DNA from a particular gene Actual size (6,400 genes) Nonfluorescent spot Fluorescent spot cDNA DNA of an expressed gene DNA of an unexpressed gene Figure 12.9

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Gel electrophoresis sorts DNA molecules by size ++ – – Power source Gel Mixture of DNA molecules of different sizes Longer molecules Shorter molecules Completed gel Figure 12.10

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 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 sceneSuspect w x yy z Cut DNA from chromosomes C C G G G G C C A C G G T G C C C C G G G G C C C C G G G G C C Figure 12.11A

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings After digestion by restriction enzymes –The fragments are run through a gel – + Longer fragments Shorter fragments x w y z y 12 Figure 12.11B

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 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 Restriction fragment preparation Gel electrophoresis Blotting Radioactive probe Detection of radioactivity (autoradiography) IIIIII IIIIII Restriction fragments Filter paper Probe Radioactive, single- stranded DNA (probe) Film I II III I II III Figure 12.11C

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings DNA technology is used in courts of law CONNECTION

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 Figure 12.12B

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 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 1248 Initial DNA segment Number of DNA molecules Figure 12.14

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Figure GENOMICS CONNECTION The Human Genome Project is an ambitious application of DNA technology The Human Genome Project, begun in 1990 and now largely completed, involved –Genetic and physical mapping of chromosomes, followed by DNA sequencing

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 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 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 CONNECTION

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 are transforming agriculture GENETICALLY MODIFIED ORGANISMS CONNECTION

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 Ti plasmid 1 Insertion of gene into plasmid using restriction enzyme and DNA ligase Recombinant Ti plasmid 2 Introduction into plant cells in culture 3 Regeneration of plant Plant with new trait T DNA carrying new gene within plant chromosome Plant cell T DNA Restriction site Figure 12.18A

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Transgenic organisms –Are those that 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 Could GM organisms harm human health or the environment? Development of GM organisms –Requires significant safety measures CONNECTION 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