© 2010 Pearson Education, Inc. Lectures by Chris C. Romero, updated by Edward J. Zalisko PowerPoint ® Lectures for Campbell Essential Biology, Fourth Edition – Eric Simon, Jane Reece, and Jean Dickey Campbell Essential Biology with Physiology, Third Edition – Eric Simon, Jane Reece, and Jean Dickey Chapter 12 DNA Technology

Biology and Society: DNA, Guilt, and Innocence DNA profiling is the analysis of DNA samples that can be used to determine whether the samples come from the same individual. DNA profiling can therefore be used in courts to indicate if someone is: –Guilty –Innocent © 2010 Pearson Education, Inc.

Figure 12.00

© 2010 Pearson Education, Inc. DNA technology has led to other advances in the: –Creation of genetically modified crops –Identification and treatment of genetic diseases

© 2010 Pearson Education, Inc. RECOMBINANT DNA TECHNOLOGY Biotechnology: –Is the manipulation of organisms or their components to make useful products –Has been used for thousands of years to –Make bread using yeast –Selectively breed livestock for desired traits

© 2010 Pearson Education, Inc. Biotechnology today means the use of DNA technology, methods for: –Studying and manipulating genetic material –Modifying specific genes –Moving genes between organisms

© 2010 Pearson Education, Inc. Recombinant DNA is formed when scientists combine nucleotide sequences (pieces of DNA) from two different sources to form a single DNA molecule. Recombinant DNA technology is widely used in genetic engineering, the direct manipulation of genes for practical purposes.

Figure 12.1

© 2010 Pearson Education, Inc. Applications: From Humulin to Foods to “Pharm” Animals By transferring the gene for a desired protein into a bacterium or yeast, proteins that are naturally present in only small amounts can be produced in large quantities.

© 2010 Pearson Education, Inc. Making Humulin In 1982, the world’s first genetically engineered pharmaceutical product was sold. Humulin, human insulin: –Was produced by genetically modified bacteria –Was the first recombinant DNA drug approved by the FDA –Is used today by more than 4 million people with diabetes

Figure 12.2

© 2010 Pearson Education, Inc. Today, humulin is continuously produced in gigantic fermentation vats filled with a liquid culture of bacteria.

Figure 12.3

© 2010 Pearson Education, Inc. DNA technology is used to produce medically valuable molecules, including: –Human growth hormone (HGH) –The hormone EPO, which stimulates production of red blood cells –Vaccines, harmless variants or derivatives of a pathogen used to prevent infectious diseases

© 2010 Pearson Education, Inc. Genetically Modified (GM) Foods Today, DNA technology is quickly replacing traditional plant- breeding programs. Scientists have produced many types of genetically modified (GM) organisms, organisms that have acquired one or more genes by artificial means. A transgenic organism contains a gene from another organism, typically of another species.

© 2010 Pearson Education, Inc. In the United States today, roughly one-half of the corn crop and over three-quarters of the soybean and cotton crops are genetically modified. Corn has been genetically modified to resist insect infestation, such as this damage caused by the European corn borer.

Figure 12.4

© 2010 Pearson Education, Inc. “Golden rice” has been genetically modified to produce beta- carotene used in our bodies to make vitamin A.

Figure 12.5

© 2010 Pearson Education, Inc. “Pharm” Animals In 2009 the FDA approved the first drug produced by livestock that has been engineered to carry a human gene. This product is a human anti-clotting protein collected from goats milk.

Figure 12.6

© 2010 Pearson Education, Inc. DNA technology: –May eventually replace traditional animal breeding but –Is not currently used to produce transgenic animals sold as food Meat may come from livestock that receive genes that produce: –Larger muscles or –Healthy omega-3 fatty acids instead of less healthy fatty acids (already done in 2006 in pigs)

© 2010 Pearson Education, Inc. Recombinant DNA Techniques Bacteria are the workhorses of modern biotechnology. To work with genes in the laboratory, biologists often use bacterial plasmids, small, circular DNA molecules that are separate from the much larger bacterial chromosome.

Plasmids Bacterial chromosome Remnant of bacterium Colorized TEM Figure 12.7

© 2010 Pearson Education, Inc. Plasmids: –Can easily incorporate foreign DNA –Are readily taken up by bacterial cells –Can act as vectors, DNA carriers that move genes from one cell to another –Are ideal for gene cloning, the production of multiple identical copies of a gene-carrying piece of DNA

© 2010 Pearson Education, Inc. Recombinant DNA techniques can help biologists produce large quantities of a desired protein. Blast Animation: Genetic Recombination in Bacteria Animation: Cloning a Gene

Plasmid Bacterial cell Isolate plasmids. Figure

Plasmid Bacterial cell Isolate plasmids. DNA Isolate DNA. Cell containing the gene of interest Figure

Plasmid Bacterial cell Isolate plasmids. DNA Isolate DNA. DNA fragments from cell Cut both DNAs with same enzyme. Gene of interest Other genes Cell containing the gene of interest Figure

Plasmid Bacterial cell Isolate plasmids. Gene of interest Recombinant DNA plasmids DNA Isolate DNA. DNA fragments from cell Cut both DNAs with same enzyme. Gene of interest Other genes Mix the DNAs and join them together. Cell containing the gene of interest Figure

Plasmid Bacterial cell Isolate plasmids. Recombinant bacteria Gene of interest Recombinant DNA plasmids Bacteria take up recombinant plasmids. DNA Isolate DNA. DNA fragments from cell Cut both DNAs with same enzyme. Gene of interest Other genes Mix the DNAs and join them together. Cell containing the gene of interest Figure

Plasmid Bacterial cell Isolate plasmids. Clone the bacteria. Recombinant bacteria Bacterial clone Gene of interest Recombinant DNA plasmids Bacteria take up recombinant plasmids. DNA Isolate DNA. DNA fragments from cell Cut both DNAs with same enzyme. Gene of interest Other genes Mix the DNAs and join them together. Cell containing the gene of interest Figure

Plasmid Bacterial cell Isolate plasmids. Find the clone with the gene of interest. Clone the bacteria. Recombinant bacteria Bacterial clone Gene of interest Recombinant DNA plasmids Bacteria take up recombinant plasmids. DNA Isolate DNA. DNA fragments from cell Cut both DNAs with same enzyme. Gene of interest Other genes Mix the DNAs and join them together. Cell containing the gene of interest Figure

Plasmid Bacterial cell Isolate plasmids. Some uses of genes Gene for pest resistance Gene for toxic-cleanup bacteria Genes may be inserted into other organisms. Find the clone with the gene of interest. The gene and protein of interest are isolated from the bacteria. Clone the bacteria. Recombinant bacteria Bacterial clone Gene of interest Recombinant DNA plasmids Bacteria take up recombinant plasmids. Harvested proteins may be used directly. Some uses of proteins Protein for “stone-washing” jeans DNA Cell containing the gene of interest Protein for dissolving clots Isolate DNA. DNA fragments from cell Cut both DNAs with same enzyme. Gene of interest Other genes Mix the DNAs and join them together. Figure

A Closer Look: Cutting and Pasting DNA with Restriction Enzymes Recombinant DNA is produced by combining two ingredients: –A bacterial plasmid –The gene of interest To combine these ingredients, a piece of DNA must be spliced into a plasmid. © 2010 Pearson Education, Inc.

This splicing process can be accomplished by: –Using restriction enzymes, which cut DNA at specific nucleotide sequences, and –Producing pieces of DNA called restriction fragments with “sticky ends” important for joining DNA from different sources DNA ligase connects the DNA pieces into continuous strands by forming bonds between adjacent nucleotides. Animation: Restriction Enzymes

Recognition sequence for a restriction enzyme Restriction enzyme Sticky end Sticky end DNA A restriction enzyme cuts the DNA into fragments. Figure

Recognition sequence for a restriction enzyme Restriction enzyme Sticky end Sticky end DNA A DNA fragment is added from another source. A restriction enzyme cuts the DNA into fragments. Figure

Recognition sequence for a restriction enzyme Restriction enzyme Sticky end Sticky end DNA A DNA fragment is added from another source. A restriction enzyme cuts the DNA into fragments. Fragments stick together by base pairing. Figure

Recognition sequence for a restriction enzyme Restriction enzyme Sticky end Sticky end DNA ligase Recombinant DNA molecule A DNA fragment is added from another source. A restriction enzyme cuts the DNA into fragments. Fragments stick together by base pairing. DNA ligase joins the fragments into strands. Figure

© 2010 Pearson Education, Inc. A Closer Look: Obtaining the Gene of Interest How can a researcher obtain DNA that encodes a particular gene of interest? –A “shotgun” approach yields millions of recombinant plasmids carrying many different segments of foreign DNA. –A collection of cloned DNA fragments that includes an organism’s entire genome (a complete set of its genes) is called a genomic library.

© 2010 Pearson Education, Inc. Once a genomic library is created, the bacterial clone containing the desired gene is identified using a specific sequence of radioactive nucleotides matching those in the desired gene, called a nucleic acid probe.

Radioactive probe (single-stranded DNA) Single-stranded DNA Mix with single-stranded DNA from various bacterial clones Base pairing indicates the gene of interest Figure 12.10

© 2010 Pearson Education, Inc. Another way to obtain a gene of interest is to: –Use reverse transcriptase and –Synthesize the gene by using an mRNA template

Cell nucleus DNA of eukaryotic gene Test tube Transcription Exon Intron Exon Intron Figure

Cell nucleus DNA of eukaryotic gene RNA transcript mRNA Test tube Transcription Introns removed and exons spliced together Exon Intron Exon Intron Figure

Cell nucleus DNA of eukaryotic gene RNA transcript mRNA Test tube Reverse transcriptase Transcription Introns removed and exons spliced together Isolation of mRNA from cell and addition of reverse transcriptase Exon Intron Exon Intron Figure

Cell nucleus DNA of eukaryotic gene RNA transcript mRNA Test tube Reverse transcriptase cDNA strand being synthesized Transcription Introns removed and exons spliced together Isolation of mRNA from cell and addition of reverse transcriptase Synthesis of cDNA strand Exon Intron Exon Intron Figure

Cell nucleus DNA of eukaryotic gene RNA transcript mRNA Test tube cDNA of gene without introns Reverse transcriptase cDNA strand being synthesized Transcription Introns removed and exons spliced together Isolation of mRNA from cell and addition of reverse transcriptase Synthesis of cDNA strand Synthesis of second DNA strand by DNA polymerase Exon Intron Exon Intron Figure

© 2010 Pearson Education, Inc. Another approach is to: –Use an automated DNA-synthesizing machine and –Synthesize a gene of interest from scratch

Figure 12.12

© 2010 Pearson Education, Inc. DNA PROFILING AND FORENSIC SCIENCE DNA profiling: –Can be used to determine if two samples of genetic material are from a particular individual –Has rapidly revolutionized the field of forensics, the scientific analysis of evidence from crime scenes To produce a DNA profile, scientists compare genetic markers, sequences in the genome that vary from person to person. Video: Biotechnology Lab

DNA isolated Crime scene Suspect 1Suspect 2 Figure

DNA isolated DNA amplified Crime scene Suspect 1Suspect 2 Figure

DNA isolated DNA amplified DNA compared Crime scene Suspect 1Suspect 2 Figure

© 2010 Pearson Education, Inc. Investigating Murder, Paternity, and Ancient DNA DNA profiling can be used to: –Test the guilt of suspected criminals –Identify tissue samples of victims –Resolve paternity cases –Identify contraband animal products –Trace the evolutionary history of organisms

Figure 12.14

Figure 12.14a

Figure 12.14b

DNA Profiling Techniques The Polymerase Chain Reaction (PCR) The polymerase chain reaction (PCR): –Is a technique to copy quickly and precisely any segment of DNA and –Can generate enough DNA, from even minute amounts of blood or other tissue, to allow DNA profiling © 2010 Pearson Education, Inc.

Initial DNA segment Number of DNA molecules Figure 12.15

© 2010 Pearson Education, Inc. Short Tandem Repeat (STR) Analysis How do you test if two samples of DNA come from the same person? Repetitive DNA: –Makes up much of the DNA that lies between genes in humans and –Consists of nucleotide sequences that are present in multiple copies in the genome

© 2010 Pearson Education, Inc. Short tandem repeats (STRs) are: –Short sequences of DNA –Repeated many times, tandemly (one after another), in the genome STR analysis: –Is a method of DNA profiling –Compares the lengths of STR sequences at certain sites in the genome Blast Animation: DNA Fingerprinting

Crime scene DNA Suspect’s DNA Same number of short tandem repeats Different numbers of short tandem repeats STR site 1STR site 2 AGAT GATA Figure 12.16

© 2010 Pearson Education, Inc. Gel Electrophoresis STR analysis: –Compares the lengths of DNA fragments –Uses gel electrophoresis, a method for sorting macromolecules—usually proteins or nucleic acids—primarily by their –Electrical charge –Size Blast Animation: Gel Electrophoresis

Mixture of DNA fragments of different sizes Power source Gel Figure

Mixture of DNA fragments of different sizes Power source Gel Figure

Mixture of DNA fragments of different sizes Power source Gel Completed gel Band of longest (slowest) fragments Band of shortest (fastest) fragments Figure

© 2010 Pearson Education, Inc. The DNA fragments are visualized as “bands” on the gel. The differences in the locations of the bands reflect the different lengths of the DNA fragments.

Amplified crime scene DNA Amplified suspect’s DNA Longer fragments Shorter fragments Figure 12.18

© 2010 Pearson Education, Inc. Gel electrophoresis may also be used for RFLP analysis, in which DNA molecules are exposed to a restriction enzyme, which produces fragments that are compared and made visible by gel electrophoresis. RFLP Analysis

Crime scene DNA Suspect’s DNA Fragment w Fragment x Fragment y Longer fragments Shorter fragments Fragment z Fragment y Crime scene DNA Suspect’s DNA Cut Restriction enzymes added x w y y z Figure 12.19

© 2010 Pearson Education, Inc. GENOMICS AND PROTEOMICS Genomics is the science of studying complete sets of genes (genomes). –The first targets of genomics were bacteria. –As of 2009, the genomes of nearly one thousand species have been published, including: –Baker’s yeast –Mice –Fruit flies –Rice

Table 12.1

© 2010 Pearson Education, Inc. The Human Genome Project Begun in 1990, the Human Genome Project was a massive scientific endeavor: –To determine the nucleotide sequence of all the DNA in the human genome and –To identify the location and sequence of every gene

© 2010 Pearson Education, Inc. At the completion of the project in 2004: –Over 99% of the genome had been determined to % accuracy –3.2 billion nucleotide pairs were identified –About 21,000 genes were found –About 98% of the human DNA was identified as noncoding

© 2010 Pearson Education, Inc. The Human Genome Project can help map the genes for specific diseases such as: –Alzheimer’s disease –Parkinson’s disease

Figure 12.20

© 2010 Pearson Education, Inc. Tracking the Anthrax Killer In October 2001: –A Florida man died after inhaling anthrax –By the end of the year, four other people had also died from anthrax In 2008, investigators: –Completed a whole-genome analysis of the spores used in the attack –Found four unique mutations –Traced the mutations to a single flask at an Army facility

Anthrax spore Envelope containing anthrax spores Figure 12.21

© 2010 Pearson Education, Inc. The anthrax investigation is just one example of the new field of comparative genomics, the comparison of whole genomes. Comparative genomics has also provided strong evidence that: –A Florida dentist transmitted HIV to several patients –The West Nile virus outbreak in 1999 was a single natural strain of virus infecting birds and humans –Our closest living relative, the chimpanzee (Pan troglodytes), shares 96% of our genome

© 2010 Pearson Education, Inc. Genome-Mapping Techniques Genomes are most often sequenced using the whole-genome shotgun method in which: –The entire genome is chopped into fragments using restriction enzymes –The fragments are cloned and sequenced –Computers running specialized mapping software reassemble the millions of overlapping short sequences into a single continuous sequence for every chromosome—an entire genome

Chromosome Figure

Chromosome Chop up with restriction enzyme DNA fragments Figure

Chromosome Chop up with restriction enzyme Sequence fragments DNA fragments Figure

Chromosome Chop up with restriction enzyme Sequence fragments DNA fragments Align fragments Figure

Chromosome Chop up with restriction enzyme Sequence fragments DNA fragments Align fragments Reassemble full sequence Figure

Figure 12.22a

© 2010 Pearson Education, Inc. Begun in 2006, the Human Variome Project: –Seeks to collect information on all of the genetic variations that affect human health

The Process of Science: Can Genomics Cure Cancer? Observation: A few patients responded quite dramatically to a new drug, gefitinib, which: –Targets a protein called EGFR found on the surface of cells that line the lungs –Is used to treat lung cancer Question: Are genetic differences among lung cancer patients responsible for the differences in gefitinib’s effectiveness? © 2010 Pearson Education, Inc.

Hypothesis: Mutations in the EGFR gene were causing the different responses to gefitinib. Prediction: DNA profiling that focuses on the EGFR gene would reveal different DNA sequences in the tumors of responsive patients compared with the tumors of unresponsive patients.

© 2010 Pearson Education, Inc. Experiment: The EGFR gene was sequenced in the cells extracted from the tumors of: –Five patients who responded to the drug –Four who did not Results: The results were quite striking. –All five tumors from gefitinib-responsive patients had mutations in EGFR. –None of the other four tumors did.

Figure 12.23

© 2010 Pearson Education, Inc. Proteomics Success in genomics has given rise to proteomics, the systematic study of the full set of proteins found in organisms. To understand the functioning of cells and organisms, scientists are studying: –When and where proteins are produced and –How they interact

© 2010 Pearson Education, Inc. HUMAN GENE THERAPY Human gene therapy: –Is a recombinant DNA procedure –Seeks to treat disease by altering the genes of the afflicted person –Often replaces or supplements the mutant version of a gene with a properly functioning one

Normal human gene isolated and cloned Healthy person Figure

Normal human gene isolated and cloned Normal human gene inserted into virus Healthy person Harmless virus (vector) Virus containing normal human gene Figure

Normal human gene isolated and cloned Normal human gene inserted into virus Virus injected into patient with abnormal gene Healthy person Harmless virus (vector) Virus containing normal human gene Bone marrow Bone of person with disease Figure

© 2010 Pearson Education, Inc. SCID is a fatal inherited disease caused by a single defective gene that prevents the development of the immune system. SCID patients quickly die unless treated with: –A bone marrow transplant or –Gene therapy

© 2010 Pearson Education, Inc. Since the year 2000, gene therapy has: –Cured 22 children with inborn SCID but –Unfortunately, caused four of the patients to develop leukemia, killing one of these children

© 2010 Pearson Education, Inc. SAFETY AND ETHICAL ISSUES As soon as scientists realized the power of DNA technology, they began to worry about potential dangers such as the: –Creation of hazardous new pathogens –Transfer of cancer genes into infectious bacteria and viruses

© 2010 Pearson Education, Inc. Strict laboratory safety procedures have been designed to: –Protect researchers from infection by engineered microbes –Prevent microbes from accidentally leaving the laboratory

Figure 12.25

© 2010 Pearson Education, Inc. The Controversy over Genetically Modified Foods GM strains account for a significant percentage of several agricultural crops in the United States.

Figure 12.26

© 2010 Pearson Education, Inc. Advocates of a cautious approach are concerned that: –Crops carrying genes from other species might harm the environment –GM foods could be hazardous to human health –Transgenic plants might pass their genes to close relatives in nearby wild areas

© 2010 Pearson Education, Inc. In 2000, negotiators from 130 countries (including the United States) agreed on a Biosafety Protocol that: –Requires exporters to identify GM organisms present in bulk food shipments

© 2010 Pearson Education, Inc. In the United States, all projects are evaluated for potential risks by a number of regulatory agencies, including the: –Food and Drug Administration –Environmental Protection Agency –National Institutes of Health –Department of Agriculture

© 2010 Pearson Education, Inc. Ethical Questions Raised by DNA Technology DNA technology raises legal and ethical questions—few of which have clear answers. –Should genetically engineered human growth hormone be used to stimulate growth in HGH-deficient children? –Do we have any right to alter an organism’s genes—or to create new organisms? –Should we try to eliminate genetic defects in our children and their descendants? –Should people use mail-in kits that can tell healthy people their relative risk of developing various diseases?

Figure 12.27

© 2010 Pearson Education, Inc. DNA technologies raise many complex issues that have no easy answers. We as a society and as individuals must become educated about DNA technologies to address the ethical questions raised by their use.

Evolution Connection: Profiling the Y Chromosome Barring mutations, the human Y chromosome passes essentially intact from father to son. By comparing Y DNA, researchers can learn about the ancestry of human males. © 2010 Pearson Education, Inc.

DNA profiling of the Y chromosome has revealed that: –Nearly 10% of Irish men were descendants of Niall of the Nine Hostages, a warlord who lived during the 5th century –The Lemba people of southern Africa are descended from ancient Jews –8% of males currently living in central Asia may be descended from Genghis Khan

Figure 12.28

DNA isolated from two sources and cut by same restriction enzyme Gene of interest (could be obtained from a library or synthesized) Recombinant DNA Plasmid (vector) Transgenic organisms Useful products Figure 12.UN1

Crime sceneSuspect 1Suspect 2 DNA Polymerase chain reaction (PCR) amplifies STR sites Longer DNA fragments Shorter DNA fragments DNA fragments compared by gel electrophoresis Gel Figure 12.UN2

Normal human gene Virus Bone marrow Normal human gene is transcribed and translated in patient, potentially curing genetic disease permanently Figure 12.UN3