Chapter 12: DNA Technology PDF

CHAPTER CONTENTS CHAPTER THREAD Genetic Engineering 252 DNA Profiling DNA Profiling and Forensic Science 259...

CHAPTER CONTENTS CHAPTER THREAD Genetic Engineering 252 DNA Profiling DNA Profiling and Forensic Science 259 BIOLOGY AND SOCIETY Using DNA to Establish Guilt Bioinformatics 263 and Innocence 251 Safety and Ethical Issues 268 THE PROCESS OF SCIENCE Can Genomics Cure Cancer? 266 EVOLUTION CONNECTION The Y Chromosome as a Window on History 271 DNA Profiling BIOLOGY AND SOCIETY Using DNA to Establish Guilt and Innocence It was a horrific crime in the heart of the nation’s capital: On February 24, 1981, a man broke into an apart- ment, just miles from the White House, and attacked a 27-year-old woman. After binding and raping her, the perpetrator stole traveler’s checks and ran off. The victim had only glimpses of her attacker. Several weeks later, a police officer thought that 18-year-old Kirk Odom resembled a sketch of the perpetrator. A month later, the victim picked Odom out of a police lineup. At trial, a Special Agent of the FBI testified that hair found in the victim’s clothing, when viewed under a microscope, was “indistinguishable” from Odom’s hair. Despite Odom having an alibi for his whereabouts on the night of the crime, after just a few hours of deliberation the jury convicted Odom of several charges, including rape while armed. He was sentenced to 20 to 66 years in prison. Odom served more than 20 years before being released on lifelong parole as a registered sex offender. However, in February 2011, prompted by recent discoveries that indicated that the FBI’s microscopic hair analysis technique was flawed—and subsequent overturned convictions in other cases—a motion was filed to reopen the Odom case for DNA testing. Modern forensic DNA analysis hinges on a simple fact: The cells of every person (except identical twins) contain unique DNA. DNA profiling is the anal- ysis of DNA samples to determine whether they come from the same individual. In the Odom case, the government located sheets, clothing, and hair evidence from the 1981 crime scene. In the more recent analysis, the results were con- clusive and irrefutable: The DNA left at the crime scene did not match Odom’s. In fact, the DNA matched a different man, a convicted sex offender (who was never charged because the statute of limitations on the crime had expired). On July 13, 2012—Odom’s 50th birthday—the court acknowledged that the original hair analysis data was erroneous and that Kirk Odom had “suffered a terrible injustice.” ­After 30 years, Odom was officially declared innocent by the court. A DNA profile. Even minuscule bits of evidence A steady stream of stories such as this one demonstrates that DNA tech- can provide a DNA profile. nology can provide evidence of either guilt or innocence. Beyond the court- room, DNA technology has led to some of the most remarkable scientific advances in recent years: Crop plants have been genetically modified to produce their own insecticides; human genes are being compared with those of other animals to help shed light on what makes us distinctly human; and significant advances have been made toward detecting and curing fatal genetic diseases. This chapter will describe these and other uses of DNA technology and explain how various DNA techniques are performed. We’ll also examine some of the social, legal, and ethical issues that lie at the intersection of biology and society. 251 M12_SIMO2368_05_GE_CH12.indd 251 25/09/15 10:23 AM  Genetic Engineering Chapter 12 DNA Technology You may think of biotechnology, the manipulation of mass-produce a variety of useful chemicals, from ­ organisms or their components to make useful prod- cancer drugs to pesticides. Scientists have also ucts, as a modern phenomenon, but it actually dates transferred genes from bacteria to plants and from back to the dawn of civilization. Consider such ancient one ­animal species to another (Figure 12.1). Such practices as using yeast to make bread and beer and ­engineering can serve a variety of purposes, from the selective breeding of livestock. But when people use basic research (What does this gene do?) to ­medical the term biotechnology today, they are usually referring ­applications (Can we create animal models for this to DNA technology, modern laboratory techniques for ­human ­disease?). studying and manipulating genetic material. Using the methods of DNA technology, scientists can modify spe- cific genes and move them between organisms as dif- Recombinant DNA ferent as bacteria, plants, and animals. Organisms that have acquired one or more genes by artificial means are Techniques called genetically modified (GM) organisms. If the Although genetic engineering can be performed on newly acquired gene is from another organism, typi- a variety of organisms, bacteria (Escherichia coli, cally of another ­species, the recombinant organism is in ­particular) are the workhorses of modern bio- called a transgenic organism. technology. To manipulate genes in the laboratory, In the 1970s, the field of biotechnology exploded ­biologists often use bacterial plasmids, which are small, CHECKPOINT with the invention of methods for making recombi- circular DNA molecules that duplicate separately from nant DNA in the laboratory. Scientists can construct the larger bacterial chromosome (­Figure 12.2). Because What is biotechnology? What is recombinant DNA? ­recombinant DNA by combining pieces of DNA from plasmids can carry virtually any gene and are passed sources, often different species two different sources—often from different species— from one generation of bacteria to the next, they are ­containing DNA from two different to form a single DNA molecule. Recombinant DNA key tools for gene cloning, the production of multiple technology is widely used in genetic engineering, the identical copies of a gene-carrying piece of DNA. Gene a useful product; a molecule ­organisms or their parts to ­produce Answer: the manipulation of direct manipulation of genes for practical purposes. cloning methods are central to the ­production of useful Scientists have genetically engineered bacteria to products from genetically engineered organisms. ▼ Figure 12.1 Genetic engineers produced glowing fish by transferring a gene for a fluorescent protein originally obtained from jellies (“jellyfish”). Plasmids Bacterial Colorized TEM 2,700× chromosome Remnant of bacterium ▲ Figure 12.2 Bacterial plasmids. The micrograph shows a bacterial cell that has been ruptured, revealing one long chro- mosome and several smaller ­plasmids. The inset is an enlarged view of a single plasmid. 252 M12_SIMO2368_05_GE_CH12.indd 252 25/09/15 10:23 AM  How to Clone a Gene recombinant DNA plasmids. The recombinant Genetic Engineering Consider a typical genetic engineering challenge: plasmids are then mixed with bacteria. Under the A genetic engineer at a pharmaceutical company identi- right conditions, the bacteria take up the recombinant fies a gene of interest that codes for a valuable protein, plasmids. Each bacterium, carrying its recombi- such as a potential new drug. The biologist wants to nant plasmid, is allowed to reproduce via cell division manufacture the protein on a large scale. Figure 12.3 to form a clone, a group of identical cells descended ­illustrates a way to accomplish this by using recombi- from a single original cell. As the bacteria multiply, nant DNA techniques. the foreign gene carried by the recombinant plasmid is To start, the biologist isolates two kinds of DNA: also copied. The transgenic bacteria with the gene CHECKPOINT bacterial plasmids that will serve as vectors (gene of interest can then be grown in large tanks, producing Why are plasmids valuable ­carriers, shown in blue in the figure) and DNA from the protein in marketable quantities. The end products tools for the production of another organism that includes the gene of interest of gene cloning may be copies of the gene itself, to be recombinant DNA? (shown in yellow). This foreign DNA may be from used in additional genetic engineering projects, or the by their bacterial host cells. any foreign gene and are replicated any type of organism, even a human. The DNA protein product of the cloned gene, to be harvested Answer: Plasmids can carry virtually from the two sources is joined together, resulting in and used. ◀ Figure 12.3 Using ­ recombinant DNA Cell containing gene Bacterium of interest technology to produce useful products. 1 Gene inserted into plasmid Bacterial Plasmid chromosome Gene of interest DNA of Recombinant chromosome DNA (plasmid) (”foreign” DNA) 2 Plasmid put into bacterial cell Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the cloned gene of interest A gene for pest A protein is resistance is used to dissolve inserted into blood clots in plants. heart attack therapy. Some uses Some uses of genes of proteins 4 The gene and protein A gene is used to Genes may be of interest Bacteria produce A protein is used to alter bacteria for inserted into are isolated proteins, which prepare ”stone-washed“ cleaning up toxic other organisms. from the can be harvested blue jeans. waste. bacteria. and used directly. 253 M12_SIMO2368_05_GE_CH12.indd 253 25/09/15 10:23 AM  Chapter 12 Cutting and Pasting DNA specific points within the sequence, like a pair of highly DNA Technology specific molecular scissors. with Restriction Enzymes As shown in Figure 12.3, recombinant DNA is created The top of ­Figure 12.4 shows a piece of DNA (blue) by combining two ingredients: a bacterial plasmid and that contains one ­restriction site for a particular re- the gene of interest. To understand how these DNA striction enzyme. The restriction enzyme cuts the molecules are spliced together, you need to learn how DNA strands b ­ etween the bases A and G within the enzymes cut and paste DNA. recognition sequence, producing pieces of DNA called The cutting tools used for making recombinant DNA ­restriction fragments. The staggered cuts yield two are bacterial enzymes called restriction enzymes. Biolo- double-stranded DNA fragments with single-stranded gists have identified hundreds of restriction enzymes, ends, called “sticky ends.” Sticky ends are the key to each recognizing a particular short DNA sequence, joining DNA restriction fragments originating from usually four to eight nucleotides long. For example, one ­different sources. Next, a piece of DNA from another restriction enzyme only recognizes the DNA sequence source ­(yellow) is added. N ­ otice that the yellow DNA CHECKPOINT GAATTC, whereas another recognizes GGATCC. The has single-stranded ends ­identical in base sequence If you mix a restriction DNA sequence recognized by a particular restriction to the sticky ends on the blue DNA because the same enzyme that cuts within ­restriction enzyme was used to cut both types of DNA. enzyme is called a ­restriction site. After a restric- the sequence AATC with tion ­enzyme binds to its restriction site, it cuts the two The complementary ends on the blue and yellow DNA of the sequence CGAATCTAGCAATCGCGA, strands of the DNA by breaking chemical bonds at fragments stick together by base pairing. The union how many restriction between the blue and yellow fragments is then made fragments will result? (For permanent by the “pasting” enzyme DNA ligase. This Recognition site (recognition sequence) simplicity, the sequence of enzyme connects the DNA pieces into continuous for a restriction enzyme only one of the two DNA strands by forming bonds between adjacent nucleotides. strands is listed.) The final outcome is a single molecule of recombinant will yield three restriction fragments. GA AT T C Answer: Cuts at two restriction sites C T T A AG DNA DNA. The process just described explains what happens in step 1 of Figure 12.3. 1 A restriction enzyme cuts the DNA into fragments. Restriction Gel Electrophoresis enzyme To separate and visualize DNA fragments of ­different lengths, researchers carry out a technique Stick y called gel electrophoresis, a method for ­sorting end AAT ­macromolecules—usually proteins or nucleic TC G G ­acids—primarily by their electrical charge and size. TAA ­Figure 12.5 shows how gel electrophoresis separates CT Stick y end DNA f­ ragments obtained from different sources. A sample with many copies of the DNA from each source 2 A DNA fragment is added from AA another source. T TC is placed in a separate well (hole) at one end of a flat, G rectangular gel. The gel is a thin slab of jellylike mate- G CT rial that acts as a molecular sieve. A negatively charged T AA electrode is then attached to the DNA-containing end of the gel and a positive electrode to the other end. 3 Fragments stick together by base pairing. ­Because the phosphate (PO4−) groups of nucleotides give DNA f­ ragments a negative charge, the ­fragments TC G A AT T move through the gel toward the positive pole. How- G A AT G C T TA A C C T TA A G ever, longer DNA fragments move more slowly through the thicket of polymer fibers in the gel than do shorter DNA fragments. To help you visualize the process, 4 DNA ligase joins the DNA imagine a small animal scampering quickly through fragments into strands. ligase a thicket of jungle vines, while a large animal plods along the same distance much more slowly. Similarly, ▶ Figure 12.4 Cutting and pasting DNA. over time, shorter molecules move farther through a The production of ­recombinant DNA requires two enzymes: a restriction enzyme, which cuts gel than longer molecules. Gel electrophoresis thus the original DNA molecules into pieces, and ­separates DNA fragments by length. When the cur- DNA ligase, which pastes the pieces together. Recombinant DNA molecule rent is turned off, a series of bands—­visible as blue 254 M12_SIMO2368_05_GE_CH12.indd 254 25/09/15 10:23 AM  ▼Figure 12.5 Gel electrophoresis of DNA molecules. The photo shows Genetic Engineering DNA fragments of various sizes visibly stained on the gel. Mixture of DNA fragments of different sizes – – Band of longest (slowest) fragments Power source Gel Band of shortest (fastest) fragments + + Completed gel smudges in the photograph of the gel—is left in each pancreas. Insulin functions as a hormone and helps column of the gel. Each band is a collection of DNA regulate the level of glucose in the blood. If the body CHECKPOINT fragments of the same length. The bands can be made fails to produce enough insulin, the result is type 1 dia- You use a restriction visible by staining, by exposure onto photographic betes. There is no cure, so people with this disease must enzyme to cut a long DNA molecule that has three film (if the DNA is radioactively labeled), or by mea- inject themselves daily with doses of insulin for the rest copies of the enzyme’s suring fluorescence (if the DNA is labeled with a of their lives. recognition sequence ­fluorescent dye). Because human insulin is not readily available, clustered near one end. diabetes was historically treated using insulin When you separate the Millions of from cows and pigs. This treatment was restriction fragments by gel Pharmaceutical individuals with diabetes live problematic, however. Pig and cow insu- electrophoresis, how do you expect the bands to Applications healthier lives lins can cause allergic reactions in people because their chemical structures differ appear? thanks to insulin of the gel (large fragment) By transferring the gene for a desired pro- slightly from that of human insulin. In ad- near the negative pole at the top made by tein into a bacterium, yeast, or other kind dition, by the 1970s, the supply of beef and (small fragments) and one band bacteria. tive pole at the bottom of the gel of cell that is easy to grow in culture, scien- pork pancreas available for insulin extraction Answer: three bands near the posi- tists can produce large quantities of useful could not keep up with the demand. proteins that are present naturally only In 1978, scientists working at a biotechnology in small amounts. In this section, you’ll company chemically synthesized DNA fragments and learn about some applications of recom- linked them to form the two genes that code for the binant DNA technology. two polypeptides that make up human insulin (see Humulin is human insulin pro- Figure 11.7). They then inserted these artificial genes duced by genetically modified bacte- into E. coli host cells. Under proper growing conditions, ria (­Figure 12.6). In humans, insulin the transgenic bacteria cranked out large quantities of is a protein normally made by the the human protein. In 1982, Humulin hit the market as the world’s first genetically engineered pharmaceuti- ▶ Figure 12.6 Humulin, human cal product. Today, it is produced around the clock in insulin produced by genetically gigantic fermentation vats filled with a liquid culture modified bacteria. of bacteria. Each day, more than 4 million people with 255 M12_SIMO2368_05_GE_CH12.indd 255 25/09/15 10:23 AM  Chapter 12 DNA Technology ▲ Figure 12.7 A factory that produces genetically ▲ Figure 12.8 A genetically modified goat. ­engineered insulin. diabetes use the insulin collected, purified, and pack- for an enzyme called lysozyme. This enzyme, found aged at such facilities (Figure 12.7). naturally in breast milk, has antibacterial properties. In Insulin is just one of many human proteins produced another example, the gene for a human blood protein by genetically modified bacteria. Another example is has been inserted into the genome of a goat so that the human growth hormone (HGH). Abnormally low lev- protein is secreted in the goat’s milk. The protein is then els of this hormone during childhood and adolescence purified from the milk. Because transgenic animals are can cause dwarfism. Because growth hormones from difficult to produce, researchers may create a single trans- other animals are not effective in people, HGH was an genic animal and then breed or clone it. The resulting early target of genetic engineers. Before genetically en- herd of transgenic animals could then serve as a grazing gineered HGH became available in 1985, children with pharmaceutical factory—“pharm” animals. an HGH deficiency could only be treated with scarce DNA technology is also helping medical researchers and expensive supplies of HGH obtained from human develop vaccines. A vaccine is a harmless variant or deriv- cadavers. Another important pharmaceutical product ative of a disease-causing microbe—such as a bacterium produced by genetic engineering is tissue plasminogen or virus—that is used to prevent an infectious disease. activator (abbreviated as tPA), a natural human protein When a person is inoculated, the vaccine stimulates the that helps dissolve blood clots. If administered shortly immune system to develop lasting defenses against the after a stroke, tPA reduces the risk of additional strokes microbe. For many viral diseases, the only way to prevent and heart attacks. serious harm from the illness is to use vaccination to pre- Besides bacteria, yeast and mammalian cells can also vent the illness in the first place. For example, the vaccine be used to produce medically valuable human proteins. against hepatitis B, a disabling and sometimes fatal liver For example, genetically modified mammalian cells disease, is produced by genetically engineered yeast cells growing in laboratory cultures are currently used to that secrete a protein found on the microbe’s outer surface. produce a hormone called erythropoietin (EPO) that stimulates production of red blood cells. EPO is used to treat anemia; unfortunately, some athletes abuse the Genetically Modified drug to seek the advantage of artificially high levels of oxygen-carrying red blood cells (a practice called “blood Organisms in Agriculture doping”). In recent decades, genetic engineers have Since ancient times, people have selectively bred even developed transgenic plant cells that can ­agricultural crops to make them more useful produce human drugs. Because they are eas- (see Figure 1.13). Today, DNA technology Someday, ily grown in culture and are unlikely to be genetically modified is quickly replacing traditional breeding contaminated by human pathogens (such potatoes could programs as scientists work to improve the as ­viruses), some scientists believe that car- prevent tens productivity of agriculturally important rots may be the drug factories of the future! of thousands of plants and animals. Genetically modified whole animals children from dying In the United States today, more than are also used to produce drugs. Figure 12.8 of cholera. 80% of the corn crop, more than 90% of the shows a transgenic goat that carries a gene soybean crop, and about 75% of the cotton crop 256 M12_SIMO2368_05_GE_CH12.indd 256 25/09/15 10:23 AM  are genetically modified. Figure 12.9 shows corn that has Genetic Engineering been genetically engineered to resist attack by an insect called the European corn borer. Growing insect-resistant plants reduces the need for chemical insecticides. In another example, modified strawberry plants produce bacterial proteins that act as a natural antifreeze, protect- ing the delicate plants from the damages of cold weather. Potatoes and rice have been engineered to produce harmless proteins derived from the cholera bacterium; researchers hope that these modified foods will one day serve as an edible vaccine against cholera, a disease that kills thousands of children in developing nations every year. In India, the insertion of a natural but rare saltwater- resistance gene has enabled new varieties of rice to thrive in water three times as salty as seawater, allowing food to be grown in drought-stricken or flooded regions. Scientists are also using genetic engineering to im- prove the nutritional value of crop plants (Figure 12.10). One example is “golden rice 2,” a transgenic variety of rice that carries genes from daffodils and corn. This rice could help prevent vitamin A deficiency and resulting blind- ness, especially in developing nations that depend on ▲ Figure 12.10 Genetically modified staple crops. rice as a staple crop. Cassava, a starchy root crop that is “Golden rice 2,” the yellow grains shown here (top) alongside a staple for nearly 1 billion people in developing ­nations, ordinary rice, has been genetically modified to produce high has similarly been modified to produce increased levels of levels of beta-carotene, a molecule that the body converts to iron and beta-carotene (which is ­converted to vitamin A vitamin A. Transgenic cassava (bottom), a starchy root crop that serves as the main food source for nearly a billion people, has in the body). However, controversy surrounds the use of been modified to produce extra nutrients. GM foods, as we’ll discuss at the end of the chapter. Genetic engineers are targeting agricultural animals as well as plant crops. Although no transgenic animals are currently being sold as food, the Food and Drug Admin- istration (FDA) has issued regulatory guidelines for their eventual introduction. Scientists might, for example, ▼ Figure 12.9 Genetically modified corn. The corn plants identify in one variety of cattle a gene that in this field carry a bacterial gene that helps prevent infestation causes the development of larger muscles by the European corn borer (inset). (which make up most of the meat we eat) and transfer it to other cattle or even to chickens. Researchers have genetically modified pigs to carry a roundworm gene whose protein converts less healthy fatty acids to omega-3 fatty acids. Meat from the modified pigs contains four to five times as much healthy omega-3 fat as regular pork. AquAdvantage is a trade name for Atlantic salmon that have been genetically modified to reach market size in half the normal time (18 months vs. 3 years). The FDA is CHECKPOINT currently reviewing AquAdvantage What is a genetically salmon; it may become the first GM modified organism? animal approved for consumption in duced through artificial means the United States. As of late 2014, the Answer: one that carries DNA intro- review continues. 257 M12_SIMO2368_05_GE_CH12.indd 257 25/09/15 10:24 AM  Chapter 12 DNA Technology Human Gene Therapy We’ve seen that bacteria, plants, and nonhuman animals can be genetically modified—so what about humans? Human gene therapy is intended to treat disease by introducing new genes into an afflicted person. In cases where a single defective gene causes a disorder, the mu- tant version of a gene may be replaced or supplemented Normal with the normal allele. This could potentially correct a human gene genetic disorder, perhaps permanently. In other cases, genes are inserted and expressed only long enough to 1 An RNA version is inserted into a virus treat a medical problem. Figure 12.11 summarizes one approach to human gene therapy. The procedure closely resembles the gene cloning process shown in steps 1 through 3 of RNA genome of virus Figure 12.3, but in this instance human cells, rather Inserted human RNA than bacteria, are the targets. A gene from a nor- mal individual is cloned, converted to an RNA version, Healthy person 2 Bone marrow and then inserted into the RNA genome of a harmless cells are infected ­virus. Bone marrow cells are taken from the patient and infected with the recombinant virus. The virus inserts a DNA copy of its genome, including the nor- mal human gene, into the DNA of the patient’s cells. The ­engineered cells are then injected back into the 3 Viral DNA inserts patient. The normal gene is transcribed and translated into the cell’s chromosome within the patient’s body, producing the desired protein. Ideally, the nonmutant version of the gene would be in- Human chromosome serted into cells that multiply throughout a person’s life. Bone marrow Bone marrow cells, which include the stem cells that cell from the patient give rise to all the types of blood cells, are prime candi- Bone of person dates. If the procedure succeeds, the cells will multiply with disease permanently and produce a steady supply of the missing protein, ­curing the patient. The promise of gene therapy thus far exceeds actual Bone marrow results, but there have been some successes. In 2009, an international research team conducted a trial that fo- 4 The engineered cused on a form of progressive blindness linked to a de- cells are injected fect in a gene responsible for producing light-detecting ▲ Figure 12.11 One approach to human gene therapy. pigments in the eye. The researchers found that a single injection of a virus carrying the normal gene into one eye of affected children improved vision in that eye, periodically removed immune system cells from sometimes enough to allow normal functioning, without the patients’ blood, infected them with a virus engi- significant side effects. The other eye was left untreated neered to carry the normal allele of the defective gene, as a control. then ­reinjected the blood into the patient. The treat- From 2000 to 2011, gene therapy was used to cure ment cured the ­patients of SCID, but there have been 22 ­children with severe combined immunodeficiency some serious side effects: Four of the treated patients CHECKPOINT (SCID), a fatal inherited disease caused by a defective ­developed leukemia, and one died after the inserted Why are bone marrow stem gene that prevents development of the immune system, gene activated an ­oncogene (see Chapter 11), creating cells ideally suited as targets requiring patients to remain isolated within protective cancerous blood cells. Gene therapy remains promis- for gene therapy? “bubbles.” Unless treated with a bone marrow trans- ing, but there is very little evidence to date of safe and plant, which is effective only 60% of the time, SCID effective ­application. Active research continues, with person’s life stem cells multiply throughout a Answer: because bone marrow patients quickly die from infections by microbes that new, tougher safety guidelines in place that are meant to most of us ­easily fend off. In these cases, researchers minimize dangers. 258 M12_SIMO2368_05_GE_CH12.indd 258 25/09/15 10:24 AM  DNA Profiling and Forensic Science DNA Profiling and Forensic Science When a crime is committed, body fluids (such as blood or semen) or small pieces of tissue (such as skin beneath DNA Profiling Techniques a victim’s fingernails) may be left at the scene or on the In this section, you’ll learn about techniques for making victim or assailant. As discussed in the Biology and Soci- a DNA profile. ety section at the start of the chapter, such evidence can be examined by DNA profiling, the analysis of DNA The Polymerase Chain Reaction (PCR) samples to determine whether they come from the same The polymerase chain reaction (PCR) is a technique by individual. Indeed, DNA profiling has rapidly trans- which a specific segment of DNA can be amplified: tar- formed the field of forensics, the scientific analysis of geted and copied quickly and precisely. Through PCR, evidence for crime scene investigations and other legal a scientist can obtain enough DNA from even minute proceedings. To produce a DNA profile, scientists com- amounts of blood or other tissue to allow a DNA profile pare DNA sequences that vary from person to person. to be constructed. In fact, a microscopic sample with as Figure 12.12 presents an overview of a typical investi- few as 20 cells can be sufficient for PCR amplification. gation using DNA profiling. First, DNA samples are In principle, PCR is simple. A DNA sample is mixed isolated from the crime scene, suspects, victims, or other with nucleotides, the DNA replication enzyme DNA evidence. Next, selected sequences from each DNA polymerase, and a few other ingredients. The solution sample are amplified (copied many times) to produce­ is then exposed to cycles of heating (to separate the a large sample of DNA fragments. Finally, the am- DNA strands) and cooling (to allow double-stranded plified DNA fragments are compared. All together, these DNA to re-form). During these cycles, specific regions ▼ Figure 12.13 DNA steps provide data about which samples are from the of each molecule of DNA are replicated, doubling the ­ mplification by PCR. The a same individual and which samples are unique. amount of that DNA (Figure 12.13). The result of this polymerase chain reaction (PCR) is a method for mak- ▼ Figure 12.12 Overview of DNA profiling. In this example, ing many copies of a specific DNA from suspect 1 does not match DNA found at the crime ­segment of DNA. Each round scene, but DNA from suspect 2 does match. of PCR, performed on a ­tabletop thermal cycler (shown at top), doubles the total ­quantity of DNA. Crime scene Suspect 1 Suspect 2 1 DNA isolated 2 DNA amplified Initial DNA segment 3 DNA compared 1 2 4 8 Number of DNA molecules 259 M12_SIMO2368_05_GE_CH12.indd 259 25/09/15 10:24 AM  Chapter 12 chain reaction is an exponentially growing population forensic scientists typically compare about a dozen DNA Technology of identical DNA molecules. The key to automated short s­ egments of noncoding repetitive DNA that are PCR is an unusually heat-stable DNA polymerase, known to vary ­between people. Have you ever seen a first isolated from prokaryotes living in hot springs puzzle in a ­magazine that presents two nearly identical (such as those shown in Figure 13.15B). Unlike most photos and asks you to find the few differences between proteins, this enzyme can withstand the heat at the them? In a similar way, scientists can focus on the few start of each cycle. areas of difference in the human genome, ignoring the A DNA molecule within a starting sample is likely identical majority. to be very long. But, most often, only a very small Repetitive DNA, which makes up much of the target region of that large DNA molecule needs to DNA that lies between genes in humans, consists of be amplified. The key to amplifying one particular nucleotide sequences that are present in multiple cop- ­segment of DNA and no others is the use of p ­ rimers, ies in the genome. Some of this DNA consists of short short (usually 15–20 nucleotides long), chemically sequences repeated many times tandemly (one after synthesized single-stranded DNA molecules. For another); such a series of repeats in the genome is called each experiment, specific primers are chosen that are a short tandem repeat (STR). For example, one person ­complementary to sequences found only at each end might have the sequence AGAT repeated 12 times in of the target sequence. The primers thus bind to se- a row at one place in the genome, the sequence GATA quences that flank the target sequence, marking the repeated 35 times at a second place, and so on; another start and end points for the segment of DNA to be person is likely to have the same ­sequences at the same amplified. Beginning with a single DNA molecule and places but with a different number of repeats. Like genes the appropriate primers, automated PCR can generate that cause physical traits, these stretches of repetitive hundreds of billions of copies of the desired sequence DNA are more likely to be an exact match between rela- in a few hours. tives than b­ etween unrelated individuals. In addition to forensic applications, PCR can be STR analysis is a method of DNA profiling that used in the treatment and diagnosis of disease. For compares the lengths of STR sequences at specific example, because the sequence of the genome of HIV sites in the genome. The standard STR analysis proce- (the virus that causes AIDS) is known, PCR can be dure used by law enforcement compares the number used to a­ mplify, and thus detect, HIV in blood or tis- of repeats of specific four-nucleotide DNA sequences sue samples. In fact, PCR is often the best way to detect at 13 sites scattered throughout the genome. Each this otherwise elusive virus. Medical scientists can now repeat site, which typically contains from 3 to 50 four-­ diagnose hundreds of human genetic disorders by us- nucleotide repeats in a row, varies widely from person ing PCR with primers that target the genes associated to person. In fact, some STRs used in the standard with these disorders. The amplified DNA product is procedure have up to 80 variations in the number of then studied to reveal the presence or absence of the repeats. In the United States, the number of repeats disease-causing mutation. Among the genes for human at each site is entered into a database called CODIS diseases that have been identified are those for sickle- (Combined DNA Index System) administered by the cell disease, hemophilia, cystic fibrosis, Huntington’s Federal ­Bureau of Investigation. Law enforcement disease, and Duchenne muscular dystrophy. Individu- agencies around the world can access CODIS to search CHECKPOINT als ­afflicted with such diseases can often be identi- for matches to DNA samples they have obtained from Why is only the slightest fied ­before the onset of symptoms, even before birth, crime scenes or suspects. trace of DNA at a crime allowing for preventative medical care to begin. PCR Consider the two samples of DNA shown in scene often sufficient for can also be used to identify symptomless carriers of ­Figure 12.14. Imagine that the top DNA segment forensic analysis? potentially harmful recessive alleles (see Figure 9.14). was obtained at a crime scene and the bottom from Parents may thus be informed of whether they have a a suspect’s blood. The two segments have the same analysis to produce enough molecules for Answer: because PCR can be used risk of bearing a child with a rare disease that they do number of repeats at the first site: 7 repeats of the four-­ not themselves display. nucleotide DNA sequence AGAT (in orange). Notice, however, that they differ in the number of repeats at Short Tandem Repeat (STR) Analysis the second site: 8 repeats of GATA (in purple) in the How do you prove that two samples of DNA come crime scene DNA, compared with 12 repeats in the from the same person? You could compare the en- suspect’s DNA. To create a DNA profile, a scientist uses tire genomes found in the two samples. But such PCR to specifically amplify the regions of DNA that an approach is impractical because the DNA of two ­include these STR sites. The resulting fragments are humans of the same sex is 99.9% identical. Instead, then compared. 260 M12_SIMO2368_05_GE_CH12.indd 260 25/09/15 10:24 AM  STR site 1 STR site 2 ◀ Figure 12.14 Short tandem DNA Profiling and r­ epeat (STR) sites. Scattered Forensic Science AGAT GATA throughout the genome, STR sites Crime scene DNA contain tandem repeats of four-­ nucleotide sequences. The number of repetitions at each site can vary Same number of Different numbers of from individual to individual. In this short tandem repeats short tandem repeats figure, both DNA ­samples have the same number of repeats (7) at the first STR site, but ­different numbers Suspect‘s DNA (8 versus 12) at the second. AGAT GATA Figure 12.15 shows the gel that would result from proved a convicted man innocent and also helped using gel electrophoresis to separate the DNA frag- identify the true perpetrator. ments from the example in Figure 12.14. (This figure Just how reliable is a genetic profile? In forensic CHECKPOINT simplifies the process; an actual STR analysis uses more cases using STR analysis with the 13 standard mark- sites than 2 and uses a different method to visualize the What are STRs, and why ers, the probability of two people having identical DNA are they useful for DNA results.) The differences in the locations of the bands profiles is somewhere between one chance in 10 bil- profiling? reflect the different lengths of the DNA fragments. This lion and one in several trillion. (The exact probability STR sites. gel would provide evidence that the crime scene DNA depends on the frequency of the individual’s particular numbers of repeats at the various did not come from the suspect. markers in the general population.) Thus, despite prob- different people have different valuable for DNA profiling because As happened in the case discussed in the Biol- lems that can still arise from insufficient data, human within the human genome. STRs are ogy and Society section, DNA profiling can provide error, or flawed evidence, genetic profiles are now repeated many times in a row peats) are nucleotide sequences ­evidence of either guilt or innocence. As of 2014, ­accepted as compelling evidence by legal experts and Answer: STRs (short tandem re- lawyers at the Innocence Project, a nonprofit legal or- scientists alike. ganization located in New York City, have helped to ex- onerate more than 310 convicted criminals in 35 states, including 18 who were on death row. ­The ­average ▼ Figure 12.16 DNA profiling: proof of innocence and guilt. In 1984, Earl Washington was convicted and sentenced sentence served by those who were ­exonerated was 14 to death for a 1982 rape and murder. In 2000, STR analysis years. In nearly half of these cases, DNA profiling has showed conclusively that he was innocent. Because every per- also identified the true perpetrators. Figure 12.16 pres- son has two chromosomes, each STR site is represented by two ents some data from a real case in which STR analysis numbers of repeats. The table shows the number of repeats for three STR markers in three samples: from semen found on the Amplified Amplified victim, from Washington, crime scene suspect’s and from another man DNA DNA who was in prison after an unrelated conviction. These and other STR data – (not shown) exonerated Washington and led the Longer other man to plead guilty fragments to the murder. Source of STR STR STR sample marker 1 marker 2 marker 3 Shorter + fragments Semen on victim 17,19 13,16 12,12 ▲ Figure 12.15 Visualizing STR fragment patterns. This figure shows the bands that would result from gel electropho- Earl Washington 16,18 14,15 11,12 resis of the STR sites illustrated in Figure 12.14. Notice that one of the bands from the crime scene DNA does not match one Kenneth Tinsley 17,19 13,16 12,12 of the bands from the suspect’s DNA. 261 M12_SIMO2368_05_GE_CH12.indd 261 25/09/15 10:24 AM  Chapter 12 DNA Technology Investigating Murder, Paternity, and Ancient DNA Since its introduction in 1986, DNA profiling has ­become a standard tool of forensics and has provided crucial evidence in many famous investigations. After the death of terrorist leader Osama bin Laden in 2011, U.S. Special Forces members obtained a sample of his DNA. Within hours, a military laboratory in Afghani- stan compared the tissue against samples previously ­obtained from several of bin Laden’s relatives, includ- ing his sister who had died of brain cancer in a Boston ­hospital in 2010. Although facial recognition and an eyewitness identification provided preliminary evidence, it was DNA that provided a conclusive match, officially ending the long hunt for the notorious terrorist. DNA profiling can also be used to identify murder victims. The largest such effort in history took place after the World Trade Center attack on September 11, 2001. Forensic scientists in New York City worked for years to identify more than 20,000 samples of victims’ remains. DNA profiles of tissue samples from the disaster site were matched to DNA profiles from tissue known to be from the victims or their relatives. More than half of the victims identified at the World Trade Center site were recognized solely by DNA evidence, providing closure ▲ Figure 12.17 Marie Antoinette. DNA profiling proved that to many grieving families. Since that time, the victims of Louis (depicted with his mother in this 1785 painting), the son other atrocities, such as mass killings during civil wars in of the Queen of France, did not survive the French Revolution. Europe and Africa, have been identified using DNA pro- filing techniques. In 2010, for example, DNA analysis was DNA profiling can also help protect endangered species used to identify the remains of war crime victims who by conclusively proving the origin of contraband animal had been buried in mass graves in Bosnia 15 years earlier. products. For example, analysis of seized elephant tusks can DNA profiling can also be used to identify victims of nat- pinpoint the location of the poaching, allowing enforce- ural disasters. After a tsunami devastated southern Asian ment officials to increase surveillance and prosecute those the day after Christmas 2004, DNA profiling was used to responsible. In 2014, three tiger poachers in India were sen- identify hundreds of victims, mostly foreign tourists. tenced to five years in jail after DNA profiling matched the Comparing the DNA of a mother, her child, and the pur- dead tigers’ flesh to tissue under the poachers’ fingernails. ported father can settle a question of paternity. Sometimes Modern methods of DNA profiling are so specific paternity is of historical interest: DNA profiling proved that and powerful that the DNA samples can be in a partially Thomas Jefferson or a close male relative fathered a child ­degraded state. Such advances are revolutionizing the study with an enslaved woman, Sally Hemings. In another his- of ancient remains. For example, a 2014 study of DNA ex- torical case, researchers wished to investigate whether any tracted from five mummified Egyptian heads (dating from descendants of Marie Antoinette (Figure 12.17), one-time 800 b.c. to 100 a.d.) was able to deduce the geographic queen of France, survived the French Revolution. DNA origins of the individuals, as well as identify DNA from the extracted from a preserved heart said to belong to her son pathogens that cause the diseases malaria and toxoplasmo- was compared to DNA ­extracted from a lock of Marie’s hair. sis. Another study determined that DNA extracted from A DNA match proved that her last known heir had, in fact, a 27,000-year-old Siberian mammoth was 98.6% identical died in jail during the revolution. More recently, a former to DNA from modern African elephants. Other studies in- backup singer for the “Godfather of Soul” James Brown volving a large collection of mammoth samples suggested sued his estate ­after his death, claiming that her child was that the last populations of the huge beasts migrated from Brown’s son. A DNA paternity test proved her claim, and North America to Siberia, where separate species interbred 25% of Brown’s estate was awarded to the mother and child. and, eventually, died out several thousand years ago. 262 M12_SIMO2368_05_GE_CH12.indd 262 25/09/15 10:24 AM  Bioinformatics Bioinformatics In the past decade, new experimental techniques The idea is that each type of base interrupts the current have generated enormous volumes of data related to for a slightly different length of time. Such techniques, DNA sequences. The need to make sense of an ever- if perfected, may usher in a new era of faster, even more increasing flood of information has spawned the field ­affordable sequencing. of ­bioinformatics, the application of computational methods to the storage and analysis of biological data. In this section, we’ll explore some of the methods by which Genomics sequence data are accumulated, as well as many of the Improved DNA sequencing techniques have transformed practical ways such knowledge can be put to use. the way in which we can explore fundamental biological questions about evolution and how life works. A major leap forward occurred in 1995 when a team of scientists DNA Sequencing announced that it had determined the nucleotide se- Researchers can exploit the principle of complemen- quence of the entire genome of Haemophilus influenzae, tary base pairing to determine the complete nucleotide a bacterium that can cause several human diseases, in- sequence of a DNA molecule. This process is called cluding pneumonia and meningitis. Genomics, the study DNA sequencing. In one standard procedure, the DNA of complete sets of genes (genomes), was born. is first cut into fragments, and then each fragment is The first targets of genomics research were bacteria,­ sequenced (Figure 12.18). In the past decade, “next- which have relatively little DNA (as you can see in generation sequencing” techniques have been developed ­Table 12.1). But soon the attention of genomics researchers that can simultaneously sequence thousands or hun- dreds of thousands of fragments, each of which can be Table 12.1 Some Important Sequenced Genomes* ­400–1,000 ­nucleotides long. This technology makes it Approximate possible to ­sequence nearly a million nucleotides per Year Size of Genome Number hour! This is an example of “high-throughput” DNA Organism Completed (in base pairs) of Genes technology, which is currently the method of choice for Haemophilus influenzae (bacterium) 1995 1.8 million 1,700 studies where massive numbers of DNA samples—even Saccharomyces cerevisiae (yeast) 1996 12 million 6,300 representing an entire genome—are being sequenced. In “third-­generation sequencing,” a single, very long Escherichia coli 1997 4.6 million 4,400 DNA molecule is sequenced on its own. Several groups (bacterium) of ­scientists have been working on the idea of moving a Caenorhabditis elegans (roundworm) 1998 100 million 20,100 single strand of a DNA molecule through a very small Drosophila melanogaster 2000 165 million 14,000 pore in a membrane (a nanopore), detecting the bases (fruit fly) one by one by their interruption of an electrical current. Arabidopsis thaliana (mustard plant) 2000 120 million 25,500 ▼ Figure 12.18 A DNA sequencer. This high-throughput Oryza sativa (rice) 2002 430 million 42,000 DNA sequencing machine can process half a billion bases in a single 10-hour run. Homo sapiens (human) 2003 3.0 billion 21,000 Rattus norvegicus 2004 2.8 billion 20,000 (lab rat) Pan troglodytes 2005 3.1 billion 20,000 (chimpanzee) Macaca mulatta (macaque) 2007 2.9 billion 22,000 Ornithorhynchus anatinus 2008 1.8 billion 18,500 (duck-billed platypus) Prunus persica (peach) 2013 227 million 27,900 *Some of the values listed are likely to be revised as genome analysis continues. 263 M12_SIMO2368_05_GE_CH12.indd 263 25/09/15 10:24 AM  Chapter 12 turned toward more complex organisms with much larger Any sequence in the database can be ­retrieved and ana- DNA Technology genomes. Baker’s yeast ­(Saccharo­myces ­cerevisiae) was lyzed. For example, software can compare a collection the first eukaryote to have its full sequence determined, of sequences from different species and diagram them and the roundworm Caenorhabditis elegans was the first as an evolutionary tree based on the sequence relation- multicellular organism. Other sequenced animals include ships. Bioinformatics has thereby revolutionized evolu- the fruit fly (Drosophila ­melanogaster) and lab rat (Rattus tionary biology by opening a vast new reservoir of data norvegicus), both model organisms for genetics research. that can test evolutionary hypotheses. Next we’ll discuss Among the sequenced plants are Arabidopsis thaliana, a particularly notable ­example of a sequenced animal a type of mustard plant used as a model organism, and ­genome—our own. rice (Oryza sativa), one of the world’s most economically important crops. As of 2014, the genomes of thousands of species The Human Genome have been published, and tens of thousands more are in The Human Genome Project was a massive scientific progress. The majority of organisms sequenced to date endeavor to determine the nucleotide sequence of all the CHECKPOINT are prokaryotes, including more than 4,000 bacterial DNA in the human genome and to identify the location Approximately how many species and nearly 200 archaea. Hundreds of eukaryotic and sequence of every gene. The project began in 1990 as nucleotides and genes are genomes—­including protists, fungi, plants, and animals contained in the human both invertebrate and vertebrate—have also been com- ▼ Figure 12.19 Genome sequencing. In the photo at the genome? and 21,000 genes pleted. Genome sequences have been determined for bottom, a technician performs a step in the whole-genome Answer: about 3 billion nucleotides cells from several cancers, for ancient