Honors Bio Chapter 12 - DNA Technology

What are some of the later organisms to have their entire genome sequenced ?

Baker's yeast (Saccharomyces cerevisiae) was the first eukaryote to have its full sequence determined, and the roundworm Caenorhabditis elegans was the first multicellular organism. Other sequenced animals include the fruit fly (Drosophila melanogaster) and lab rat (Ratus norvegicus), both model organisms for genetics research. Among the sequenced plants are Arabidopsis thaliana, a type of mustard plant used as a model organism, and rice (Oryza sativa) and potato (Solanum tuberosum), two of the world's most economically important crops.

Name three ways a gene of interest can be obtained.

Can be isolated from genomic library, produced from mRNA using reverse transcriptose or synthesized from scrath.

What Biosafety Protocol did negotiators from 130 countries (including the United States) agreed on?

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 and allows importing countries to decide whether the shipments pose environmental or health risks. This agreement has been hailed as a breakthrough by environmentalists.

Who was Cheddar Man?

One of the strangest cases of DNA profiling is that of Cheddar Man, a 9,000-year-old skeleton found in a cave near Cheddar, England (Figure 12.14). DNA was extracted from his tooth and used to construct a DNA profile. The results suggested that Cheddar Man was a direct ancestor (through approximately 300 generations) of a present-day schoolteacher who lived only half a mile from the cave!

How have scientists used Recombinant DNA?

Scientists have genetically engineered bacteria to massproduce a variety of useful chemicals, from cancer drugs to pesticides. Scientists have also transferred genes from bacteria to plants and from one animal species to another (Figure 12.1 ). Such engineering can serve a variety of purposes, from basic research (What does this gene do?) to medical applications (Can we create animal models for this human disease?).

Explain the ethical question raised by DNA technology that has to do with privacy.

Similarly, advances in genetic profiling raise privacy issues. If we were to create a DNA profile of every person at birth, then theoretically we could match nearly every violent crime to a perpetrator because it is virtually impossible for someone to commit a violent crime without leaving behind DNA evidence. Furthermore, having a complete profile database could help with the identification of crime victims or remains. But are we, as a society, prepared to sacrifice our genetic privacy, even for such worthwhile goals?

What was The Human Genome Project?

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. The project began in 1990 as an effort by government-funded researchers from six countries. Several years into the project, private companies, chiefly Celera Genomics, joined the effort. At the completion of the project, more than 99% of the genome had been determined to 99.999% accuracy. (There remain a few hundred gaps of unknown sequence that will require special methods to figure out.) This ambitious project has provided a wealth of data that may illuminate the genetic basis of what it means to be human.

How do scientists cut DNA in order to make recombinant DNA?

The cutting tools used for making recombinant DNA are bacterial enzymes called restriction enzymes. Biologists have identified hundreds of restriction enzymes, each recognizing a particular short DNA sequence (usually four to eight nucleotides long). For example, one restriction enzyme only recognizes the DNA sequence GAATTC, whereas another recognizes GGATCC. The DNA sequence recognized by a particular restriction enzyme is called a restriction site. After a restriction enzyme binds to its restriction site, it cuts the two strands of the DNA at specific points within the sequence, like a pair of highly specific molecular scissors.

What is the polymerase chain reaction (PCR)?

The polymerase chain reaction (PCR) is a technique by which a specific segment of DNA can be targeted and copied quickly and precisely. Through PCR, a scientist can obtain enough DNA from even minute amounts of blood or other tissue to allow a DNA profile to be constructed.

What is DNA profiling?

the analysis of DNA samples to determine whether they come from the same individual, known as DNA profiling,

What is forensics?

the scientific analysis of evidence for crime scene investigations and other legal proceedings.

How do we really feel about wielding one of nature's powers-the evolution of new organisms?

A much broader ethical question is how do we really feel about wielding one of nature's powers-the evolution of new organisms? Some might ask if we have any right to alter an organism's genes-or to create new organisms. The benefits to people and the environment must also be considered. For example, bacteria are being engineered to clean up mining wastes and other pollutants that threaten the soil, water, and air. Since these organisms may be the only feasible solutions to some of our most pressing environmental problems, many people think this type of genetic engineering should be encouraged.

List some examples of famous cases where DNA profiling was used to identify the bad guys or exonerate good guys.

DNA profiling first came to widespread attention during the 0. J. Simpson murder trial. In this case, DNA analysis proved that blood in Simpson's car belonged to the victims and that blood at the crime scene belonged to Simpson. (The jury did not find the DNA evidence alone to be sufficient to convict the suspect, and Simpson was found not guilty.) And during the investigation that led up to his impeachment, President Bill Clinton repeatedly denied that he had sexual relations with Monica Lewinsky-until DNA profiling proved that his semen was on her dress. Of course, DNA evidence can prove innocence as well as guilt. As of 2011, lawyers at the Innocence Project, a nonprofit legal organization located in New York City, have helped to exonerate more than 270 convicted criminals in 34 states, including 17 who were on death row. In more than a third of these cases, DNA profiling has also identified the true perpetrators .

How was DNA profiling used to identify murder victims after the World trade Center attack?

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. If no sample of a victim's DNA was available, blood samples from close relatives were used to confirm identity through near matches. More than half of the victims identified at the World Trade Center site were recognized solely by DNA evidence, providing closure to many grieving families.

How do the US and Europe differ in their reaction to GM foods?

GM strains account for a significant percentage of several staple crops. Controversy about the safety of these foods is an important political issue (Figure 12.26). For example, the European Union has suspended the introduction of new GM crops and considered banning the import of all GM foodstuffs. In the United States and other countries where the GM revolution has proceeded more quietly than in Europe, the labeling of GM foods is now being debated but has not yet become law.

What was the biggest surprise from the Human Genome Project?

However, the biggest surprise from the Human Genome Project is the relatively small number of human genes-currently estimated to be about 21,000-very close to the number found in a roundworm! This number of genes is well below estimates made before the results of the project were known.

What is genomics?

In 1995, a team of scientists announced that it had determined the nucleotide sequence of the entire genome of Haemophilus influenzae, a bacterium that can cause several human diseases, including pneumonia and meningitis. Genomics, the study of complete sets of genes (genomes), was born.

What did the U.S. National Academy of Sciences study on transgenic crops find?

The U.S. National Academy of Sciences released a study finding no scientific evidence that transgenic crops pose any special health or environmental risks. But the authors of the study also recommended more stringent long-term monitoring to watch for unanticipated environmental impacts.

How does a DNA probe tags a gene. ?

The probe is a short, radioactive, single-stranded molecule of DNA or RNA. When it is mixed with single-stranded DNA from a gene with a complementary sequence, it attaches by hydrogen bonds (shown by the small red dots), "labeling" the gene.

How did scientists attempt to address concerns of DNA technology?

To address such concerns, scientists developed a set of guidelines that have become formal government regulations in the United States and some other countries.

How do scientists produce a DNA profile?

To produce a DNA profile, scientists compare sequences in the genome that vary from person to person. Like a gene, a noncoding genetic sequence is more likely to be a match between relatives than between unrelated individuals.

How far back does Biotechnology date?

You may think of biotechnology, the manipulation of organisms or their components to make useful products, as a modern phenomenon, but it actually dates back to the dawn of civilization. Consider such ancient practices as using yeast to make bread and the selective breeding of livestock.

What is gene cloning?

the production of multiple identical copies of a gene-carrying piece of DNA. Gene-cloning methods are central to the production of useful products from genetically engineered organisms. Consider a typical genetic engineering challenge: A molecular biologist at a pharmaceutical company identifies a gene of interest that codes for a valuable protein, such as a potential new drug. The biologist wants to manufacture the protein on a large scale. Figure 12.8 illustrates a way to accomplish this by using recombinant DNA techniques.

How would a scientist synethesize a gene of interest from scratch?

A final approach is to synthesize a gene of interest from scratch. Automated DNA-synthesizing machines can accurately and rapidly produce customized DNA molecules of any sequence up to lengths of a few hundred nucleotides

What is a vaccine?

A vaccine is a harmless variant or derivative of a disease-causing microbe-such as a bacterium or virus-that is used to prevent an infectious disease. When a person is inoculated, the vaccine stimulates the immune system to develop lasting defenses against the microbe. For many viral diseases, the only way to prevent serious harm from the illness is to use vaccination to prevent the illness in the first place.

What concerns to people who advocate for a cautious approach to GM foods have?

Advocates of a cautious approach fear that crops carrying genes from other species might harm the environment or be hazardous to human health (by, for example, introducing new allergens, molecules that can cause allergic reactions, into foods). A major concern is that transgenic plants might pass their new genes to close relatives in nearby wild areas. We know that lawn and crop grasses, for example, commonly exchange genes with wild relatives via pollen transfer. If domestic plants carrying genes for resistance to herbicides, diseases, or insect pests pollinated wild plants, the offspring might become "superweeds" that would be very difficult to control. However, researchers may be able to prevent the escape of such plant genes in various ways-for example, by engineering plants so that they cannot breed. Concern has also been raised that the widespread use of GM seeds may reduce natural genetic diversity, leaving crops susceptible to catastrophic die-offs in the event of a sudden change to the environment or introduction of a new pest.

How would a scientist use a nucleic acid probe to identify the bacterial clone containing a desired gene after using the "shotgun" approach to cloning a gene?

After you've created a genomic library, you have to find the right "book"-that is, you must identify the bacterial clone containing a desired gene (step 7 in Figure 12.8). Methods for detecting a gene depend on base pairing between the gene and a complementary sequence on another nucleic acid molecule, either DNA or RNA. When at least part of the nucleotide sequence of a gene is known, this information can be used to advantage. For example, if we know that a gene contains the sequence TAGGCT, a biologist can synthesize a short single strand of DNA with a complementary sequence (ATCCGA) and label it with a radioactive isotope or fluorescent dye. This labeled complementary molecule is called a nucleic acid probe because it is used to find a specific gene or other nucleotide sequence within a mass of DNA. (In actual practice, probe molecules are considerably longer than six nucleotides.) When a radioactive DNA probe is added to the DNA of various clones, it tags the correct molecule-finds the right book in the library-by base-pairing to the complementary sequence in the gene of interest (Figure 12.10). After a probe detects the desired clone within a library, more of the tagged cells can be grown, resulting in the production of large quantities of the gene of interest.

Why are bacterial plasmids useful in gene-cloning?

Although recombinant DNA techniques can be performed using many types of cells, bacteria are the workhorses of modern biotechnology. To manipulate genes in the laboratory, biologists often use bacterial plasmids, which are small, circular DNA molecules that replicate (duplicate) separately from the larger bacterial chromosome (Figure 12.7). Because plasmids can carry virtually any gene and are passed from one generation of bacteria to the next, they are key tools for gene cloning, the production of multiple identical copies of a gene-carrying piece of DNA.

How would a scientist synthesize a gene of interest using reverse transcriptase?

Another approach to obtaining a gene of interest is to synthesize it. One method uses reverse transcriptase, a viral enzyme that can synthesize DNA by using an mRNA template (see Chapter 10). Figure 12.11 shows the steps involved. A eukaryotic cell (1) transcribes the gene of interest and (2) processes the transcript, removing intrans and splicing exons together to produce mRNA. A researcher then (3) isolates the mRNA in a test tube and (4) makes single-stranded DNA from it using reverse transcriptase. (5) DNA polymerase is then used to synthesize a second DNA strand. When the researcher starts with an mRNA mixture from a particular cell type, the DNA that results from this procedure, called complementary DNA (cDNA), represents only those genes that were actually transcribed in the starting cells. Because cDNA molecules lack intrans, they are shorter than the full version of the genes and therefore easier to work with.

Explain the ethical question raised by DNA technology that has to do with having too much information.

As more information becomes available about our personal genetic makeup, many people question whether greater access to this information is always beneficial. For example, mail-in kits (Figure 12.27) have become available that can tell healthy people their relative risk of developing various diseases (such as Parkinson's and Crohn's) later in life. Some argue that such information helps families to prepare. Others say that the tests prey on our fears without offering any real benefit because certain diseases, such as Parkinson's, are not currently preventable or treatable. Other tests, however, such as for breast cancer risk, may help a person make changes that can prevent disease. How can we identify truly useful tests?

The Y Chromosome as a Window on History

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. DNA profiling can thus provide data about recent human evolution. In 2003, geneticists discovered that about 8% of males currently living in central Asia have Y chromosomes of striking genetic similarity. Further analysis traced their common genetic heritage to a single man living about 1,000 years ago. In combination with historical records, the data led to the speculation that the Mongolian ruler Genghis Khan (Figure 12.28) may have been responsible for the spread of the chromosome to nearly 16 million men living today. A similar study of Irish men in 2006 suggested that nearly 10% of them were descendants of Niall of the Nine Hostages, a warlord who lived during the 1400s. Another study ofY DNA seemed to confirm the claim by the Lemba people of southern Africa that they are descended from ancient Jews. Sequences of Y DNA distinctive of the Jewish priestly caste called Kohanim are found at high frequencies among the Lemba. Comparison ofY chromosome DNA profiles is part of a larger effort to learn more about the human genome. Other research efforts are extending genomic studies to many more species. These studies will advance our understanding of all aspects of biology, including health and ecology, as well as evolution. In fact, comparisons of the completed genome sequences of bacteria, archaea, and eukaryotes first supported the theory that these are the three fundamental domains of life-a topic we discuss further in the next unit, "Evolution and Diversity."

Why was there demand for human insulin?

Because human insulin is not readily available, diabetes was historically treated using insulin from cows and pigs. This treatment was problematic, however. Pig and cow insulins can cause allergic reactions in people because their chemical structures differ slightly from that of human insulin. In addition, by the 1970s, the supply of beef and pork pancreas available for insulin extraction could not keep up with the demand.

Besides bacteria, what else can be used to produce medically valuable human proteins?

Besides bacteria, yeast and mammalian cells can also be used to produce medically valuable human proteins. For example, genetically modified mammalian cells growing in laboratory cultures are currently used to produce a hormone called erythropoietin (EPO) that stimulates production of red blood cells. EPO is used to treat anemia; unfortunately, some athletes abuse the drug to seek the advantage of artificially high levels of oxygen-carrying red blood cells (a practice ca!Jed "blood doping").

How similar is your DNA to the DNA of a chimp?

Biojnformatics can also reveal similarities and differences in organisms more closely related to humans. In 2005, researchers completed the genome sequence for our closest living relative on the evolutionary tree of life, the chimpanzee (Pan troglodytes). Comparisons with human DNA revealed that we share 96% of our genome with our closest animal relative. Genomic scientists are currently finding and studying the important differences, shedding scientific light on the age-old question of what makes us human.

How can scientists use Recombinant DNA to produce large quantities of useful proteins that are present naturally only in small amounts?

By transferring the gene for a desired protein into a bacterium, yeast, or other kind of cell that is easy to grow in culture, scientists can produce large quantities of useful proteins that are present naturally only in small amounts.

Give an example of STR Analysis

Consider the two samples of DNA shown in Figure 12.16. Imagine that the top DNA segment was obtained at a crime scene and the bottom from a suspect' s blood. The two segments have the same number of repeats at the first site: 7 repeats of the four-nucleotide DNA sequence AGAT (in orange). Notice, however, that they differ in the number of repeats at the second site: 8 repeats of GATA (in purple) in the crime scene DNA, compared with 13 repeats in the suspect's DNA. To create a DNA profile, a scientist uses PCR to specifically amplify the regions of DNA that include these STR sites. The resulting fragments are then compared.

Explain the ethical question raised by DNA technology that has to do with who can get certain treatments.

DNA technology raises legal and ethical questions-few of which have clear answers. Consider, for example, the treatment of dwarfism with injections of human growth hormone (HGH) produced by genetically engineered cells. Should parents of short but hormonally normal children be able to seek HGH treatment to make their kids taller? If not, who decides which children are "tall enough" to be excluded from treatment?

when people use the term biotechnology today, what are they usually referring to?

DNA technology, modern laboratory techniques for studying and manipulating genetic material. Using these methods, scientists can modify specific genes and move them between organisms as different as bacteria, plants, and animals.

What was one early concern about DNA technology?

Early concerns focused on the possibility of creating hazardous new disease-causing organisms. What might happen, for instance, if cancer-causing genes were transferred into infectious bacteria or viruses?

How is DNA used besides forensic science?

Even beyond the courtroom, DNA technology has led to some of the most remarkable scientific advances in recent years: Corn and other crops, such as soybeans and tomatoes, 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.

Enumerate the steps in an investigation using DNA profiling?

Figure 12.13 presents an overview of a typical investigation using DNA profiling. (1) First, DNA samples are isolated from the crime scene, suspects, victims, or other evidence. (2) Next, selected sequences from each DNA sample are amplified (copied many times) to produce a large sample of DNA fragments. (3) Finally, the amplified DNA regions are compared using a gel (a method we'll discuss later). All together, these steps provide data about which samples are from the same individual and which samples are unique

Enumerate the steps in human gene therapy

Figure 12.24 summarizes one approach to human gene therapy. (1) A gene from a normal individual is cloned, converted to an RNA version, and then inserted into the RNA genome of a harmless virus. (2) Bone marrow cells are taken from the patient and infected with the recombinant virus. (3) The virus inserts a DNA copy of its genome, including the normal human gene, into the DNA of the patient's cells. (4) The engineered cells are then injected back into the patient. The normal gene is transcribed and translated within the patient's body, producing the desired protein. Ideally, the nonmutant version of the gene would be inserted into cells that multiply throughout a person's life. Bone marrow cells, which include the stem cells that give rise to all the types of blood cells, are prime candidates. If the procedure succeeds, the cells will multiply permanently and produce a steady supply of the missing protein, curing the patient.

What are "Pharm" animals?

Figure 12.6 shows a transgenic pig that carries a gene for human hemoglobin. The pig-produced hemoglobin can be isolated and used in human blood transfusions. Because transgenic animals are difficult to produce, researchers may create a single transgenic animal and then breed or clone it. The resulting herd of transgenic animals could then serve as a grazing pharmaceutical factory-"pharm" animals. DNA technology may eventually replace traditional animal breeding. Scientists might, for example, identify a gene that causes the development of larger muscles (which make up most of the meat we eat) in one variety of cattle and transfer it to other cattle or even to chickens. In 2006, University of Pittsburgh researchers 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. Unlike transgenic plants, however, transgenic animals are currently used only to produce potentially useful proteins; as of 2011, no transgenic animals are sold as food, although the Food and Drug Administration has issued regulatory guidelines for their eventual introduction.

How is Gel electrophoresis used for RFLP (pronounced ''rif-lip")?

Gel electrophoresis has many uses besides STR analysis. One application is RFLP analysis. RFLP (pronounced ''rif-lip") stands for restriction fragment length polymorphism. In this method, the DNA molecules to be compared are exposed to a restriction enzyme (Figure 12.19). The resulting restriction fragments are separated and made visible on a gel. In this case, both the number and location of bands indicate whether the original DNA samples had identical nucleotide sequences at the sites shown.

Explain the ethical question raised by DNA technology that has to do with eliminating genetic defects from children.

Genetic engineering of gametes (sperm or ova) and zygotes has been accomplished in lab animals. It has not been attempted in humans because such a procedure would raise very difficult ethical questions. Should we try to eliminate genetic defects in our children and their descendants? Should we interfere with evolution in this way? From a long-term perspective, the elimination of unwanted versions of genes from the gene pool could backfire. Genetic variety is a necessary ingredient for the adaptation of a species as environmental conditions change with time. Genes that are damaging under some conditions may be advantageous under others (one example is the sickle-cell allele-see the Evolution Connection section at the end of Chapter 13). Are we willing to risk making genetic changes that could be detrimental to our species in the future? We may have to face such questions soon.

How are genomics and proteomics changing how biologists study life?

Genomics and proteomics are enabling biologists to approach the study of life from an increasingly holistic (whole-system) perspective. Biologists are now compiling catalogs of genes and proteins-that is, listings of all the "parts" that contribute to the operation of cells, tissues, and organisms. As such catalogs become complete, researchers are shifting their attention from the individual parts to how these parts function as a whole in biological systems. Such analyses may have many practical applications. For example, proteins associated with specific diseases may be used to aid diagnosis (by developing tests that search for a particular combination of proteins) and treatment (by designing drugs that interact with the proteins involved).

What are some ways scientists have genetically modified food crops?

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, protecting the plants from cold weather, which can harm the delicate crop. Potatoes and rice have been experimentally modified 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 improve the nutritional value of crop plants. One example is "golden rice 2," a transgenic variety of rice that carries genes from daffodils and corn (Figure 12.5). This rice could help prevent vitamin A deficiency and resulting blindness, especially in developing nations that depend on rice as a staple crop.

What is Human gene therapy?

Human gene therapy is a recombinant DNA procedure intended to treat disease by altering an afflicted person's genes. In some cases, a mutant version of a gene may be replaced or supplemented with the normal allele. This could potentially correct a genetic disorder, perhaps permanently. In other cases, genes are inserted and expressed only long enough to treat a medical problem.

What is Humulin?

Humulin is human insulin produced by genetically modified bacteria (Figure 12.2). In humans, insulin is a protein normally made by the pancreas. Insulin functions as a hormone and helps regulate the level of glucose in the blood. If the body fails to produce enough insulin, the result is type l diabetes. There is no cure, so people with this disease must inject themselves daily with doses of insulin for the rest of their lives.

Where did Humulin come from?

In 1978, scientists working at the biotechnology company Genentech chemically synthesized two genes, one for each of the polypeptides of the active form of human insulin (see Figure 11.7). Because the amino acid sequences of the two insulin polypeptides were already known, it was easy to use the genetic code (see Figure 10.11) to determine nucleotide sequences that would encode for them. Researchers synthesized DNA fragments and linked them to form the insulin genes. In 1979, they succeeded in inserting these artificial genes into Escherichia coli host cells. Under proper growing conditions, these bacteria cranked out large quantities of the human protein. In 1982, Humulin hit the market as the world's first genetically engineered pharmaceutical product. Today, it is produced around the clock in gigantic fermentation vats filled with a liquid culture of bacteria. Each day, more than 4 million people with diabetes use the insulin collected, purified, and packaged at such facilities

How did the FBI track down the (alleged) anthrax killer?

In October 2001, a 63-year-old Florida man died from inhalation anthrax, a disease caused by breathing spores of the bacterium Bacillus anthracis. As the first victim of this disease in the United States since 1976 (and coming so soon after the 9/11 terrorist attacks the month before), his death was immediately suspicious. By the end of the year, four more people had died from anthrax. Law enforcement officials realized that someone was sending anthrax spores through the mail {Figure 12.21). The United States was facing an unprecedented bioterrorist attack. In the investigation that followed, one of the most helpful clues turned out to be the anthrax spores themselves. Investigators compared the genomes of the mailed anthrax spores with several laboratory strains. They quickly established that all of the mailed spores were genetically identical, suggesting that a single perpetrator was behind all the attacks. Furthermore, they were able to match the deadly spores with a laboratory subtype isolated at the U.S. Army Medical Research Institute oflnfectious Diseases in Fort Detrick, Maryland. A second, more comprehensive analysis of the spores used in the attack was completed in 2008. This analysis found four unique mutations in the mailed anthrax and traced the mutations to a single flask at the army facility. Based in part on this evidence, the FBI named an army research scientist as a suspect in the case. Although never charged, that suspect committed suicide in 2008; the case remains officially unsolved.

How does a scientist do PCR?

In principle, PCR is simple. A DNA sample is mixed with nucleotides, the DNA replication enzyme DNA polymerase, and a few other ingredients. The solution is then exposed to cycles of heating (to separate the DNA strands) and cooling (to allow double-stranded DNA to re-form). During these cycles, specific regions of each molecule of DNA are replicated, doubling the amount of that DNA (Figure 12.15). The key to automated PCR is an unusually heat-stable DNA polymerase, first isolated from prokaryotes living in hot springs. Unlike most proteins, this enzyme can withstand the heat at the start of each cycle. Beginning with a single DNA molecule, automated PCR can generate hundreds of billions of copies in a few hours.

What % of corn, soybean and cotton crops in the US are genetically modified?

In the United States today, roughly half the corn crop and more than three-quarters of the soybean and cotton crops are genetically modified

In the US, who evaluates genetic engineering projects for potential risks?

In the United States, all genetic engineering projects are evaluated for potential risks by a number of regulatory agencies, including the Food and Drug Administration, the Environmental Protection Agency, the National Institutes of Health, and the Department of Agriculture.

What is HGH, and why do scientists genetically engineer it?

Insulin is just one of many human proteins produced by genetically modi6.ed bacteria. Another example is human growth hormone (HGH). Abnormally low levels of this hormone during childhood and adolescence can cause dwarfism. Because growth hormones from other animals are not effective in people, HGH was an early target of genetic engineers. Before genetically engineered HGH became available in 1985, children with an HGH deficiency could only be treated with scarce and expensive supplies of HGH obtained from human cadavers.

Name three types of DNA that do not code for another molecule.

Introns, repetitive DNA and gene control sequences

Story of DNA used to prove guilt / innocence of a crime

It was a horrific crime: Eady in the morning of May 3, 1992, a 3-year-old girl sleeping in her Mississippi home was abducted, raped, murdered, and thrown into a creek. A tiny semen sample was recovered at the crime scene. Police arrested the mother's boyfriend, who had been babysitting the girl that night. Although police could not conclusively match the crime scene sample to the suspect, the jury found the circumstantial evidence compelling. He was convicted of the crime and sentenced to death. Although the semen sample from the 1992 crime scene was insufficient for DNA profiling at the time, new methods allowed a DNA profile to be determined in 2001. The results this time were conclusive: The semen at the crime scene did not match the man originally convicted of the crime. Several years later, the DNA profile obtained from the crime scene was matched to a different man. The second man soon confessed to the crime and to the nearly identical 1990 murder of another 3-year-old girl, for which a third man was serving jail time. After years of legal maneuvering, the two wrongly convicted men were freed in 2008, exonerated based on the DNA evidence.

Can Genomics Cure Cancer?

Lung cancer, which kills more Americans ever:y year than any other type of cancer, has long been the target of searches for effective chemotherapy drugs. One drug used to treat lung cancer, called gefitinib, targets the protein encoded by the EGFR gene. This protein is found on the surface of cells that line the lungs and is also found in lung cancer tumors. Unfortunately, treatment with gefitinib is ineffective for many patients. While studying the effectiveness of gefitinib, researchers at the Dana-Farber Cancer Institute in Boston made the observation that a few patients actually responded quite positively to the drug. This posed a question: Are genetic differences among lung cancer patients responsible for the differences in gefitinib's effectiveness? The researchers' hypothesis was that mutations in the EGFR gene were causing the different responses to gefitinib. The team made the prediction that DNA profiles focusing on the EGFR gene would reveal different DNA sequences in the tumors of responsive patients compared with the tumors of unresponsive patients. The researchers' experiment involved sequencing the EGFR gene in cells extracted from the tumors of five patients who responded to the drug and four who did not. The results were quite striking: All five tumors from gefitinib-responsive patients had mutations in EGFR, whereas none of the other four tumors did (Figure 12.23). These results suggest that doctors can use DNA profiling techniques to screen lung cancer patients for those who are most likely to benefit from treatment with this drug. In broader terms, this work suggests that genomics may bring about a revolution in the treatment of disease by allowing therapies to be customtailored to the genetic makeup of each patient.

How can a biological sample be used to provide evidence of guilt?

Modern forensic investigations hinge on a simple fact: The cells of every person (except identical twins) contain unique DNA. DNA profiling is the analysis of DNA samples to determine whether they come from the same individual.

Can DNA profiling work if the DNA is partially degraded?

Modern methods of DNA profiling are so specific and powerful that the DNA samples can be in a partially degraded state. This allows DNA analysis to be applied in a great number of ways. In evolution research, the technique has been used to study DNA recovered from an ancient mummified human and from a 30-million-year-old plant fossil. A 2005 study determined that DNA extracted from a 27,000-year-old Siberian mammoth was 98.6% idel).tical to DNA from modern African elephants.

How is genetic engineering used to create some vaccines?

One approach to vaccine production is to use genetically engineered yeast cells to make large amounts of a protein found on the microbe's outer surface. The vaccine against hepatitis B, a disabling and sometimes fatal liver disease, is made in this way.

What are some safety measures that are taken to prevent the release of a super germ?

One safety measure is a set of strict laboratory procedures to protect researchers from infection by engineered microbes and to prevent microbes from accidentally leaving the laboratory (Figure ll.25). In addition, strains of microbes to be used in recombinant DNA experiments are genetically crippled to ensure that they cannot survive outside the laboratory. Finally, certain obviously dangerous experiments have been banned. Today, most public concern about possible hazards centers not on recombinant microbes but on genetically modified (GM) foods.

What made sequencing the human genome a major challenge?

Our genome was a major challenge to sequence because, like the genomes of most complex eukaryotes, only a small amount of our total DNA consists of genes that code for proteins, tRNAs, or rRNAs. Most complex eukaryotes have a huge amount of noncoding DNAabout 98% of human DNA is of this type.

What is Recombinant DNA?

Recombinant DNA is constructed when scientists combine pieces of DNA from two different sources-often from different species to form a single DNA molecule. Recombinant DNA technology is widely used in genetic engineering, the direct manipulation of genes for practical purposes.

What is STR analysis?

STR analysis is a method of DNA profiling that compares the lengths of STR sequences at specific sites in the genome. Most commonly, STR analysis compares the number of repeats of specific four-nucleotide DNA sequences at 13 sites scattered throughout the genome. Each repeat site, which typically contains from 3 to 50 fournucleotide repeats in a row, varies widely from person to person. In fact, some STRs used in the standard procedure have up to 80 variations in the number of repeats. In the United States, the number of repeats at each site is entered into a database called CODIS (Combined DNA Index System) administered by the Federal Bureau of Investigation. Law enforcement agencies around the world can access CODIS to search for matches to DNA samples they have obtained from crime scenes or suspects.

About 98% of human DNA is noncoding DNA. What is it made up of?

Some of this noncoding DNA is made up of gene control sequences such as promoters, enhancers, and microRNAs (see Chapter 11). Other noncoding regions include introns and repetitive DNA (some of which is used in DNA profiling). Some noncoding DNA is important to our health, with certain regions known to carry diseasecausing mutations. But the function (if there is any) of most noncoding DNA remains unknown. Biologists used to refer to noncoding DNA as "junk DNA," but it is now generally accepted that much of this DNA plays some (still mysterious) role.

Where can you find the DNA sequences determined by the Human Genome Project? What are scientists doing with the information?

The DNA sequences determined by the Human Genome Project have been deposited in a database that is available on the World Wide Web. (You can browse it yourself at the website for the National Center for Biotechnology Information.) Scientists use software to scan and analyze the sequences for genes, control elements, and other features. The result is a genetic map containing all the genes and their locations on chromosomes. Next comes the most exciting challenge: figuring out the functions of the genes and other sequences and how they work together to direct the structure and function of a living organism. This challenge and the applications of the new knowledge should keep scientists busy well into the twenty-first century.

What is bioinformatics?

The anthrax investigation is just one example of the new field of bioinformatics, the application of computational tools to molecular biology. In 1991, sequence data provided strong evidence that a Florida dentist transmitted HIV to several patients. In 1993, after a cult released anthrax spores in downtown Tokyo, genomic analysis showed why their attack didn't kill anyone: They had used a harmless veterinary vaccine strain. And investigation of the West Nile virus outbreak in 1999 proved that a single natural strain of virus was infecting both birds and people. Bioinformatics has even allowed geneticists to produce a family tree of dog breeds (see the Evolution Connection section in Chapter 9).

How many chromosomes in the human genome? How many nucleotide pairs of DNA?

The chromosomes in the human genome (22 autosomes plus the X and Y sex chromosomes) contain approximately 3 billion nucleotide pairs of DNA.

What were the first targets of genomics?

The first targets of genomics research were bacteria, which have relatively little DNA. The H. influenzae genome, for example, contains 1.8 million nucleotides and 1,709 genes. But soon the attention of genomics researchers turned toward more complex organisms with much larger genomes (Table 12.1). As of 2011, the genomes of more than 1,700 species have been published, and more than 8,000 are in progress. The majority of organisms sequenced to date are prokaryotes, including E. coli and more than 1,000 other bacteria (some of medical importance) and more than 100 archaea. Over 100 eukaryotic genomes have also been completed.

Who's genome did they sequence when they sequenced the human genome?

The human genome sequenced by governmentfunded scientists was actually a reference genome compiled from a group of individuals. The genome sequenced by Celera consisted primarily of DNA from scientist Craig Venter, the company's president. As of 2011, the complete genomes of a handful of other individualsincluding James Watson, codiscoverer of the structure of DNA-have also been sequenced. Scientists have even begun to gather sequence data from our extinct relatives, such as a nearly complete genome, published in 2010, from a Neanderthal. The more human genomes we have, the more insight we can gain into what makes each person unique. We may soon enter an age of "personal genomics," where individual genetic differences among people will be put to routine medical use. Some of these seemingly minuscule differences can actually be a matter of life and death, as we'll see next.

How can the lengths of DNA fragments be compared?

The lengths of DNA fragments can be compared using gel electrophoresis, a method for sorting macromoleculesusually proteins or nucleic acids-primarily by their electrical charge and size. Figure 12.17 shows how gel electrophoresis separates DNA fragments obtained from different sources. A DNA sample from each source is placed in a separate well (hole) at one end of a flat, rectangular gel, a thin slab of jellylike material that acts as a molecular sieve. A negatively charged electrode is then attached to the DNA-containing end of the gel and a positive electrode to the other end. Because the phosI?hate (P04-) groups of nucleotides give DNA fragments a negative charge, the fragments move through the gel toward the positive pole. However, longer DNA fragments move more slowly through the thicket of polymer fibers in the gel than do shorter DNA fragments. Over time, shorter molecules move farther through the gel than longer molecules. Gel electrophoresis thus separates DNA fragments by length. When the current is turned off, a series of bands is left in each "lane" of the gel. Each band is a collection of DNA fragments of the same length. The bands can be made visible by staining, by exposure onto photographic film (if the DNA is radioactively labeled), or by measuring fluorescence (if the DNA is labeled with a fluorescent dye).

What are some of the potential benefits of having a complete map of the human genome?

The potential benefits of having a complete map of the human genome are enormous. Already, hundreds of disease-associated genes have been identified. One example is the gene that is mutated in an inherited type of Parkinson's disease, a debilitating brain disorder that causes tremors of increasing severity (Figure 12.20). Data from the Human Genome Project mapped some cases of Parkinson's disease to a specific gene. Interestingly, an altered version of the p rotein encoded by this gene has also been tied to Alzheimer's disease, suggesting a link between these two brain disorders. Moreover, the same gene is also found in rats, where it plays a role in the sense of smell, and in zebra finches, where it is thought to be involved in song learning. Cross-species comparisons such as these may uncover clues about the role played by the normal version of the protein in the human brain. And such knowledge could eventually lead to treatment for Parkinson's disease

Why is the method that has been described of creating recombinant DNA called a "shotgun" approach? What is a genomic library?

The procedure shown in Figure 12.8 can yield millions of recombinant plasmids carrying many different segments of foreign DNA. Such a procedure is called a "shotgun" approach to gene cloning because it "hits" an enormous number of different pieces of DNA. A typical cloned DNA fragment is big enough to carry one or a few genes. A collection of cloned DNA fragments that includes an organism's entire genome (a completer set of its genes) is called a genomic library.

How have the results of human gene therapy trials been?

The promise of gene therapy thus far exceeds actual results, but there have been some successes. In 2009, an international research team conducted a trial that focused on a form of progressive blindness linked to a defect in a gene responsible for producing lightdetecting 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, sometimes enough to allow normal functioning. The other eye was left untreated as a control. From 2000 to 2011, gene therapy also cured 22 children with severe combined immunodeficiency (SCID), a fatal inherited disease caused by a defective gene that prevents development of the immune system, requiring patients to remain isolated within protective "bubbles." Unless treated with a bone marrow transplant, which is effective only 60% of the time, SCID patients quickly die from infections by microbes that most of us easily fend off. In these cases, researchers periodically removed immune system cells from the patients' blood, infected them with a virus engineered to carry the normal allele of the defective gene, then reinjected the blood into the patient. The treatment cured the patients of SCID, but there have been some serious side effects: Four of the treated patients developed leukemia, and one died after the inserted gene activated an oncogene (see Chapter 11), creating cancerous blood cells. Gene therapy remains promising, but there is very little evidence to date of safe and effective application. Active research continues, with new, tougher safety guidelines in place that are meant to prevent the problems of the past.

What is proteomics?

The successes in the field of genomics have encouraged scientists to begin similar systematic studies of the full protein sets (proteomes) that genomes encode, an approach called proteomics. The number of different proteins in humans far exceeds the number of different genes (about 100,000 proteins versus about 21,000 genes). And since proteins, not genes, actually carry out the activities of the cell, scientists must study when and where proteins are produced and how they interact to understand the functioning of cells and organisms.

Enumerate the steps of how DNA is cut and pasted together

The top of Figure 12.9 shows a piece of DNA (blue) that contains one recognition sequence for a particular restriction enzyme. (1) The restriction enzyme cuts the DNA strands between the bases A and G within the recognition sequence, producing pieces of DNA called restriction fragments. The staggered cuts yield two double-stranded DNA fragments with single-stranded ends, called "sticky ends." Sticky ends are the key to joining DNA restriction fragments originating from different sources. (2) Next, a piece of DNA from another source (green) is added. Notice that the green DNA has single-stranded ends identical in base sequence to the sticky ends on the blue DNA because the same restriction enzyme was used to cut both types of DNA. (3) The complementary ends on the blue and green fragments stick together by base pairing. (4) The union between the blue and green fragments is then made permanent by the "pasting" enzyme DNA ligase. This enzyme, which is one of the proteins the cell normally uses in DNA replication, connects the DNA pieces into continuous strands by forming bonds between adjacent nucleotides. The final outcome is a single molecule of recombinant DNA.

List some ways DNA profiling is used outside of crime investigations?

The use of DNA profiling extends beyond crimes. For instance, comparing the DNA of a mother, her child, and the purported father can settle a question of paternity. Sometimes paternity is of historical interest: DNA profiling proved that Thomas Jefferson or a close male relative fathered a child with one of his slaves, Sally Hemings. DNA profiling can also help protect endangered species by conclusively proving the origin of contraband animal products. For example, analysis of seized elephant tusks can pinpoint the location of the poaching, allowing enforcement officials to increase surveillance and prosecute those responsible.

What is the Genetic Information Nondiscrimination Act of 2008?

There is also a danger that information about disease-associated genes could be abused. One issue is the possibility of discrimination and stigmatization. In response, Congress passed the Genetic Informa- tion Nondiscrimination Act of 2008. Title I of the act prohibits insurance companies from requesting or requiring genetic information during an application for health insurance. Title II provides similar wotections in employment. But even with such safeguards in place, can we fully prevent genetic information from being used in a discriminatory manner?

Enumerate the steps to clone a gene

To start, the biologist isolates two kinds of DNA: (1) bacterial plasmids that will serve as vectors (gene carriers) and (2) DNA from another organism that includes the gene of interest (along with other unwanted genes). This other DNA may be from any type of organism, even a human. (3) The researcher uses an enzyme to cut the two kinds of DNA. Each plasmid is cut in only one place; the other DNA is cut into many fragments, one of which carries the gene of interest. The figure shows the processing of just three of these DNA fragments and three plasmids, but actually millions of plasmids and millions of DNA fragments (most of which do not contain the gene of interest) are treated simultaneously. (4) Next, the DNA fragments are mixed in a test tube with the cut plasmids. The two kinds of DNA join together, resulting in recombinant DNA plasmids, some of which contain the gene of interest. (5) The recombinant plasmids are then mixed with bacteria. Under the right conditions, the bacteria take up the recombinant plasmids. (6) Each bacterium, with its recombinant plasmid, is allowed to reproduce. This step is the actual gene cloning. As the bacterium forms a clone (a group of identical cells descended from a single ancestral cell), any genes carried by the recombinant plasmid are also copied. (7) The biologist locates and isolates the few bacterial clones that contain the desired gene. (8) The transgenic bacteria with the gene of interest can then be grown in large tanks, producing the protein in marketable quantities. The bottom of the figure shows several useful end products that may be created via gene cloning. In the examples on the left of the figure, copies of the gene itself are the immediate product, to be used in further genetic engineering projects. In the examples on the right, the protein product of the cloned gene is harvested and used.

What is a GMO? What is a transgenic organism?

Today, DNA technology is quickly replacing traditional breeding programs as scientists work to improve the productivity of agriculturally important plants and animals. Scientists have produced many varieties of genetically modified (GM) organisms, organisms that have acquired one or more genes by artificial means. If the newly acquired gene is from another organism, typically of another species, the recombinant organism is called a transgenic organism.

What method is most-often used to map entire genome sequences?

Today, entire genomes are most often sequenced using the whole-genome shotgun method. The first step in this method is to chop the entire genome into fragments using restriction enzymes. Next, all the fragments are cloned and sequenced. Finally, computers running specialized mapping software reassemble the millions of overlapping short sequences into a single continuous sequence for every chromosome-an entire genome

How do you prove that two samples of DNA come from the same person?

You could compare the entire genomes found in the two samples. But such an approach would be extremely impractical, requiring a lot of time and money. Instead, forensic scientists typically compare about a dozen short segments of noncoding repetitive DNA. Repetitive DNA, which makes up much of the DNA that lies between genes in humans, consists of nucleotide sequences that are present in multiple copies in the genome. Some of this DNA consists of short sequences repeated many times tandemly ( one after another); such a series of repeats is called a short tandem repeat (STR). For example, one person might have the sequence AGAT repeated 12 times in a row at one place in the genome, the sequence GATA repeated 35 times at a second place, and so on; another person is likely to have the same sequences at the same places but with a different number of repeats. These stretches of repetitive DNA are more likely to be an exact match between relatives than between unrelated individuals.