Introduction Hawaii’s papaya industry seemed doomed just a few decades ago. A deadly pathogen called the papaya ringspot virus (PRV) had spread throughout the islands. It appeared poised to completely decimate the papaya plant population. Scientists from the University of Hawaii were able to rescue the industry by creating new, genetically engineered PRV-resistant strains of papaya. Today, the papaya industry is once again vibrant, and the vast majority of Hawaii’s papayas are genetically modified organisms (GMOs).

GMO Genetically Modified Organisms (GMO): When a gene from one organism is purposely moved to improve or change another organism in a laboratory, the result is a genetically modified organism (GMO). It is also sometimes called "transgenic" for transfer of genes. 84% of corn grown today is genetically modified. Most is used in processed foods and some are used for animal feed. Some strains are drought resistant, others are pest resistant, some are herbicide resistant. 94% of the soy crop in the US is genetically modified. Very few fresh fruits and vegetables in your local grocery store are genetically modified. Potatoes are one of the genetically engineered vegetables available in the United States. Other genetically modified vegetables that have been approved for sale in the U.S. are tomatoes, radicchio, zucchini and yellow squash.

Isn’t everything genetically modified????

Genes can be cloned in recombinant plasmids Biotechnology- For thousands of years, humans have used microbes to make wine and cheese and selectively bred stock, dogs, and other animals. DNA technology – Genetic engineering- Student Misconceptions and Concerns  Student comprehension of restriction enzymes, nucleic acid probes, and many other aspects of recombinant DNA techniques depends upon a comfortable understanding of basic molecular genetics. Consider addressing Chapter 12 after an exam that covers the content in Chapters 10 and 11.  Students might bring some awareness and/or concerns about biotechnology to the classroom, for example, in their reactions to the controversies regarding genetically modified (GM) foods. This experience can be used to generate class interest and to highlight the importance of good information when making judgments. Consider starting class with a headline addressing one of these issues. The recent process of FDA approval for genetically engineered salmon raised for food might be particularly useful and relevant. Teaching Tips  Figure 12.1B is a synthesis of the techniques discussed in further detail in Modules 12.2–12.5. Figure 12.1 is therefore an important integrative piece that lays the foundation of most of the biotechnology discussion. Repeatedly referring to this figure in class helps students relate the text to your lecture.  The general genetic engineering challenge discussed in Module 12.1 begins with the need to insert a gene of choice into a plasmid. This process is very similar to film or video editing. What do we need to do to insert a minute of one film into another? We will need techniques to (a) cut and remove the minute of film to be inserted, (b) cut cut the new film apart, and (c) insert the new minute. In general, this is also like removing one boxcar from one train, and transferring the boxcar to another train. Students can become confused by the details of gene cloning through misunderstanding this basic editing relationship. Active Lecture Tips  As you begin to address genetic engineering and GMO foods, ask students to work in small groups to brainstorm about what they know about genetically engineered foods. Have each group create a list of statements that can be short or long, reflecting their impressions. The results may surprise you, and help you address some common misunderstandings and concerns.

Genes can be cloned in recombinant plasmids Gene cloning- Recombinant DNA – One source contains the gene that will be cloned. Another source is a gene carrier, called a ______. _________ are small, circular DNA molecules that replicate separately from the much larger bacterial chromosome; they are often used as vectors.

Genes can be cloned in recombinant plasmids The steps in cloning a gene: _____________is isolated. DNA containing the gene of interest is isolated. Plasmid DNA is treated with a _____________ that cuts in one place, opening the circle. DNA with the target gene is treated with the same enzyme, and many fragments are produced. Plasmid and target DNA __________________. Student Misconceptions and Concerns  Student comprehension of restriction enzymes, nucleic acid probes, and many other aspects of recombinant DNA techniques depends upon a comfortable understanding of basic molecular genetics. Consider addressing Chapter 12 after an exam that covers the content in Chapters 10 and 11.  Students might bring some awareness and/or concerns about biotechnology to the classroom, for example, in their reactions to the controversies regarding genetically modified (GM) foods. This experience can be used to generate class interest and to highlight the importance of good information when making judgments. Consider starting class with a headline addressing one of these issues. The recent process of FDA approval for genetically engineered salmon raised for food might be particularly useful and relevant. Teaching Tips  Figure 12.1B is a synthesis of the techniques discussed in further detail in Modules 12.2–12.5. Figure 12.1 is therefore an important integrative piece that lays the foundation of most of the biotechnology discussion. Repeatedly referring to this figure in class helps students relate the text to your lecture.  The general genetic engineering challenge discussed in Module 12.1 begins with the need to insert a gene of choice into a plasmid. This process is very similar to film or video editing. What do we need to do to insert a minute of one film into another? We will need techniques to (a) cut and remove the minute of film to be inserted, (b) cut the new film apart, and (c) insert the new minute. In general, this is also like removing one boxcar from one train, and transferring the boxcar to another train. Students can become confused by the details of gene cloning through misunderstanding this basic editing relationship. Active Lecture Tips  As you begin to address genetic engineering and GMO foods, ask students to work in small groups to brainstorm about what they know about genetically engineered foods. Have each group create a list of statements that can be short or long, reflecting their impressions. The results may surprise you, and help you address some common misunderstandings and concerns.

A cell with DNA containing the gene of interest E. coli bacterium 2 The cell’s DNA is isolated. 1 A plasmid is isolated. Bacterial chromosome Plasmid DNA Gene of interest (gene V) 3 The plasmid is cut with an enzyme 4 The cell’s DNA is cut with the same enzyme. Gene of interest 5 The targeted fragment and plasmid DNA are combined. Figure 12.1b-1-4 An overview of gene cloning (part 1, step 4) 6 DNA ligase is added, which joins the two DNA molecules. Recombinant DNA plasmid Gene of interest

Genes may be inserted into other organisms. Gene of interest Recombinant DNA plasmid The recombinant plasmid is taken up by a bacterium through trans- formation. 7 9 Recombinant bacterium Harvested proteins may be used directly. The bacterium reproduces. 8 Figure 12.1b-2-3 An overview of gene cloning (part 2, step 3) Clone of cells

VISUALIZING THE CONCEPT: Enzymes are used to “cut and paste” DNA Restriction enzymes recognize a particular short DNA sequence, called a restriction site, and cut both strands of the DNA at precise points in the sequence, yielding pieces of DNA called restriction fragments. Once cut, fragments of DNA can be pasted together by the enzyme DNA ligase. Student Misconceptions and Concerns  Student comprehension of restriction enzymes, nucleic acid probes, and many other aspects of recombinant DNA techniques depends upon a comfortable understanding of basic molecular genetics. Consider addressing Chapter 12 after an exam that covers the content in Chapters 10 and 11.  Students might bring some awareness and/or concerns about biotechnology to the classroom, for example, in their reactions to the controversies regarding genetically modified (GM) foods. This experience can be used to generate class interest and to highlight the importance of good information when making judgments. Consider starting class with a headline addressing one of these issues. The recent process of FDA approval for genetically engineered salmon raised for food might be particularly useful and relevant. Teaching Tips  In nature, restriction enzymes protect bacterial cells against foreign DNA. Thus, these enzymes “restrict” the invasion of foreign genetic material.  A genomic library of the sentence you are now reading would be all of the sentence fragments that made up the sentence. One could string together all of the words of this first sentence, without spaces between letters, and then conduct a word processing edit, placing a space between any place where the letter “e” is followed by the letter “n.” The resulting fragments of this original sentence would look like this and would be similar to a genomic library. ‑Age nomiclibraryofthese nte nceyouare nowreadingwouldbeallofthese nte ncefragme ntsthatmadeupthese nte nce. Active Lecture Tips  As you begin to address genetic engineering and GMO foods, ask students to work in small groups to brainstorm about what they know about genetically engineered foods. Have each group create a list of statements that can be short or long, reflecting their impressions. The results may surprise you, and help you address some common misunderstandings and concerns.

Animation: Restriction Enzymes

Restriction site Sticky end Every restriction enzyme recognizes one specific nucleotide sequence (its restriction site). Restriction site DNA GAATTC CTTAAG A restriction enzyme always cuts DNA sequences at its restriction site in an identical manner. Restriction enzyme G AATTC CTTAA G Sticky end A piece of DNA from another source (the gene of interest) is cut by the same restriction enzyme. Gene of interest AATTC G G CTTAA Sticky end The DNA fragments from the two sources stick together by hydrogen bonding of base pairs. Figure 12.2-5 Creating recombinant DNA using a restriction enzyme and DNA ligase (step 5) G AATT C G AATT C C TTAA G C TTAA G The enzyme DNA ligase creates new covalent bonds that join the backbones of the DNA strands. The result is a piece of recombinant DNA. DNA ligase Recombinant DNA

Recombinant cells and organisms can mass-produce gene products Recombinant cells and organisms constructed by DNA technologies are used to manufacture many useful products, chiefly proteins. Bacteria are often the best organisms for manufacturing a protein product because bacteria have plasmids and phages available for use as gene- cloning vectors, can be grown rapidly and cheaply, can be engineered to produce large amounts of a particular protein, and often secrete the proteins directly into their growth medium. Student Misconceptions and Concerns  The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet many debates about scientific issues are confused by misinformation. This provides an opportunity for you to ask students to research an issue before taking firm positions. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent is evidence of evolution that may be missed by your students. Active Lecture Tips  If you have already addressed basic transcription and translation in your course, consider asking small groups of students in your class to explain the following. DNA technology is primarily used to produce proteins. Why aren’t lipids and carbohydrates typically produced by these processes?

Recombinant cells and organisms can mass-produce gene products Yeast cells are eukaryotes, are easy to grow, have long been used to make bread and beer, can take up foreign DNA and integrate it into their genomes, and are often better than bacteria at synthesizing and secreting eukaryotic proteins. Student Misconceptions and Concerns  The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet many debates about scientific issues are confused by misinformation. This provides an opportunity for you to ask students to research an issue before taking firm positions. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent is evidence of evolution that may be missed by your students. Active Lecture Tips  If you have already addressed basic transcription and translation in your course, consider asking small groups of students in your class to explain the following. DNA technology is primarily used to produce proteins. Why aren’t lipids and carbohydrates typically produced by these processes?

Recombinant cells and organisms can mass-produce gene products Mammalian cells must be used to produce glycoproteins, proteins with chains of sugars attached. Examples include human erythropoietin (EPO), which stimulates the production of red blood cells, factor VIII to treat hemophilia, and tissue plasminogen activator (TPA), used to treat heart attacks and strokes. Student Misconceptions and Concerns  The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet many debates about scientific issues are confused by misinformation. This provides an opportunity for you to ask students to research an issue before taking firm positions. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent is evidence of evolution that may be missed by your students. Active Lecture Tips  If you have already addressed basic transcription and translation in your course, consider asking small groups of students in your class to explain the following. DNA technology is primarily used to produce proteins. Why aren’t lipids and carbohydrates typically produced by these processes?

Table 12.6-0 Some Protein Products of Recombinant DNA Technology

Recombinant cells and organisms can mass-produce gene products Pharmaceutical researchers are currently exploring the mass production of gene products by whole animals or plants. Recombinant animals are difficult and costly to produce and may be cloned to produce more animals with the same traits. Student Misconceptions and Concerns  The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet many debates about scientific issues are confused by misinformation. This provides an opportunity for you to ask students to research an issue before taking firm positions. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent is evidence of evolution that may be missed by your students. Active Lecture Tips  If you have already addressed basic transcription and translation in your course, consider asking small groups of students in your class to explain the following. DNA technology is primarily used to produce proteins. Why aren’t lipids and carbohydrates typically produced by these processes?

CONNECTION: DNA technology has changed the pharmaceutical industry and medicine DNA technology, including gene cloning, is widely used to produce medicines and to diagnose diseases. Therapeutic hormones produced by DNA technology include insulin to treat diabetes, human growth hormone to treat dwarfism, and tissue plasminogen activator (TPA), a protein that helps dissolve blood clots and reduces the risk of subsequent heart attacks. Student Misconceptions and Concerns  The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet many debates about scientific issues are confused by misinformation. This provides an opportunity for you to ask students to research an issue before taking firm positions. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent is evidence of evolution that may be missed by your students. Teaching Tips  Annual flu vaccinations are a common way to prevent diseases that cannot be easily treated. However, students might not understand why people receive the vaccine every year. A new annual vaccine is necessary because the flu viruses keep evolving, another lesson in evolution that may be missed by your students.

CONNECTION: DNA technology has changed the pharmaceutical industry and medicine DNA technology is used to test for inherited diseases, detect infectious agents such as HIV, and produce vaccines, harmless variants (mutants) or derivatives of a pathogen that stimulate the immune system to mount a lasting defense against that pathogen, thereby preventing disease. Student Misconceptions and Concerns  The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet many debates about scientific issues are confused by misinformation. This provides an opportunity for you to ask students to research an issue before taking firm positions. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent is evidence of evolution that may be missed by your students. Teaching Tips  Annual flu vaccinations are a common way to prevent diseases that cannot be easily treated. However, students might not understand why people receive the vaccine every year. A new annual vaccine is necessary because the flu viruses keep evolving, another lesson in evolution that may be missed by your students.

CONNECTION: Genetically modified organisms are transforming agriculture Since ancient times, people have selectively bred agricultural crops to make them more useful. DNA technology is quickly replacing traditional breeding programs to improve the productivity of agriculturally important plants and animals. Genetically modified organisms (GMOs) contain one or more genes introduced by artificial means. Transgenic organisms contain at least one gene from another species. Student Misconceptions and Concerns  The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet many debates about scientific issues are confused by misinformation. This provides an opportunity for you to ask students to research an issue before taking firm positions. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent is evidence of evolution that may be missed by your students. Teaching Tips  Roundup Ready corn, a product of the agricultural biotechnology corporation Monsanto, is resistant to the herbicide Roundup. The general strategy for farmers is to spray fields of Roundup Ready corn with the herbicide Roundup, killing weeds but not the corn. A search of the Internet will quickly reveal the controversy associated with this and other genetically modified organisms (GMOs), which can encourage interesting discussions and promote critical thinking skills. Module 12.9 discusses some of the issues related to the concerns over the use of GM organisms.

CONNECTION: Genetically modified organisms are transforming agriculture The most common vector used to introduce new genes into plant cells is a plasmid from the soil bacterium Agrobacterium tumefaciens called the Ti plasmid. Student Misconceptions and Concerns  The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet many debates about scientific issues are confused by misinformation. This provides an opportunity for you to ask students to research an issue before taking firm positions. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent is evidence of evolution that may be missed by your students. Teaching Tips  Roundup Ready corn, a product of the agricultural biotechnology corporation Monsanto, is resistant to the herbicide Roundup. The general strategy for farmers is to spray fields of Roundup Ready corn with the herbicide Roundup, killing weeds but not the corn. A search of the Internet will quickly reveal the controversy associated with this and other genetically modified organisms (GMOs), which can encourage interesting discussions and promote critical thinking skills. Module 12.9 discusses some of the issues related to the concerns over the use of GM organisms.

Agrobacterium tumefaciens DNA containing the gene for a desired trait Plant cell Ti plasmid 1 2 3 The gene is inserted into the plasmid. The recombinant plasmid is introduced into a plant cell. The plant cell grows into a plant. Recombinant Ti plasmid DNA carrying the new gene Restriction site Figure 12.8a-3 Using the Ti plasmid to genetically engineer plants (step 3) A plant with the new trait

CONNECTION: Genetically modified organisms are transforming agriculture GMO crops may be able to help a great many hungry people by improving food production, shelf life, pest resistance, and the nutritional value of crops. Golden Rice, a transgenic variety created in 2000 with a few daffodil genes, produces yellow grains containing beta-carotene, which our body uses to make vitamin A. Student Misconceptions and Concerns  The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet many debates about scientific issues are confused by misinformation. This provides an opportunity for you to ask students to research an issue before taking firm positions. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent is evidence of evolution that may be missed by your students. Teaching Tips  Roundup Ready corn, a product of the agricultural biotechnology corporation Monsanto, is resistant to the herbicide Roundup. The general strategy for farmers is to spray fields of Roundup Ready corn with the herbicide Roundup, killing weeds but not the corn. A search of the Internet will quickly reveal the controversy associated with this and other genetically modified organisms (GMOs), which can encourage interesting discussions and promote critical thinking skills. Module 12.9 discusses some of the issues related to the concerns over the use of GM organisms.

CONNECTION: Genetically modified organisms are transforming agriculture Genetic engineers are now creating plants that make human proteins for medical use. Pharmaceutical trials currently under way involve using modified rice to treat infant diarrhea, corn to treat cystic fibrosis, safflower to treat diabetes, and duckweed to treat hepatitis. Although promising, no plant-made drugs intended for use by humans have been approved or sold. Student Misconceptions and Concerns  The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet many debates about scientific issues are confused by misinformation. This provides an opportunity for you to ask students to research an issue before taking firm positions. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent is evidence of evolution that may be missed by your students. Teaching Tips  Roundup Ready corn, a product of the agricultural biotechnology corporation Monsanto, is resistant to the herbicide Roundup. The general strategy for farmers is to spray fields of Roundup Ready corn with the herbicide Roundup, killing weeds but not the corn. A search of the Internet will quickly reveal the controversy associated with this and other genetically modified organisms (GMOs), which can encourage interesting discussions and promote critical thinking skills. Module 12.9 discusses some of the issues related to the concerns over the use of GM organisms.

CONNECTION: Gene therapy may someday help treat a variety of diseases Gene therapy is the alteration of a diseased individual’s genes for therapeutic purposes. One possible procedure is the following: A gene from a healthy person is cloned, converted to an RNA version, and then inserted into the RNA genome of a harmless virus. Bone marrow cells are taken from the patient and infected with the recombinant virus. The virus inserts a DNA version of its genome, including the normal human gene, into the cells’ DNA. The engineered cells are then injected back into the patient. Teaching Tips  In 2008, the Genetic Information Nondiscrimination Act (GINA) was signed into law. The following link to a related U.S. government website characterizes the effect of the act as follows. GINA “…prohibits U.S. insurance companies and employers from discriminating on the basis of information derived from genetic tests.” The website can be found at www.ornl.gov/sci/techresources/Human_Genome/elsi/legislat.shtml.  As gene therapy technology expands, our ability to modify the genome in human embryos through in vitro fertilization permits genetic modification at the earliest stages of life. Future generations of humans, like our crops today, may include those with and without a genetically modified ancestry. The benefits and challenges of these technologies raise issues many students have never considered. Our students, and the generations soon to follow, may face the potential of directed human evolution.

Cloned gene (normal allele) 1 An RNA version of a healthy human gene is inserted into a retrovirus. RNA genome of virus Healthy person Retrovirus 2 Bone marrow cells are infected with the virus. 3 Viral DNA carrying the human gene inserts into the cell’s chromosome. Figure 12.10-0 One type of gene therapy procedure Bone marrow cell from the patient Bone marrow 4 The engineered cells are injected into the patient.

The analysis of genetic markers can produce a DNA profile DNA profiling is the analysis of DNA samples to determine whether they came from the same individual. In a typical investigation involving a DNA profile: DNA samples are isolated from the crime scene, suspects, victims, or other evidence, selected markers from each DNA sample are amplified (copied many times), producing a large sample of DNA fragments, and the amplified DNA markers are compared, providing data about which samples are from the same individual. Student Misconceptions and Concerns  Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete.  Students might expect DNA profiling for criminal investigations to involve an analysis of the entire human genome. Consider explaining why such an analysis is unrealistic and unnecessary. Modules 12.12–12.16 describe methods used to describe specific portions of the genome of particular interest. Teaching Tips  Figure 12.11 describes the general steps of DNA profiling. This overview is a useful reference to employ while the details of each step are discussed. Active Lecture Tips • See the Media Review: “Learn.Genetics” Genetic Science Learning Center from the University of Utah on the Instructor Exchange. Visit the Instructor Exchange in the MasteringBiology instructor resource area for a description of this activity.

The DNA of selected markers is amplified. Crime scene Suspect 1 Suspect 2 1 DNA is isolated. 2 The DNA of selected markers is amplified. Figure 12.11 An overview of DNA profiling 3 The amplified DNA is compared.

The PCR method is used to amplify DNA sequences Polymerase chain reaction (PCR) is a technique by which a specific segment of a DNA molecule can be targeted and quickly amplified in the laboratory. Student Misconceptions and Concerns  Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete.  Students might expect DNA profiling for criminal investigations to involve an analysis of the entire human genome. Consider explaining why such an analysis is unrealistic and unnecessary. Modules 12.12–12.16 describe methods used to describe specific portions of the genome of particular interest. Teaching Tips  In PCR, the product becomes another master copy. Imagine that while you are photocopying, every copy is used as a master at another copy machine. This would require many copy machines. However, it would be very productive! Active Lecture Tips • See the Media Review: “Learn.Genetics” Genetic Science Learning Center from the University of Utah on the Instructor Exchange. Visit the Instructor Exchange in the MasteringBiology instructor resource area for a description of this activity.

The PCR method is used to amplify DNA sequences PCR relies upon a pair of short primers, which are chemically synthesized, single-stranded DNA molecules with sequences that are complementary to sequences at each end of the target sequence. One primer is complementary to one strand at one end of the target sequence. The second primer is complementary to the other strand at the other end of the sequence. The primers thus bind to sequences that flank the target sequence, marking the start and end points for the segment of DNA being amplified. Student Misconceptions and Concerns  Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete.  Students might expect DNA profiling for criminal investigations to involve an analysis of the entire human genome. Consider explaining why such an analysis is unrealistic and unnecessary. Modules 12.12–12.16 describe methods used to describe specific portions of the genome of particular interest. Teaching Tips  In PCR, the product becomes another master copy. Imagine that while you are photocopying, every copy is used as a master at another copy machine. This would require many copy machines. However, it would be very productive! Active Lecture Tips • See the Media Review: “Learn.Genetics” Genetic Science Learning Center from the University of Utah on the Instructor Exchange. Visit the Instructor Exchange in the MasteringBiology instructor resource area for a description of this activity.

The PCR method is used to amplify DNA sequences The basic steps of PCR are as follows: The reaction mixture is heated to separate the strands of the DNA double helices. The strands are cooled. As they cool, primer molecules hydrogen-bond to their target sequences on the DNA. A heat-stable DNA polymerase builds new DNA strands by extending the primers in the 5→3  direction. These three steps are repeated over and over, doubling the amount of DNA after each three-step cycle. Student Misconceptions and Concerns  Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete.  Students might expect DNA profiling for criminal investigations to involve an analysis of the entire human genome. Consider explaining why such an analysis is unrealistic and unnecessary. Modules 12.12–12.16 describe methods used to describe specific portions of the genome of particular interest. Teaching Tips  In PCR, the product becomes another master copy. Imagine that while you are photocopying, every copy is used as a master at another copy machine. This would require many copy machines. However, it would be very productive! Active Lecture Tips • See the Media Review: “Learn.Genetics” Genetic Science Learning Center from the University of Utah on the Instructor Exchange. Visit the Instructor Exchange in the MasteringBiology instructor resource area for a description of this activity.

Figure 12.12-0 DNA amplification by PCR Cycle 1 yields two molecules Cycle 2 yields four molecules Cycle 3 yields eight molecules Additional Cycles… Sample DNA 3′ 5′ 3′ 5′ 3′ 5′ 5′ 5′ 3′ 1 Heat separates DNA strands. 2 Primers bond with ends of target sequences. 3 DNA polymerase adds nucleotides. 3′ 5′ 5′ 3′ Target sequence 5′ 3′ 5′ 5′ 3′ 5′ 3′ 5′ 3′ Figure 12.12-0 DNA amplification by PCR Primer New DNA

The PCR method is used to amplify DNA sequences Devised in 1985, PCR has had a major impact on biological research and biotechnology. PCR has been used to amplify DNA from fragments of ancient DNA from a mummified human, a 40,000-year-old frozen woolly mammoth, a 30-million-year-old plant fossil, and DNA from fingerprints or from tiny amounts of blood, tissue, or semen found at crime scenes. Student Misconceptions and Concerns  Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete.  Students might expect DNA profiling for criminal investigations to involve an analysis of the entire human genome. Consider explaining why such an analysis is unrealistic and unnecessary. Modules 12.12–12.16 describe methods used to describe specific portions of the genome of particular interest. Teaching Tips  In PCR, the product becomes another master copy. Imagine that while you are photocopying, every copy is used as a master at another copy machine. This would require many copy machines. However, it would be very productive! Active Lecture Tips • See the Media Review: “Learn.Genetics” Genetic Science Learning Center from the University of Utah on the Instructor Exchange. Visit the Instructor Exchange in the MasteringBiology instructor resource area for a description of this activity.

Gel electrophoresis sorts DNA molecules by size Many DNA technology applications rely on gel electrophoresis, a method that separates macromolecules, usually proteins or nucleic acids, on the basis of size, electrical charge, or other physical properties. Student Misconceptions and Concerns  Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete.  Students might expect DNA profiling for criminal investigations to involve an analysis of the entire human genome. Consider explaining why such an analysis is unrealistic and unnecessary. Modules 12.12–12.16 describe methods used to describe specific portions of the genome of particular interest. Teaching Tips  Separating ink using paper chromatography is a simple experiment that approximates some of what occurs in gel electrophoresis. Consider doing this as a class demonstration while addressing electrophoresis. Cut a large piece of filter paper into a rectangle or square. Use markers to color large dots about 2 cm away from one edge of the paper. Separate the dots from each other by 3–4 cm. Place the paper on edge, dots down, into a beaker containing about 1 cm of ethanol or isopropyl alcohol (50% or higher will do). The dots should not be in contact with the pool of alcohol in the bottom of the beaker. As the alcohol is drawn up the filter paper by capillary action, the alcohol will dissolve the ink dots. As the alcohol continues up the paper, the ink follows. Not all of the ink components move at the same speed, based upon their size and chemical properties. If you begin the process at the start of class, you should have some degree of separation by the end of a 50-minute period. Experiment with the technique a day or two before class to fine-tune the demonstration. (Save and air-dry these samples for your class.) Consider using brown, green, and black markers, because these colors are often made by color combinations. Active Lecture Tips • See the Media Review: “Learn.Genetics” Genetic Science Learning Center from the University of Utah on the Instructor Exchange. Visit the Instructor Exchange in the MasteringBiology instructor resource area for a description of this activity.

Gel electrophoresis sorts DNA molecules by size Gel electrophoresis can be used to separate DNA molecules based on size as follows: A DNA sample is placed at one end of a porous gel. Current is applied, and DNA molecules move from the negative electrode toward the positive electrode. Shorter DNA fragments move through the gel matrix more quickly and travel farther through the gel. DNA fragments appear as bands, visualized through staining or detecting radioactivity or fluorescence. Each band is a collection of DNA molecules of the same length. Student Misconceptions and Concerns  Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete.  Students might expect DNA profiling for criminal investigations to involve an analysis of the entire human genome. Consider explaining why such an analysis is unrealistic and unnecessary. Modules 12.12–12.16 describe methods used to describe specific portions of the genome of particular interest. Teaching Tips  Separating ink using paper chromatography is a simple experiment that approximates some of what occurs in gel electrophoresis. Consider doing this as a class demonstration while addressing electrophoresis. Cut a large piece of filter paper into a rectangle or square. Use markers to color large dots about 2 cm away from one edge of the paper. Separate the dots from each other by 3–4 cm. Place the paper on edge, dots down, into a beaker containing about 1 cm of ethanol or isopropyl alcohol (50% or higher will do). The dots should not be in contact with the pool of alcohol in the bottom of the beaker. As the alcohol is drawn up the filter paper by capillary action, the alcohol will dissolve the ink dots. As the alcohol continues up the paper, the ink follows. Not all of the ink components move at the same speed, based upon their size and chemical properties. If you begin the process at the start of class, you should have some degree of separation by the end of a 50-minute period. Experiment with the technique a day or two before class to fine-tune the demonstration. (Save and air-dry these samples for your class.) Consider using brown, green, and black markers, because these colors are often made by color combinations. Active Lecture Tips • See the Media Review: “Learn.Genetics” Genetic Science Learning Center from the University of Utah on the Instructor Exchange. Visit the Instructor Exchange in the MasteringBiology MasteringBiology instructor resource area for a description of this activity.

A mixture of DNA fragments of different sizes Longer (slower) molecules Power source Gel Shorter (faster) molecules Figure 12.13-0 Gel electrophoresis of DNA Completed gel

Short tandem repeat analysis is commonly used for DNA profiling Repetitive DNA consists of nucleotide sequences that are present in multiple copies in the genome. Short tandem repeats (STRs) are short nucleotide sequences that are repeated in tandem, composed of different numbers of repeating units in individuals, that are used in DNA profiling. STR analysis compares the lengths of STR sequences at specific sites in the genome and typically analyzes 13 sites scattered in the genome. Student Misconceptions and Concerns  Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete.  Students might expect DNA profiling for criminal investigations to involve an analysis of the entire human genome. Consider explaining why such an analysis is unrealistic and unnecessary. Modules 12.12–12.16 describe methods used to describe specific portions of the genome of particular interest. Teaching Tips  In most legal cases, the probability of two people having identical DNA profiles can be one in 10 billion or more. However, eyewitness testimony has been a standard part of the justice system. If you want to make the point about the unreliability of eyewitnesses in a trial, compared to techniques such as genetic profiling, consider this exercise. Arrange for a person who is not well known to the class to run into your classroom, take something you have placed near you (perhaps a bag, stack of papers, or box), and leave quickly. You need to take care that no one in the class is so alarmed as to do something dangerous. Once the “thief” is gone, tell the class that this was planned and do not speak. Have them each write a description of the person, including height, hair color, clothing, facial hair, behavior, etc. Many students will be accurate, but some will likely get details wrong. This is also an effective exercise to demonstrate the need for large sample sizes and accurate recording devices for good scientific technique. Active Lecture Tips • See the Media Review: “Learn.Genetics” Genetic Science Learning Center from the University of Utah on the Instructor Exchange. Visit the Instructor Exchange in the MasteringBiology instructor resource area for a description of this activity.

The number of short tandem repeats match STR site 1 STR site 2 AGAT GATA Crime scene DNA The number of short tandem repeats match The number of short tandem repeats do not match Suspect’s DNA Figure 12.14a Two representative STR sites from crime scene DNA samples AGAT GATA

Amplified crime scene DNA Amplified suspect’s DNA Longer STR fragments Figure 12.14b DNA profiles generated from the STRs in Figure 12.14a Shorter STR fragments

CONNECTION: DNA profiling has provided evidence in many forensic investigations DNA profiling is used to determine guilt or innocence in a crime, settle questions of paternity, and probe the origin of nonhuman materials. Student Misconceptions and Concerns  Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete.  Students might expect DNA profiling for criminal investigations to involve an analysis of the entire human genome. Consider explaining why such an analysis is unrealistic and unnecessary. Modules 12.12–12.16 describe methods used to describe specific portions of the genome of particular interest. Teaching Tips  Although the statistical odds of a DNA-profiling match can exceed one in 10 billion, the odds of a mistake in the collecting and testing procedures can be much greater. This is an important distinction. An error as simple as mislabeling a sample can confuse the results. Unfortunately, the odds of human error will vary and are difficult to determine. Active Lecture Tips • See the Media Review: “Learn.Genetics” Genetic Science Learning Center from the University of Utah on the Instructor Exchange. Visit the Instructor Exchange in the MasteringBiology MasteringBiology instructor resource area for a description of this activity.

RFLPs can be used to detect differences in DNA sequences Geneticists have cataloged many single-base-pair variations in the genome. Such a variation found in at least 1% of the population is called a single nucleotide polymorphism (SNP, pronounced “snip”). SNPs occur on average about once in 100 to 300 base pairs in the human genome, in the coding sequences of genes and in noncoding sequences between genes. Student Misconceptions and Concerns  Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete.  Students might expect DNA profiling for criminal investigations to involve an analysis of the entire human genome. Consider explaining why such an analysis is unrealistic and unnecessary. Modules 12.12–12.16 describe methods used to describe specific portions of the genome of particular interest. Teaching Tips  Here is another way to explain restriction fragment analysis. Consider these two words, equilibrium and equalibrium. Imagine that a mutation produced the spelling error of the second word. If we used a “restriction enzyme” that splits these words between u and i, how will the fragments compare in size and number? equilibrium = equ ilibri um (three fragments of three, six, and two letters) equalibrium = equalibri um (two fragments of nine and two letters) Active Lecture Tips • See the Media Review: “Learn.Genetics” Genetic Science Learning Center from the University of Utah on the Instructor Exchange. Visit the Instructor Exchange in the MasteringBiology instructor resource area for a description of this activity.

RFLPs can be used to detect differences in DNA sequences SNPs may alter a restriction site—the sequence recognized by a restriction enzyme. Such alterations change the lengths of the restriction fragments formed by that enzyme when it cuts the DNA. A sequence variation of this type is called a restriction fragment length polymorphism (RFLP, pronounced “rif-lip”). Thus, RFLPs can serve as genetic markers for particular loci in the genome. Student Misconceptions and Concerns  Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete.  Students might expect DNA profiling for criminal investigations to involve an analysis of the entire human genome. Consider explaining why such an analysis is unrealistic and unnecessary. Modules 12.12–12.16 describe methods used to describe specific portions of the genome of particular interest. Teaching Tips  Here is another way to explain restriction fragment analysis. Consider these two words, equilibrium and equalibrium. Imagine that a mutation produced the spelling error of the second word. If we used a “restriction enzyme” that splits these words between u and i, how will the fragments compare in size and number? equilibrium = equ ilibri um (three fragments of three, six, and two letters) equalibrium = equalibri um (two fragments of nine and two letters) Active Lecture Tips • See the Media Review: “Learn.Genetics” Genetic Science Learning Center from the University of Utah on the Instructor Exchange. Visit the Instructor Exchange in the MasteringBiology instructor resource area for a description of this activity.

Restriction enzymes added DNA sample 1 DNA sample 2 w Cut C C G G G G C C A C G G T G C C z x Cut C C G G G G C C Cut C C G G G G C C y y Figure 12.16-0 RFLP analysis Sample 1 Sample 2 Longer fragments z x w Shorter fragments y y

You should now be able to Explain how plasmids are used in gene cloning. Explain how restriction enzymes are used to “cut and paste” DNA into plasmids. Explain how DNA technology has helped to produce insulin, growth hormone, and vaccines. Explain how genetically modified organisms (GMOs) are transforming agriculture. Describe the benefits and risks of gene therapy in humans.

You should now be able to Describe the benefits and risks of gene therapy in humans. Describe the basic steps of DNA profiling. Explain how PCR is used to amplify DNA sequences. Explain how gel electrophoresis is used to sort DNA and proteins. Explain how short tandem repeats are used in DNA profiling. Explain how restriction fragment analysis is used to detect differences in DNA sequences.

DNA is attracted to + pole due to PO4− groups Figure 12.UN02 A mixture of DNA fragments Longer fragments move slower A “band” is a collection of DNA fragments of one particular length Power source Shorter fragments move faster Figure 12.UN02 Reviewing the concepts, 12.13 DNA is attracted to + pole due to PO4− groups

(a) (b) (c) (d) (e) Figure 12.UN03 DNA amplified via Bacterial plasmids DNA sample treated with treated with (b) DNA fragments sorted by size via (c) Recombinant plasmids are inserted into bacteria Figure 12.UN03 Connecting the concepts, question 1 Add (d) Particular DNA sequence highlighted are copied via (e)