Recombinant DNA Technology

      Recombinant DNA Technology This chapter examines how genetic researchers have adopted the natural enzymes and processes of DNA recombination, replication, transcription, transformation, transduction, and conjugation to manipulate genes for industrial, medical, and agricultural purposes. Together these techniques are termed recombinant DNA technology. We end the chapter with a discussion of the ethics and safety of these techniques. The Role of Recombinant DNA Technology in Biotechnology LE A R N I N G O U TC O M E S 8.1 Define biotechnology and recombinant DNA technology. 8.2 List several examples of useful products made possible • To combine beneficial traits of two or more organisms to create valuable new organisms, such as laboratory animals that mimic human susceptibility to HIV. • To create organisms that synthesize products that humans need, such as paint solvents, vaccines, antibiotics, enzymes, and hormones. For instance, geneticists have successfully inserted the human gene for insulin into bacteria so that the bacteria synthesize human insulin, which is cheaper and safer than insulin derived from animals. Recombinant DNA technology is not a single procedure or technique but rather a collection of tools and techniques scientists use to manipulate the genomes of organisms. In general, they isolate a gene from a cell, manipulate it in vitro,1 and insert it into another organism. (FIGURE 8.1) illustrates the basic processes involved in recombinant DNA technology. by biotechnology. TELL ME WHY technology. Why aren’t the terms recombinant DNA technology (genetic engineering) and biotechnology synonymous? 8.3 Identify the three main goals of recombinant DNA Biotechnology—the use of microorganisms to make practical products—is not a new field. For thousands of years, humans have used microbes to make products such as bread, cheese, soy sauce, and alcohol. During the 20th century, scientists industrialized the natural metabolic reactions of bacteria to make large quantities of acetone, butanol, and antibiotics. More recently, scientists have adapted microorganisms for use in the manufacture of paper, textiles, and vitamins; to assist in cleaning up industrial wastes, oil spills, and radioactive isotopes; and to aid in mining copper, gold, uranium, and other metals. (Chapter 25 discusses some industrial and environmental applications of biotechnology.) Until recently, microbiologists were limited to working with naturally occurring organisms and their mutants for achieving such industrial and medical purposes. Since the 1990s, however, scientists have become increasingly adept at intentionally modifying the genomes of organisms, by natural processes, for a variety of practical purposes. This is recombinant DNA technology (sometimes called “genetic engineering”), and it has expanded the possibilities of biotechnology in ways that seemed like science fiction only a few years ago. Today, scientists can isolate specific genes from almost any so-called donor organism, such as a human, a plant, or a bacterium, and insert it into the genome of almost any kind of recipient organism. The result is often called a genetically modified organism, or GMO. We consider the ethics and safety of GMOs later. Scientists who manipulate genomes have three main goals: • To eliminate undesirable phenotypic traits in humans, animals, plants, and microbes. For example, scientists have inserted genes from microbes into plants to make them resistant to pests or freezing, and since 1999, they have used gene therapy to cure some children born with a fatal and previously untreatable genetic disorder called severe combined immuno­ deficiency (SCID). M08_BAUM7206_05_SE_C08.indd 238 The Tools of Recombinant DNA Technology Scientists use a variety of physical agents, naturally occurring enzymes, and synthetic molecules to manipulate genes and genomes. These tools of recombinant DNA technology include mutagens, reverse transcriptase, synthetic nucleic acids, restriction enzymes, and vectors. Scientists use these molecular tools to create gene libraries, which are a time-saving tool for genetic researchers. Mutagens LE A R N I N G O UTC O M E 8.4 Describe how gene researchers use mutagens. Mutagens are physical and chemical agents that produce mutations (heritable changes in the nucleotide sequence of a genome). Scientists deliberately use mutagens to create changes in microbes’ genomes so that the microbes’ phenotypes are changed. They then select for and culture cells with characteristics considered beneficial for a given biotechnological application. For example, scientists exposed the fungus Penicillium (pen-i-sil′ē-ŭm) to mutagenic agents and then selected strains that produce greater amounts of penicillin. In this manner, they developed a strain of Penicillium that secretes over 25 times as much penicillin as did the strain originally isolated by Alexander Fleming (1881–1955). Today, with recombinant DNA techniques (discussed shortly), researchers can isolate mutated genes rather than dealing with entire organisms. 1Latin, meaning “within glassware”; that is, in a laboratory. 11/23/16 3:02 AM The Use of Reverse Transcriptase to Synthesize cDNA Bacterial cell       R The Tools of ecombinant DNA Technology DNA containing gene of interest LE A R N I N G O U TC O M E 8.5 Explain the function and use of reverse transcriptase in synthesizing cDNA. Transcription involves the transmission of genetic information from molecules of DNA to molecules of RNA (see Chapter 7, Figure 7.9). The discovery of retroviruses, which have genomes consisting of RNA instead of DNA, led to the discovery of the enzyme reverse transcriptase. Reverse transcriptase creates a flow of genetic information in the opposite direction from the flow in conventional transcription: it uses an RNA template to transcribe a molecule of DNA, which is called complementary DNA (cDNA) because it is complementary to an RNA template. Bacterial chromosome Plasmid 1 Isolate plasmid. Gene of interest 2 Enzymatically cleave DNA into fragments. 3 Isolate fragment with the gene of interest. RNA Reverse transcriptase 4 Insert gene into plasmid. cDNA Because hundreds to millions of copies of mRNA exist for every active gene, it is frequently easier to produce a desired gene by first isolating the mRNA molecules that code for a particular polypeptide and then use reverse transcription to synthesize a cDNA gene from the mRNA template. Further, eukaryotic DNA is not normally expressible by prokaryotic cells, which cannot remove the introns (noncoding sequences) present in eukaryotic pre-mRNA. However, since eukaryotic mRNA has already been processed to remove introns, cDNA produced from processed mRNA lacks noncoding sequences. Therefore, scientists can successfully insert cDNA into prokaryotic cells, making it possible for the prokaryotes to produce eukaryotic proteins such as human growth factor, insulin, or blood-clotting factors. 5 Insert plasmid and gene into bacterium. 6 Culture bacteria. Synthetic Nucleic Acids LE A R N I N G O U TC O M E S 8.6 Explain how gene researchers synthesize nucleic acids. 8.7 Describe three uses of synthetic nucleic acids. The enzymes of DNA replication and RNA transcription function not only in vivo2 but also in vitro, making it possible for scientists to produce molecules of DNA and RNA in cell-free solutions for genetic research. In fact, scientists have so mechanized the processes of nucleic acid replication and transcription that they can produce molecules of DNA and RNA with any nucleotide sequence; all they must do is enter the desired sequence into a synthesis machine’s four-letter keyboard. A computer controls the actual synthesis, using a supply of nucleotides and other required reagents. Nucleic acid synthesis machines synthesize molecules over 100 nucleotides long in a few hours, 2Latin, meaning “in life” (i.e., within a cell). M08_BAUM7206_05_SE_C08.indd 239 Harvest copies of gene to insert into plants or animals. Eliminate undesirable phenotypic traits. Create beneficial combination of traits. Harvest proteins coded by gene. Produce vaccines, antibiotics, hormones, or enzymes. ▲ FIGURE 8.1 Overview of recombinant DNA technology. and scientists can join two or more of these molecules end to end with ligase to create even longer synthetic molecules. Researchers have used synthetic nucleic acids in many ways, including the following: • Elucidating the genetic code. Using synthetic molecules of varying nucleotide sequences and observing the amino 11/23/16 3:02 AM ­ Technology TABLE 8.1 Properties of Some Restriction Enzymes Restriction Sitea Bam I Bacillus amyloliquefaciens Gt GatCC CCtaGc G Eco I Escherichia coli Y13 Gt aattC Cttaac G Eco II E. coli 245 CCt GG GGc CC HindII Haemophilus influenzae d Gtpy t pu aC Capu c pytG HindIII H. influenzae d at aGCtt ttCGac a HinfI H. influenzae f Gt aNtC CtNac G HpaI H. parainfluenzae Gtt t aaC Caac ttG MspI Moraxella sp. CCt GG GGc CC SmaI Serratia marcescens CCCt GGG GGGc CCC An important development in recombinant DNA technology was the discovery of restriction enzymes (also called restriction endo­ nucleases) in bacterial cells. Such enzymes cut DNA molecules and are restricted in their action—they cut DNA only at locations called restriction sites. Restriction sites are specific nucleotide sequences, which are usually palindromes3—they have the same sequence when read forward or backward. In nature, bacterial cells use restriction enzymes to protect themselves from phages by cutting phage DNA into nonfunctional pieces. The bacterial cells protect their own DNA by methylation4 of some of their nucleotides, hiding the DNA from the restriction enzymes (see Chapter 7). M08_BAUM7206_05_SE_C08.indd 240 a T rrows indicate sites of cleavage: py = pyrimidine (either thymine [ ] or cytosine [C]); pu = purine (either adenine [ ] or guanine [G]); N = any nucleotide ( , , G, or C). T a methyl group, :CH3, to a chemical. A Greek palin, meaning “again,” and dramein, meaning “to run.” 4Adding A 3From R R 8.8 Explain the source and names of restriction enzymes. 8.9 Describe the importance and action of restriction enzymes. R R LE A R N I N G O U TC O M E S H Bacterial Source H Restriction Enzymes Enzyme R • Researchers name restriction enzymes with three letters (denoting the genus and specific epithet of the source bacterium) and Roman numerals (to indicate the order in which enzymes from the same bacterium were discovered). In some cases, a fourth letter denotes the strain of the bacterium. Thus, Escherichia coli (esh-ĕ-rik′ē-ə kō’lō) strain R produces the restriction enzymes EcoRI and EcoRII. HindIII is the third restriction enzyme isolated from Haemophilus influenzae (hē-mof′i-lŭs in-flu-en′zō) strain Rd. Scientists have discovered hundreds of restriction enzymes and categorize them in two basic groups on the basis of types of cuts they make. The first type, as exemplified by EcoRI, makes staggered cuts of the two strands of DNA, producing fragments that terminate in mortise-like sticky ends. Each sticky end is composed of up to four nucleotides that form hydrogen bonds with its complementary sticky end (FIGURE 8.2a). Scientists can use these bits of single-stranded DNA to combine pieces of DNA from different organisms into a single recombinant DNA molecule (the enzyme ligase unites the sugar-phosphate backbones of the pieces) (FIGURE 8.2b). Other restriction enzymes, such as HindII and SmaI (the latter from Serratia marcescens, ser-rat′ē-a marses′enz), cut both strands of DNA at the same point, resulting in blunt ends (FIGURE 8.2c). It is more difficult to make recombinant DNA from blunt-ended fragments because they are not sticky, but they have a potential advantage—blunt ends are nonspecific. This enables any two blunt-ended fragments, even those produced by different restriction enzymes, to be combined (FIGURE 8.2d). In contrast, sticky-ended fragments bind only to complementary, sticky-ended fragments produced by the same restriction enzyme. TABLE 8.1 identifies several restriction enzymes and their target DNA sequences. ▶ ANIMATIONS: Recombinant DNA R T ­ • acids in the resulting polypeptides, scientists elucidated the genetic code. For example, synthetic DNA consisting only of adenine nucleotides yields a polypeptide consisting solely of the amino acid phenylalanine. Therefore, the mRNA codon UUU (transcribed from the DNA triplet AAA) must code for phenylalanine (see Figure 7.12). Creating genes for specific proteins. Once they know the genetic code and the amino acid sequence of a protein, scientists can create a gene for that protein. In this manner, scientists synthesized a gene for human insulin. Of course, such a synthetic gene likely consists of a nucleotide sequence slightly different from that of its cellular counterpart because of the redundancy in the genetic code (see Figure 7.12). Synthesizing DNA and RNA probes to locate specific sequences of nucleotides. Probes are nucleic acid molecules with a specific nucleotide sequence that have been labeled with radioactive or fluorescent chemicals so that their locations can be detected. The use of probes to locate specific sequences of nucleotides is based on the fact that any given nucleotide sequence will preferentially bond to its complementary sequence. Thus, a probe constructed with the nucleotide sequence ATGCT will bond to a DNA strand with the sequence TACGA, and the probe’s label allows researchers to then detect the complementary site. Probes are essential tools for locating specific nucleic acid sequences, such as genes for particular polypeptides. Synthesizing antisense nucleic acid molecules. Antisense nucleic acid molecules have nucleotide sequences that bind to and interfere with genes and mRNA molecules. Scientists are developing antisense molecules to control genetic diseases. Synthesizing PCR primers. The most common usage for synthetic nucleic acid molecules is as single-stranded primers for a polymerase chain reaction (PCR) (see Figure 8.5). Primers are typically 20–30 nucleotides long. R • echnology A • A       Chapter 8   Recombinant DN 12/03/2018 13:20       R The Tools of ecombinant DNA Technology Restriction site (palindrome) 5¿ 5¿ 3¿ GAA T T C C T T AAG CCCGGG GGGC C C A T G C T TAA 5¿ TC G A T T CGA AG T T A GGG CCC Restriction enzyme 2 3¿ 5¿ GT T CAA 3¿ AAC T TG (c) Production of blunt ends (a) Production of sticky ends C CCC GGG 3¿ Blunt ends Sticky ends AG GT T AAC CAAT TG Restriction enzyme 1 Restriction enzyme A 5¿ 3¿ Ligase C A T T CGA T T A 5¿ CCCAAC GGG T T G 3¿ 5¿ 3¿ G T T GGG CAACCC Recombinant DNA molecules Restriction fragments from two different organisms cut by the same restriction enzyme (d) Recombinants using blunt ends Ligase AAGC T T T T CGAA 3¿ + 5¿ AAGC T T T T CGAA 3¿ Recombinant DNA molecules id V e Tu r to (b) Recombinants using sticky ends o 5¿ 8.10 Define vector as the term applies to genetic manipulation. One goal of recombinant DNA technology is to insert a useful gene into a cell so that the cell has a new phenotype—for example, the ability to synthesize a novel protein. To deliver a gene into a cell, researchers use vectors, which are nucleic acid molecules, such as viral genomes, transposons, and plasmids. Genetic vectors share several useful properties: • Vectors are small enough to manipulate in a laboratory. Large DNA molecules the size of entire chromosomes are generally too fragile to serve as vectors. • Vectors survive inside cells. Plasmids, which are circular DNA, make good vectors because they are more stable than are linear fragments of DNA, which are typically degraded by cellular enzymes. However, some linear vectors, such as transposons and certain viruses, can insert themselves rapidly into a host’s chromosome before they can be degraded. M08_BAUM7206_05_SE_C08.indd 241 E H indIII, and (c) SmaI and paI. LE A R N I N G O U TC O M E Figure 8.2 (a) coRI, (b) Vectors H R ▲ FIGURE 8.2 Actions of representative restriction enzymes. estriction enzymes recognize and cut both strands of a DNA molecule at a specific (usually palindromic) restriction site. (a) Certain restriction enzymes produce staggered cuts with complementary “sticky ends.” (b) When two complementary sticky-ended fragments come from different organisms, their bonding (catalyzed by ligase) produces recombinant DNA. (c) Other restriction enzymes produce blunt-ended fragments. (d) A lack of specificity enables blunt-ended fragments produced by different restriction enzymes to be combined easily into recombinant DNA. Which restriction enzymes act at the restriction sites shown? • Vectors contain a recognizable genetic marker so that researchers can identify the cells that have received the vector and thereby the specific gene of interest. Genetic markers can be either phenotypic markers, such as those that confer antibiotic resistance or code for enzymes that metabolize a unique nutrient, or radioactive or fluorescent labels. • Vectors can ensure genetic expression by providing required genetic elements, such as promoters. An example of the process used to produce a vector containing a specific gene is depicted in (FIGURE 8.3). After a given restriction enzyme cuts both the DNA molecule containing the gene of interest (in this example, the human growth hormone gene) and the vector DNA (here, a plasmid containing a gene for antibiotic resistance as a marker) into fragments with sticky ends 1 , ligase anneals the fragments to produce a recombinant plasmid 2 . After the recombinant plasmid has been inserted into a bacterial cell 3 , the bacteria are grown on a medium containing the antibiotic 4 ; only those cells that contain the recombinant plasmid (and thus the human growth hormone gene as well) can grow on the medium. Generally, viruses and transposons are able to carry larger genes than can plasmids. Researchers are developing vectors 11/23/16 3:02 AM a t mRNA for human growth hormone (HGH) AAG T TC T T C AA G Antibioticresistance gene Restriction site enzyme was used in this example? Reverse transcription cDNA for HGH Plasmid (vector) 1 Restriction enzyme A T TC Restriction enzyme Gene Libraries G A AG C Gene for human growth hormone AG HGH A A G T GH T T C C G C T AH A T T A A T TC G T T A A Ligase A HGH A G C T T T AHGH T C G A AAG T TC C G T T A A Recombinant plasmid 3 from adenoviruses, poxviruses, and a genetically modified form of the human immunodeficiency virus (HIV). HIV in particular might make an excellent vector because HIV inserts itself directly into human chromosomes; however, scientists must ensure that viral vectors do not insert DNA into the middle of a necessary gene, leading to mutation and possibly causing cancer. Sticky ends A G C T T HGH A A HGH T T C G A 2 ◀ FIGURE 8.3 An example of the process for producing a recombinant vector. In this case, the vector is a plasmid.Which restriction Figure 8.3 HindIII.       Ch p er 8   Recombinant DNA Technology Introduce recombinant plasmid into bacteria. LE A R N I N G O UTC O M E 8.11 Explain the significance of gene libraries. Suppose you were a scientist investigating the effects of the genes for 24 different kinds of interleukins (proteins that mediate certain aspects of immunity). Having to isolate the specific genes for each type of interleukin would require much time, labor, and expense. Your task would be made much easier if you could obtain the genes you need from a gene library, a collection of bacterial or phage clones—identical descendants— each of which contains a portion of the genetic material of interest. In effect, each clone is like one book in a library in that it contains one fragment (typically a single gene) of an organism’s entire genome. Alternatively, a gene library may contain clones with all the genes of a single chromosome or the full set of cDNA that is complementary to an organism’s mRNA. As depicted in (FIGURE 8.4), genetic researchers can create each of the clones in a gene library by using restriction enzymes to generate fragments of the DNA of interest and then using ligase to synthesize recombinant vectors. They insert the vectors into bacterial cells, which are then grown on culture media. Once a scientist isolates a recombinant clone and places it in a gene library, the gene that the clone carries becomes available to other investigators, saving them the time and effort required to isolate that gene. Many gene libraries are now commercially available. For example, one library contains every gene from E. coli strain K-12 controlled by inducible promoters, so scientists can control gene expression at will. TELL ME WHY Bacterial chromosome 4 Recombinant plasmid Inoculate bacteria on media containing antibiotic. Bacteria containing the plasmid with HGH gene survive because they also have resistance gene. M08_BAUM7206_05_SE_C08.indd 242 Why did the discovery and development of restriction enzymes speed up the study of recombinant DNA technology? Techniques of Recombinant DNA Technology Scientists use the tools of recombinant DNA technology in a number of basic techniques to multiply, identify, manipulate, isolate, map, and sequence the nucleotides of genes. 11/23/16 3:02 AM Multiplying DNA In Vitro: The Polymerase Chain Reaction Genome LE A R N I N G O UTC O M E 8.12 Describe the purpose and application of the polymerase 1 Isolate genome of organism. chain reaction. The polymerase chain reaction (PCR) is a technique by which scientists produce a large number of identical molecules of a specific segment of DNA in vitro. Using PCR, researchers start with a single molecule of DNA and generate billions of exact replicas within hours. Such rapid and specific amplification of DNA is critical in a variety of situations. For example, epidemiologists used PCR to amplify the genomes of Ebola viruses from both western and central Africa in 2014. The large number of identical DNA molecules produced by PCR allowed scientists to determine and compare the nucleotide sequences of the two viruses. Because the two viruses differed significantly, epidemiologists were able to determine that the western epidemic had not escaped their quarantine procedures and spread to central Africa; instead, the central African outbreak was a separate epidemic. ▶ ANIMATIONS: Polymerase Chain Reaction (PCR): 2 Generate fragments using restriction enzymes. 1 2 7 3 8 4 9 5 6 10 11 3 Insert each fragment into a vector. 1 2 7 3 8 4 9 5 10 6 11 Overview, Components 4 Introduce vectors into cells. PCR is a repetitive process that alternately separates and replicates the two strands of DNA. Each cycle of PCR consists of the following three steps (FIGURE 8.5a): 1 2 3 4 5 6 8 9 7 11 5 Culture recombinant cells; descendants are clones. 2 3 4 5 6 7 8 9 10 11 ▲ FIGURE 8.4 Production of a gene library. A gene library is the population of all cells or phages that together contain all of the genetic material of interest. In this figure, each clone of cells carries a portion of a bacterium’s genome. M08_BAUM7206_05_SE_C08.indd 243 1 2 10 1       R Techniques of ecombinant DNA Technology 3 Denaturation. Exposure to heat (about 94°C) separates the two strands of the target DNA by breaking the hydrogen bonds between base pairs but otherwise leaves the two strands unaltered. Priming. A mixture containing an excess of DNA primers (synthesized such that they are complementary to nucleotide sequences at the ends of the target DNA), DNA polymerase, and an abundance of the four deoxyribonucleotide triphosphates (A, T, G, and C) is added to the target DNA. Because there is an excess of primers, single strands are more likely to bind to a primer than to one another. The primers provide DNA polymerase with the 3′ hydroxyl group it requires for DNA synthesis and bind only to specific sequences of 20–30 nucleotides. Extension. Raising the temperature to about 72°C increases the rate at which DNA polymerase replicates each strand to produce more DNA. ▶ ANIMATION: PCR:The Process These steps are repeated over and over 4 , so the number of DNA molecules increases exponentially (FIGURE 8.5b). After 30 cycles—which require only a few hours to complete—PCR produces over 1 billion identical copies of the original DNA molecule. The process can be automated using a thermocycler, a device that automatically performs PCR by continuously cycling all the necessary reagents—DNA, DNA polymerase, primers, and triphosphate deoxynucleotides—through the three temperature 11/23/16 3:02 AM t a       Ch p er 8   Recombinant DNA Technology Original DNA molecule 3¿ 3¿ 5¿ First cycle Second cycle Third cycle Fourth cycle 5¿ Heat to 945C 2 DNA molecules (b) 1 Denaturation 4 DNA molecules DNA primer Deoxyribonucleotide 2 Priming triphosphates 8 DNA molecules DNA polymerase 4 Repeat 16 DNA molecules Cool to 655C ▲ FIGURE 8.5 Use of the polymerase chain reaction (PCR) to replicate DNA. The artwork is not rendered to scale; for 5¿ 3¿ 5¿ 725C regimes. A thermocycler uses DNA polymerase derived from hyperthermophilic archaea or bacteria, such as Thermus aquat­ icus (ther′mŭs a-kwa′ti-kŭs). This enzyme, called Taq DNA poly­ merase or simply Taq, is not denatured at 94°C, so the machine need not be replenished with DNA polymerase after each cycle. Investigators use a variation of PCR—called real-time PCR—to accurately measure the number of DNA sequences present in a sample. For example, health care providers can use real-time PCR to determine the number of HIV genome copies in a patient’s blood sample. This is important in monitoring the progression of disease and effectiveness of treatment of diseases such as AIDS. Investigators perform real-time PCR by adding a dye that fluoresces when bound to double-stranded DNA (dsDNA). A photometer measures the amount of fluorescence—an amount that corresponds to the number of dsDNA molecules. As the amount of fluorescence increases with each cycle of PCR, the M08_BAUM7206_05_SE_C08.indd 244 R P E P (a) R DNA primer P 3 Extension example, primers are typically 20–30 nucleotides long. (a) ach cycle of C consists of three steps: 1 During denaturation at 94°C, the hydrogen bonds between DNA strands are broken, and the strands separate. 2 Cooling to 65°C in the presence of deoxyribonucleotide triphosphates, primers, and DNA polymerase allows priming to occur. 3 During extension at 72°C, warming speeds the action of DNA polymerase in replicating strands to produce more DNA. (b) ach cycle of C doubles the amount of DNA 4 ; over 1 billion copies of the original DNA molecule are produced by 30 cycles of C . E 5¿ 3¿ 5¿ R DNA polymerase number of new molecules of dsDNA can be ascertained. The number of original molecules can then be calculated. Combined with a step of reverse transcription, researchers can use real-time PCR to accurately determine the number of copies of a specific mRNA in a cell; with this information, they can determine the expression level of the corresponding gene. Selecting a Clone of Recombinant Cells LE A R N I N G O UTC O M E 8.13 Explain how researchers use DNA probes to identify recombinant cells. Before recombinant DNA technology can have practical application, a scientist must be able to select and isolate recombinant cells that contain particular genes of interest. For example, 11/23/16 3:02 AM once researchers have created a gene library, they must find the clone containing the DNA of interest. To do so, scientists use probes—which, as explained earlier in the chapter, bind specifically and exclusively to their complementary nucleotide sequences and have either radioactive or fluorescent markers. A probe of only 30 nucleotides in length can bind to one unique site, and thus one specific gene, in the human genome. Researchers then isolate and culture cells that have the radioactive or fluorescent marker, which also helps identify the specific location of the genes of interest, as performed in a technique called gel electrophoresis. Separating DNA Molecules: Gel Electrophoresis and the Southern Blot LE A R N I N G O U TC O M E       R Techniques of ecombinant DNA Technology of a purified sugar component of agar, called agarose, which acts as a molecular sieve that retards the movement of DNA fragments down the chamber and separates the fragments by size. Smaller DNA fragments move faster and farther than larger ones. Scientists can determine the size of a fragment by comparing the distance it travels to the distances traveled by standard DNA fragments of known sizes. As we have seen, DNA probes allow a researcher to find specific DNA sequences such as genes in a cell. Scientists could try to use probes in electrophoresis gels, but because gels are flimsy and easily broken, they are difficult to probe. In 1975, Ed Southern (1938–) devised a method, called the Southern blot, to transfer DNA from gels onto nitrocellulose membranes, which are less delicate. Fluorescently labeled probes are then added, and the probes are detected using a digital camera, which reveals the presence of DNA of interest. A northern blot is a similar technique used to detect specific RNA molecules. 8.14 Describe the process and use of gel electrophoresis, par- ticularly as it is used in a Southern blot. Electrophoresis (ē-lek-trō-fōr-ē′sis) is a technique that involves separating molecules based on their electrical charge, size, and shape. In recombinant DNA technology, scientists use gel electrophoresis to isolate fragments of DNA molecules that can then be inserted into vectors, multiplied by PCR, or preserved in a gene library. In gel electrophoresis, DNA molecules, which have an overall negative charge, are drawn through a semisolid gel by an electric current toward the positive electrode within an electrophoresis chamber (FIGURE 8.6). The gel is typically composed A D C 8.15 Describe the manufacture and use of DNA microarrays. Another tool of biotechnology is a DNA microarray. An array consists of molecules of single-stranded DNA (ssDNA), either genomic DNA or cDNA, immobilized on glass slides, silicon chips, or nylon membranes. Robots, similar to those that construct computer chips, deposit PCR-derived copies of hundreds or thousands of different DNA sequences in precise locations Electrophoresis chamber filled with buffer solution E (50) (40) (35) Agarose gel (15) a DNA LE A R N I N G O UTC O M E (–) Wells B DNA Microarrays Movement of DNA (10) (+) (5) b Wire Lane of DNA fragments of known sizes (kilobase pairs) (a) (b) E E ▲ FIGURE 8.6 Gel electrophoresis. (a) After DNA is cleaved into fragments by restriction enzymes, it is loaded into wells, which are small holes cut into the agarose gel. DNA fragments of known sizes, typically in thousands (kilo) of nucleotide base pairs, are often loaded into one well (in this case, ) to serve as standards. After the DNA fragments are drawn toward the positive electrode by an electric current, they are stained with a dye. (b) thidium bromide dye fluoresces under ultraviolet illumination to reveal the locations of DNA within a gel. Compare the positions of the fragments in lanes A and B of the diagram to the positions of the fragments of known sizes. What sizes are the fragments labeled a and b? Figure 8.6 a: 40 kilobase pairs; b: 10 kilobase pairs. M08_BAUM7206_05_SE_C08.indd 245 11/23/16 3:02 AM t a       Ch p er 8   Recombinant DNA Technology on an array (FIGURE 8.7). An array may consist of DNA from a single species (e.g., DNA microarrays containing sequences from all the genes of E. coli are available commercially), or an array may contain DNA sequences from numerous species. In any case, single strands of fluorescently labeled DNA in a sample washed over an array adhere only to locations on the array where there are complementary DNA sequences. Scientists use DNA microarrays in a number of ways, including the following: cDNA sequences from reverse transcription, a microbe, or a gene library Multiple copies of each single-stranded sequence are attached to substrate in precisely defined locations. • Monitoring gene expression. One way organisms control metabolism is by controlling RNA transcription. Scientists use DNA microarrays to monitor which genes a cell is transcribing at a particular time by making fluorescently labeled, single-stranded cDNA from mRNA in the cell. These DNA strands bind to complementary DNA sequences on an array, and the location of fluorescence on the array at specific sites reveals which genes the cell was transcribing at the time. For example, activity of normal cells can be compared to activity in cancerous cells to determine how gene expression is related to oncogenesis. DNA microarray Fluorescently labeled DNA Researchers using DNA microarrays can simultaneously monitor the expression of thousands of genes and compare and contrast genetic expression under different conditions. Different colors of fluorescent dye can be used to label DNA from microbes grown in each condition. (a) ­ • Diagnosing infection. DNA microarrays made with DNA sequences of numerous pathogens reveal the presence of those pathogens in medical samples. • Identifying organisms in an environmental sample. Microbial ecologists monitor the presence or absence of microbes in an environment by using microarrays of DNA from the organisms. Health care professionals can use microarrays to simultaneously detect and identify numerous bacterial and fungal species in clinical blood samples. Inserting DNA into Cells LE A R N I N G O U TC O M E 8.16 List and explain three artificial techniques for introduc- ing DNA into cells. • Electroporation (FIGURE 8.8a). Electroporation involves using an electrical current to puncture microscopic holes through a cell’s membrane so that DNA can enter the cell from the environment. Electroporation can be used on all types of cells, though the thick-walled cells of fungi and algae must first be converted to protoplasts, which are cells whose cell walls have been enzymatically M08_BAUM7206_05_SE_C08.indd 246 (b) P ▲ FIGURE 8.7 DNA microarray. (a) Construction and use of a microarray. Multiple copies of single-stranded DNA with known sequences are affixed in precise locations on a glass slide, silicon chip, nylon membrane, or other substrate. Fluorescently labeled DNA washed over the microarray binds to complementary strands. (b) hotograph of a DNA microarray showing locations of differently labeled cDNA molecules. ­ A goal of recombinant DNA technology is the insertion of a gene into a cell. In addition to using vectors and the natural methods of transformation of competent cells, transduction, and conjugation, scientists have developed several artificial methods to introduce DNA into cells, including the following: removed. Cells treated by electroporation repair their membranes and cell walls after a time. • Protoplast fusion (FIGURE 8.8b). When protoplasts encounter one another, their cytoplasmic membranes may fuse to form a single cell that contains the genomes of both 11/23/16 3:02 AM “parent” cells. Exposure to polyethylene glycol increases the rate of fusion. The DNA from the two fused cells recombines to form a recombinant molecule. Scientists often use protoplast fusion for the genetic modification of plants. • Injection. Two types of injection are used with larger eukaryotic cells. Researchers use a gene gun powered by a blank .22-caliber cartridge or compressed gas to fire tiny tungsten or gold beads coated with DNA into a target cell (FIGURE 8.8c). The cell eventually eliminates the inert metal beads. In microinjection, a geneticist inserts DNA into a target cell with a glass micropipette having a tip diameter smaller than that of the cell or nucleus (FIGURE 8.8d). Unlike electroporation and protoplast fusion, injection can be used on intact tissues, such as in plant seeds. In every case, foreign DNA that enters a cell remains in a cell’s progeny only if the DNA is self-replicating, as in the case       R Applications of ecombinant DNA Technology of plasmid and viral vectors, or if the DNA integrates into a cellular chromosome by recombination. TELL ME WHY Why wasn’t polymerase chain reaction (PCR) practical before the discovery of hyperthermophilic bacteria? Applications of Recombinant DNA Technology The importance of recombinant DNA technology lies not in the novelty, cleverness, or elegance of its procedures but in its wide range of applications. In this section we consider how recombinant DNA technology is used to solve various problems and create research, medical, and agricultural products. Pores in wall and membrane Chromosome Cell synthesizes new wall Electrical field applied Competent cell Recombinant cell DNA from another source (a) Electroporation Cell synthesizes new wall Cell walls Enzymes remove cell walls Polyethylene glycol Recombinant cell New wall Protoplasts Fused protoplasts (b) Protoplast fusion Micropipette containing DNA Blank .22caliber shell Nylon projectile Vent Target cell Suction tube to hold target cell in place Plate to stop nylon projectile Target cell DNA-coated beads Nylon projectile (c) Gene gun Target cell’s nucleus (d) Microinjection P E ▲ FIGURE 8.8 Artificial methods of inserting DNA into cells. (a) lectroporation, in which an electrical current applied to a cell makes it competent to take up DNA. (b) rotoplast fusion, in which enzymes digest cell walls to create protoplasts that fuse at a high rate when treated with polyethylene glycol. (c) A gene gun, which fires DNA-coated beads into a cell. (d) Microinjection, in which a solution of DNA is introduced into a cell through a micropipette. M08_BAUM7206_05_SE_C08.indd 247 11/23/16 3:02 AM t a       Ch p er 8   Recombinant DNA Technology Genetic Mapping LE A R N I N G O U TC O M E S 8.17 Describe genetic mapping and fluorescent in situ hybrid- ization, and explain their usefulness. 8.18 Define genomics and functional genomics. 8.19 Contrast Sanger sequencing and next-generation sequencing. One application of the tools and techniques of recombinant DNA technology is genetic mapping, which involves locating genes on a nucleic acid molecule. Genetic maps provide scientists with useful facts, including information concerning an organism’s metabolism and growth characteristics, as well as its potential relatedness to other microbes. For example, scientists have discovered a virus with a genetic map similar to those of certain hepatitis viruses. They named the new discovery hep­ atitis G virus because it presumably causes hepatitis, though it has not been demonstrated that the virus actually causes the disease. Locating Genes Until about 1970, scientists identified the specific location of genes on chromosomes by cumbersome, time-consuming, labor-intensive methods. Recombinant DNA techniques provide simpler and universal methods for genetic mapping. One technique for locating genes, called restriction frag­ mentation, was one of the earliest applications of restriction enzymes. In this technique, which is used for mapping the relative locations of genes in plasmids and viruses, researchers compare DNA fragments resulting from cleavages by several restriction enzymes to determine each fragment’s location relative to the others. If the researchers know the locations of specific genes on specific fragments, then elucidation of the correct arrangement of the fragments will reveal the relative locations of the genes on the entire DNA molecule. Using this method, scientists first completed the entire gene map of a cellular microbe—the bacterium H. influenzae— in 1995. Since then, geneticists have elucidated complete gene maps of numerous viruses and prokaryotic and eukaryotic organisms. Often a scientist wants to know where in the environment, clinical sample, or biofilm a particular microbial species is located. When researchers know of a particular gene exclusive to that organism, they can locate the gene and thereby the microbe using fluorescent in situ hybridization (FISH). In this method, scientists use fluorescent DNA probes that hybridize with their complementary target. Scientists using fluorescent microscopes to view such probes can determine where the gene and its organism are located (FIGURE 8.9). Using a number of different colors of fluorescent probes, researchers can locate numerous genes and the microbes that carry them simultaneously. FISH is used for a variety of purposes, including diagnosing disease, identifying microbes in environmental samples, and analyzing biofilms. M08_BAUM7206_05_SE_C08.indd 248 LM 3 μm ▲ FIGURE 8.9 Fluorescent in situ hybridization (FISH). Green-fluorescing cells are Staphylococcus aureus, whereas red-fluorescing cells are another species of Staphylococcus. The absence of fluorescence in some cells indicates that at least one other species is present in this blood sample. Genomics and Nucleotide Sequencing Genomics is the sequencing and analysis of the nucleotide bases of genomes of organisms, viruses, and eukaryotic mitochondria and chloroplasts. At first, scientists sequenced DNA molecules by selectively cleaving DNA at A, T, G, or C bases, separating the fragments by gel electrophoresis, and mapping the order in which overlapping fragments occur in a complete DNA molecule. Such cumbersome sequencing was limited to short DNA molecules, such as those of plasmids. Modern DNA sequencing began when Frederick Sanger (1918–2013) began using modified versions of each of the four DNA nucleotides—versions that terminate DNA replication. By using various amounts of these modified nucleotides and then examining numerous DNA fragments produced by termination, the genetic sequence could be pieced together. Scientists improved the Sanger method by incorporating four different fluorescent dyes—a different color for each nucleotide base—and then using an automated DNA sequencer to determine the sequence of colors emitted by the dyes. Scientists using the automated Sanger method, with machines often running 24 hours a day for months, have sequenced the entire genomes of numerous viruses, bacteria, and eukaryotic organisms. The Human Genome Project reached a milestone in 2001 by using automated Sanger techniques to sequence approximately 3 billion nucleotide base pairs that constitute a human genome. This was a significant step that has launched numerous investigations into the genetic basis of disease prevention and treatment. The original and the automated Sanger sequencing methods are considered first-generation DNA sequencing. Researchers now use next-generation sequencing (NGS). Among the variety of NGS methods, a popular one is four­color reversible termi­ nation sequencing. In this method, DNA synthesis is temporarily stopped after the addition of a single modified nucleotide. Each of the four kinds of nucleotide fluoresces a color specific to its base (A, T, G, or C): one color for each of the four bases. After 11/23/16 3:02 AM a record is made, the fluorescent dyes are removed and a new nucleotide is added as the process repeats. This process is illustrated in (FIGURE 8.10): Researchers attach identical ssDNA fragments (produced by PCR) at known locations on a slide 1 . The great advantage of NGS is to not sequence one piece of DNA at a time, but hundreds of millions; 1 Attach identical ssDNA fragments at known locations on a slide. Multiple copies of ssDNA       R Applications of ecombinant DNA Technology it is massively parallel. An automated sequencer then passes modified molecules of all four types of DNA nucleotides over the slide at once 2 . Each nucleotide carries a dye that can fluoresce a specific color—one color for each of the four nucleotide bases—and a modification to the nucleotide that stops DNA elongation. Each time a nucleotide is added to a growing DNA Template strand Multiple copies of a different template strand 2 Pass all four types of modified nucleotides over the slide. Each type of nucleotide is modified to fluoresce a color and has a molecular group that causes synthesis to cease. T A C G A T G C DNA polymerase G C A T ssDNA T C C A G G C 3 Laser shines across the slide, causing each fluorescent dye to flash its color. 5 The sequencer then removes the fluorescent dye and the stop group. Synthesis restarts, but stops as soon as another modified nucleotide is added. T A C G A T G C G C A T T C 4 A light detector records the event. A G G C T A C G A T G C G C A T T A C A G G C A G Fluorescent dye and chemical group that stops DNA synthesis. T G 6 The laser stimulates the dyes and another image is captured. 7 The process repeats until a complementary DNA strand has been synthesized for each ssDNA on the slide. T A C G 9 A T G C G C A T T A C G A T G C G C C G 8 The sequence of colors recorded by the light detector at each location corresponds to the sequence of nucleotides in the ssDNA molecules at that site. ▲ FIGURE 8.10 Next-generation DNA sequencing (NGS). Shown here is four-color reversible termination sequencing. M08_BAUM7206_05_SE_C08.indd 249 11/23/16 3:02 AM t a       Ch p er 8   Recombinant DNA Technology strand, synthesis of that strand ceases. The sequencer shines a laser across the slide, causing each fluorescent dye to flash its color, each color corresponding to its particular nucleotide 3 . A light detector records the event 4 . The sequencer then chemically removes the fluorescent dye and the chemical group that stopped elongation 5 . DNA synthesis begins again but stops as soon as modified nucleotides are added. The laser stimulates the dyes, and another image is captured before the dyes and stop groups are removed 6 . The process repeats until a complementary DNA strand has been synthesized for each fragment on the slide 7 . The sequence of colors recorded by the light detector at each location corresponds to the sequence of nucleotides in the ssDNA molecules at that site 8 . NGS, four-color reversible termination sequencing instruments can sequence an astounding 80 billion nucleotide bases in a few days. In contrast, the automated Sanger method would require almost seven years of nonstop sequencing to process the same number. With various sequencing methods, particularly NGS, researchers have ascertained the complete genomes of over 20,000 bacteria, about a thousand archaea, and of numerous eukaryotes. We are well into the genomic age. Elucidation of the gene sequences of pathogens, particularly those affecting hundreds of millions of people and those with potential bioterrorist uses, is a current priority of researchers. Scientists hope to use the information to develop novel drugs and more effective therapies and vaccines. TABLE 8.2 Another use for genomics is to relate DNA sequence data to protein function. For instance, scientists are investigating the genes and proteins of Deinococcus radiodurans (dō-nō-kok′ŭs rō-dē-ō-dur′anz), a microorganism that is remarkably resistant to damage of its DNA by radiation. Such studies may lead to methods of reversing genetic damage in cancer patients undergoing radiation therapy. Researchers are also investigating the genetic basis of the enzymes of psychrophiles, which are microorganisms that thrive at temperatures below 20°C. Such enzymes have potential applications in food processing and in the manufacture of drugs. TABLE 8.2 summarizes some tools and techniques of recombinant DNA technology. Functional Genomics Currently, a major emphasis in genomics is to determine what genes’ products do—this is the field of functional genom­ ics. A classic method to determine the function of a gene is a gene knockout, which involves removing a single gene from a bacterial genome while leaving the rest of the genome intact. Phenotypic analysis of the knockout strain may then reveal the gene’s function, since the bacteria will lack this function. The E. coli Keio Knockout collection (from Keio University in Japan) is a powerful resource for functional analysis: it contains 3985 strains, each having one nonessential gene replaced by a kanamycin-resistance gene. Tools and Techniques of Recombinant DNA Technology Description Potential Application Mutagen Chemical or physical agent that creates mutations Creating novel genotypes and phenotypes Synthetic nucleic acid R Synthesizing a gene using an m NA template DNA molecule prepared in vitro Creating DNA probes to localize genes within a genome Collection of cells or viruses, each of which carries a portion of a given organism’s genome rotoplast fusion Gene gun Microinjection roduces multiple copies of a DNA molecule U U ses electrical charge to separate molecules according to their size ses electrical current to make cells competent roviding a ready source of genetic material Multiplying DNA for various applications Separating DNA fragments by size Inserting a novel gene into a cell Fuses two cells to create recombinants Inserting a novel gene into a cell Blasts genes into target cells Inserting a novel gene into a cell U E P lectroporation R P olymerase chain reaction ( C ) Gel electrophoresis P Altering the genome of a cell Gene library P Creating recombinant DNA by joining fragments Transposon, plasmid, or virus that carries DNA into cells R Bacterial enzyme that cleaves DNA at specific sites Vector P estriction enzyme nzyme from NA retrovirus that synthesizes cDNA from an NA template R E everse transcriptase R R Tool or Technique ses micropipette to inject genes into cells Inserting a novel gene into a cell Localizes specific DNA sequences on a stable membrane Identifying a strain of pathogen Nucleic acid probes NA or DNA molecules labeled with radioactive or fluorescent tags Localizing specific genes in a Southern blot Genetic mapping ses restriction enzymes to locate relative positions of restriction sites Locating genes in an organism’s genome DNA sequencing Determines the sequence of nucleotide bases in DNA Comparing genomes of organisms DNA microarray eveals presence of specific DNA or NA molecules in a sample Diagnosing infection M08_BAUM7206_05_SE_C08.indd 250 R R U R Southern blot 11/23/16 3:02 AM       R Applications of ecombinant DNA Technology Pharmaceutical and Therapeutic Applications Another basic method in functional genomics is to overexpress a particular gene by enhancing its transcription or translation, resulting in an abundance of that gene’s product in the cell. Scientists can then analyze the phenotype or purify the individual protein for further analysis. There are gene expression libraries that contain every gene in a plasmid under control of a strong promoter for this purpose. LE A R N I N G O UTC O M E S 8.20 Describe seven potential medical applications of recom- binant DNA technology. 8.21 Describe the steps and uses of genetic screening. 8.22 Define gene therapy. Microbial Community Studies Researchers now supplement traditional biotechnology with recombinant DNA technology to produce a variety of pharmaceutical and therapeutic substances and to perform a host of medically important tasks. Here, we explore the use of recombinant DNA technology to synthesize medically important proteins, produce vaccines, screen for genetic diseases, treat genetic illnesses, diagnose diseases by matching DNA specimens to the organisms from which they came, aid in organ transplantation, and make animals that respond more similarly to humans when treated with various pathogens or experimental treatments. It is estimated that more than 99% of microorganisms have never been grown in a laboratory; indeed, scientists know them only by unique DNA sequences, sometimes called signatures or DNA fingerprints,. For example, based on such unique DNA sequences, scientists have detected DNA from over 500 species of bacteria from human mouths; however, they have been able to positively identify only about 150 species. An understanding of the biology of the other 350 species may lead to a better understanding of tooth and gum decay, diagnosis of disease, and advances in oral health care. Because next-generation DNA sequencing is relatively inexpensive and rapid, investigators can now determine complete genetic sequences for and relative abundance of all the members making up a microbiome, which is all the microorganisms in a particular environment. Beneficial Microbes: Our Other “Organ” examines the microbiome of humans and the effects it may have on our health. Protein Synthesis Scientists have inserted synthetic genes for insulin, for interferon (a natural antiviral chemical), and for other proteins into bacteria and yeast cells so that the microbes synthesize these proteins in vast quantities. In the past, such proteins were isolated from donated blood or from animals—labor-intensive E R E E B N FICIAL MIC OB S Our Other “Organ” R E P H P H M08_BAUM7206_05_SE_C08.indd 251 P As expected, the researchers didn’t find obligate pathogens in the healthy humans, though they commonly found opportunistic pathogens. A surprise to many researchers was that the fecal bacterium Escherichia coli, though present in the gut of most people, is actually rare, present at less than 1 in 1000 bacteria. The diversity of microbes among people was much greater than expected. For example, before the M , microbiologists taught that anaerobic bacteria in the phylum Bacteroidetes were the most common bacteria in everyone’s gut. Now, we know this is true for only some people; the actual percentage varies among these healthy individuals from 20% to 80% of the total number of bacteria. Obviously, the normal and healthy number of bacteroids for one person can be very different from the number in another. Another surprising finding was that although the taxonomic diversity at a particular body site varied widely among individuals, the abundance of bacterial genes representing certain metabolic pathways was highly consistent, suggesting that metabolism controls what organisms are there. This could possibly be medically significant: future therapies may focus on restoring certain biochemical activities to a patient’s gut microbiome, rather than simply adding specific bacteria. H H The number of cells in an adult human body is an astounding 10–40 trillion. owever, scientists estimate that perhaps ten times as many bacteria, fungi, and other microorganisms, plus innumerable viruses, cover the surfaces of our bodies. Four hundred trillion “guests”! You support this gigantic, symbiotic ecosystem—a microbiome, which can be defined as the community of all microbes in a particular environment. A major health benefit of the microbiome is that it prevents invading pathogens from attaching or persisting on your body surfaces. What if we could identify and categorize all of the different species of microorganisms that live in and on a body? This was the ambitious goal of the uman Microbiome roject ( M ). To begin, several hundred scientists from 80 institutions collected samples from 242 healthy volunteers. ighteen different body sites were sampled, including nine different oral locations, nostrils, ears, and the gut, which was represented by fecal samples. esearchers utilized direct DNA extraction and next-generation sequencing to discover DNA from almost every microbe in each sample. Because most microorganisms have not been cultured in the lab, the majority of the members of the microbiome as determined by DNA sequences had never before been observed in humans. 11/23/16 3:02 AM t a       Ch p er 8   Recombinant DNA Technology processes that carry the risk of inducing allergies or of transferring pathogens, such as hepatitis B and HIV. “Genetically engineered” proteins are safer and less expensive than their naturally occurring counterparts. Vaccine Production Vaccines contain antigens—foreign substances such as weakened bacteria, viruses, and toxins that stimulate the body’s immune system to respond to and subsequently remember these foreign materials. In effect, a vaccine primes the immune system to respond quickly and effectively when confronted with pathogens and their toxins. However, the use of some vaccines entails a risk—they may cause the disease they are designed to prevent. Scientists now use recombinant DNA technology to produce safer vaccines. Once they have inserted the gene that codes for a pathogen’s antigens into a vector, they can inject the recombinant vector or the proteins it produces into a patient. Thus, the patient’s immune system is exposed to a subunit of the pathogen—one of the pathogen’s antigens—but not to the pathogen itself. Such subunit vaccines are especially useful in safely protecting against pathogens that either cannot be cultured or cause incurable fatal diseases. Hepatitis B vaccine is an example of a successful subunit vaccine. Scientists are also pursuing subunit vaccines against HIV. A promising future approach to vaccination involves introducing genes coding for antigenic proteins of pathogens into common fruits or vegetables, such as bananas or beans. The immune systems of people or animals eating such altered produce would be exposed to the pathogen’s antigens and theoretically would develop immunological memory against the pathogen. Such a vaccine would have the advantages of being painless and easy to administer, and vaccination would not require a visit to a health care provider. Another type of vaccination involves producing a recombinant plasmid carrying a gene from a pathogen and injecting the plasmid into a human whose body then synthesizes polypeptides characteristic of the pathogen. The polypeptides stimulate immunological memory, readying the body to mount a vigorous immune response and prevent infection should it be exposed to the real pathogen. These DNA vaccines have great potential because the production and delivery method remains the same—the pathogen providing the antigen is the only difference—which should reduce costs. Genetic Screening Genetic mutations cause some diseases, such as inherited forms of breast cancer and Huntington’s disease. Laboratory technicians use DNA microarrays to screen patients, prospective parents, and fetuses for such mutant genes. This procedure, called genetic screening, can also identify viral DNA sequences in a patient’s blood or other tissues. For instance, genetic screening can identify HIV in a patient’s cells even before the patient shows any other sign of infection. M08_BAUM7206_05_SE_C08.indd 252 Gene Therapy An exciting use of recombinant DNA technology is gene therapy, in which missing or defective genes are replaced with normal copies. Scientists remove a few genetically defective cells—for example, cells that produce a defective protein—from a patient, insert normal genes, and replace the cells into the patient, curing the disease. Alternatively, plasmid, bacterial, or viral vectors could deliver genes directly to target cells within a patient. Gene therapy is often difficult in practice, primarily because it is difficult to get functioning genes into a sufficient number of human cells to affect the disease. Nevertheless, scientists are conducting more than 1700 clinical trials that use gene therapies to treat a wide variety of diseases. Doctors have successfully treated patients for severe combined immunodeficiency (SCID); metachromatic leukodystrophy, a hereditary neurological disorder; Wiskott-Aldrich syndrome, an immune system disorder; and choroidoeremia, a disease resulting in blindness. Other conditions that may respond well to gene therapy are cystic fibrosis, sickle-cell anemia, some types of hemophilia, muscular dystrophy, and diabetes. Medical Diagnosis Clinical microbiologists use PCR, fluorescent genetic probes, and DNA microarrays in diagnostic applications. They examine specimens from patients for the presence of gene sequences unique to certain pathogens, such as particular hepatitis viruses, cytomegalovirus, human immunodeficiency virus, or the bacterial pathogens of gonorrhea, tuberculosis, trachoma, and ulcers. These tests are rapid and highly specific. For almost all viruses and for many bacteria, molecular detection has replaced culturing and identification by biochemical testing in clinical labs. Xenotransplantation Xenotransplants 5 are animal cells, tissues, or organs introduced into the human body. For years physicians have performed xenotransplants, for instance, using valves from pig hearts to repair severely damaged human hearts. However, recombinant DNA technology may expand the possibilities. It is theoretically feasible to insert functional human genes into animals to direct them to produce organs and tissues for transplantation into humans. For example, scientists could induce pigs to produce humanlike cytoplasmic membrane proteins so that entire organs from pigs would not be rejected as foreign tissue by a transplant recipient. Biomedical Animal Models Researchers use common animals such as mice or zebra fish in biomedical research to understand mechanisms of cancer, genetic disorders, infectious disease, and microbiome interactions. The animals allow scientists to develop new diagnostic and therapeutic procedures for human and veterinary 5From Greek xenos, meaning “stranger.” 11/23/16 3:02 AM       R Applications of ecombinant DNA Technology medicine. Although the most common biomedical model, the mouse, is very similar to humans, it does not always accurately predict the responses of a human body. Using recombinant DNA technology, mouse physiology and tissues can be made “more human,” thus making the genetically altered animal more useful. For example, scientists have replaced mouse genes that code for antibody proteins with their human equivalents. The genetically altered mice make human antibodies when exposed to pathogens, making them useful models for research of infectious disease and vaccines. Agricultural Applications LE A R N I N G O U TC O M E DNA technology. Recombinant DNA technology has been applied to the realm of agriculture to produce transgenic organisms—recombinant plants and animals that have been altered for specific purposes by the addition of genes from other organisms. The purposes for which transgenic organisms, also called genetically modified organisms (GMOs), have been produced are many and varied and include herbicide resistance, tolerance to salty soils, resistance to freezing and pests, and improvements in nutritional value and yield. More than 15 million farmers in 29 countries grow transgenic crops on over 2.5 billion acres worldwide— more land than in the entire United States. ▲ FIGURE 8.11 Genetically modified papaya plants. lants on the left are dying from ringspot virus infection. Those on the right are modified to produce viral protein, which prevents infection by the virus. P 8.23 Identify six agricultural applications of recombinant The biodegradable herbicide glyphosate (Roundup) normally kills all plants—weeds and crops alike—by blocking an enzyme that is essential for plants to synthesize several amino acids. After scientists discovered and isolated a gene from Agrobac­ terium tumefaciens (ag′rō-bak-tēr′ē-um tū′me-fōsh-enz) that conveys tolerance to glyphosate, they produced transgenic crop plants containing the gene. As a result of this application of recombinant DNA technology, farmers can now apply glyphosate to a field of transgenic plants to kill weeds without damaging the crop. An added benefit is that farmers do not need to till the soil to suppress weeds during the growing season, reducing soil erosion by 80%. Most of the soybeans, corn, and cotton grown in the United States are genetically modified in this manner to be “Roundup ready.” Glyphosate-tolerant rice, wheat, sugar beets, and alfalfa strains are also available, as are soybeans and corn tolerant of other herbicides. particularly organic growers, have used Bt for over 30 years to reduce insect damage to their crops. Now, genes for Bt toxin have been inserted into a variety of crop plants, including potatoes, cotton, rice, and corn, so that they produce Bt for themselves. Insects feeding on such plants are killed, while humans and other animals that eat them are unharmed. By inserting genes for more than one type of Bt, scientists hope to forestall evolution of resistant insects. Scientists have also developed crops that are resistant to microbial diseases. Two cases illustrate. The water mold Phytophthora infestans (fō-tof′tho-ră in-fes′tanz) is the most devastating potato pathogen; it caused the great Irish potato famine of the 19th century, resulting in the deaths of at least a million people. The mold still causes many billions of dollars of crop damage each year. Scientists have now cloned genes from potato species resistant to Phytophthora; the cloned genes, multiplied by PCR, can be inserted into potato crops to reduce losses to farmers and increase available food for a growing world population. Papaya ringspot virus causes devastating harm to papayas, mottling and malforming leaves, streaking fruit, and eventually killing the entire plant. Researchers centered at the University of Hawaii saved that state’s papaya industry by inserting a gene for ringspot virus capsid into the papaya’s genome. When the plant produces the viral protein, it triggers protection by an unknown mechanism against infection by the ringspot virus. Genetically modified plants grow normally (FIGURE 8.11). Pest Resistance Salt Tolerance Strains of the bacterium Bacillus thuringiensis (ba-sil′ͥs thur-in-je-en′sis) produce a protein that, when modified by enzymes in the intestinal tracts of insects, becomes Bt toxin (Bt). Bt binds to receptors lining the insect’s digestive tract and causes the tissue to dissolve. Unlike some insecticides, Bt is naturally occurring, harmful only to insects, and biodegradable. Farmers, Years of irrigation have resulted in excessive salt buildup in farmland throughout the world, rendering the land useless for farming. Though salt-tolerant plants can grow under these conditions, they are not edible. Scientists have now successfully removed the gene for salt tolerance from these plants and inserted it into tomato and canola Herbicide Tolerance M08_BAUM7206_05_SE_C08.indd 253 11/23/16 3:02 AM t a       Ch p er 8   Recombinant DNA Technology plants to create food crops that can grow in soil so salty that it would poison normal crops. Not only do such transgenic plants survive and produce fruit, but they also remove salt from the soil, restoring the soil and making it suitable to grow unmodified crops as well. Researchers are now attempting to insert the gene for salt tolerance into rice, cotton, wheat, and corn. Freeze Resistance Ice crystals form more readily when bacterial proteins (from natural bacteria present in a field) are available as crystallization nuclei. Scientists have modified strains of the bacterium Pseudomonas (soo-dō-mō′nas) with a gene for a polypeptide that prevents ice crystals from forming. Crops sprayed with genetically modified bacteria can tolerate mild freezes, so the farmers no longer lose their crops to unseasonable cold snaps. Improvements in Nutritional Value and Yield Genetic researchers have increased crop and animal yields in several ways. For example, MacGregor tomatoes remain firm after harvest because the gene for the enzyme that breaks down pectin has been suppressed. This allows farmers to let the tomatoes ripen on the vine before harvesting and increases the tomatoes’ shelf life. Scientists suppressed the gene indirectly by inserting a promoter in the noncoding DNA strand that allows transcription of antisense RNA. The antisense RNA then binds to the gene’s mRNA, forming double-stranded RNA that makes it impossible to translate the pectin-catabolizing enzyme. Another example of agricultural improvement involves bovine growth hormone (BGH), which when injected into cattle enables them to more rapidly gain weight and produce 10% more milk. Also, use of BGH reduces the fat content in beef. Though BGH can be derived from animal tissue, it is more economical to insert the BGH gene into bacteria so that they produce the hormone, which is then purified and injected into farm and ranch animals. In yet another application, scientists have improved the nutritional value of rice by adding a gene for beta-carotene, which is a precursor to vitamin A. Vitamin A is required for human embryonic development and for vision in adults, and it is an important antioxidant that plays a role in ameliorating cancer and atherosclerosis (hardening of arteries). Recombinant DNA technology has progressed to the point that scientists are now considering transplanting genes coding for entire metabolic pathways rather than merely genes encoding single proteins. For instance, researchers are attempting to transfer into corn and rice all the genes that bacteria use to convert atmospheric nitrogen into nitrogenous fertilizer, in effect allowing the recombinant plants to produce their own fertilizer. Recombinant DNA tools and techniques allow scientists to examine, compare, and manipulate the genomes of microorganisms, plants, animals, and humans for a variety of purposes, including gene mapping and forensic, medical, and M08_BAUM7206_05_SE_C08.indd 254 agricultural applications. However, as with many scientific advances, concerns arise about the ethics and safety of genetic manipulations. The next section deals with these issues. TELL ME WHY Why don’t doctors routinely insert genes into their patients to cure the common cold, flu, or tuberculosis? The Ethics and Safety of Recombinant DNA Technology LE A R N I N G O UTC O M E 8.24 Discuss the pros and cons concerning the safety and eth- ics of recombinant DNA technology. Recombinant DNA technology provides the opportunity to transfer genes among unrelated organisms, even among organisms in different kingdoms, but how safe and ethical is it? No other change in agricultural practices has generated so much controversy. “Biological Russian roulette” and “Frankenfood” are some of the terms opponents use to denigrate gene therapy and genetically modified organisms. Critics of GMOs correctly state that the long-term effects of transgenic manipulations are unknown and that unanticipated problems arise from every new technology and procedure. Recombinant DNA technology may burden society with complex and unforeseen regulatory, administrative, financial, legal, social, medical, and environmental problems. Critics also argue that natural genetic transfer through sexual reproduction and processes such as transformation and transduction could deliver genes from transgenic plants and animals into other organisms. For example, if herbicideresistant plants cross-pollinate with related weed species, we might be cursed with weeds that are more difficult to kill. Opponents further express concern that transgenic organisms might trigger allergies or cause harmless organisms to become pathogenic. Some opponents of recombinant DNA technology desire to ban all genetically modified organisms and all products derived from them. The U.S. National Academy of Sciences, the U.S. National Research Council, the European Food Safety Authority (EFSA), and 81 research projects conducted between 1985 and 2012 by the European Union have not revealed any risks to human health or the environment from genetically modified agricultural products beyond those found with conventional plant breeding. In fact, the European Union concluded in 2001 that “the use of more precise technology and the greater regulatory scrutiny probably make them [GMOs] even safer than conventional plants and foods.” Additionally, studies have shown that Bt crops spare harmless and beneficial insects that would be 11/23/16 3:02 AM H Caroline’s grandmother suspected that she has dengue fever, an infection transmitted via a mosquito bite. Dr. Watson does an initial physical exam, gets a medical history (including recent travel), and draws blood for lab analysis. In addition, Dr. Watson notices some bleeding along Caroline’s gum line. Based on Caroline’s symptoms and her recent travel to Brazil, Dr. Watson also suspects Caroline may have dengue. e admits her to the hospital, where she can be constantly monitored. The results of Caroline’s blood work confirm the dengue diagnosis. There is no cure for dengue—rest, fluid replacement, pain relievers, and time for the immune system to conquer the infection are all that can be done. Caroline remains in the hospital, and within a week, she has recovered enough to return home. Dr. Watson tells Caroline to thank her grandmother for pushing her to go to the doctor—the relatively early diagnosis allowed for Caroline’s quick and complete recovery from dengue. M08_BAUM7206_05_SE_C08.indd 255       TELL ME WHY Why don’t scientists who work with recombinant DNA know all the long-term effects of their work? 1. A sample of Caroline’s blood was sent to the Centers for Disease Control and Prevention (CDC) for confirmation of dengue. The CDC uses real-time polymerase chain reaction (PCR) to confirm the presence of dengue virus in a blood sample. Explain how real-time PCR can be used to identify the presence of a specific pathogen in a blood sample. 2. The CDC also determines the particular strain of dengue in each sample using genetic analysis. 3. Similarities in DNA sequences indicate relatedness of different viruses. What method could the CDC use to determine the DNA sequence of the dengue virus isolated from Caroline’s blood sample? Provide a rationale for your choice. Check your answers to Micro in the Clinic Follow- p questions in the MasteringMicrobiology Study Area. U Cause for Concern? those entities have access to all the genetic information that results? Should businesses be allowed to have patents on and make profits from living organisms they have genetically altered? Should governments be allowed to require genetic screening and then force genetic manipulations on individuals to correct perceived genetic abnormalities that some claim are the bases of criminality, manic depression, risk-taking behavior, and alcoholism? Should HMOs, physicians, or the government demand genetic screening and then refuse to provide services related to the birth or care of supposedly “defective” children? We as a society will have to confront these and other ethical considerations as the genomic revolution continues to affect people’s lives in many unpredictable ways.  ­ killed if pesticides were used. With fewer harmful insects to transmit fungi, Bt corn crops contain less cancer-causing fungal toxin, a potential benefit to human health. As the debate continues, governments impose additional standards on laboratories involved in recombinant DNA technology. These are intended to prevent the accidental release of altered organisms or exposure of laboratory workers to potential dangers. Additionally, genetic researchers often design organisms to lack a vital gene so that they could not survive for long outside a laboratory. Security concerns arise because terrorists could apply the procedures used to create beneficial crops and animals to engineer biological weapons that are more infective or more resistant to treatment than their natural counterparts are. Though international treaties prohibit the development of biological weapons, Bacillus anthracis (an-thrō′sis) spores were used in bioterrorist attacks in the United States in 2001. Thankfully, the strain utilized was not genetically altered to realize its deadliest potential. Emergent recombinant DNA technologies raise numerous other ethical issues. Should people be routinely screened for diseases that are untreatable or fatal? Who should pay for these procedures: individuals, employers, prospective employers, insurance companies, health maintenance organizations (HMOs), or government agencies? What rights do individuals have to genetic privacy? If entities other than individuals pay the costs involved in genetic screening, should R E The thics and Safety of ecombinant DNA Technology 11/23/16 3:02 AM t a       Ch p er 8   Recombinant DNA Technology CHAPTER SUMMARY . The Role of Recombinant DNA Technology in Biotechnology (p. 238) 1. Biotechnology is the use of microorganisms to make useful products. Historically these include bread, cheese, soy sauce, and alcohol. 2. Recombinant DNA technology, also known as “genetic engineering,” is a new type of biotechnology in which scientists change the genotypes and phenotypes of organisms to benefit humans. The Tools of Recombinant DNA Technology (pp. 238–242) 2. Mutagens are chemical and physical agents used to create changes in a microbe’s genome to effect desired changes in the microbe’s phenotype. 3. The enzyme reverse transcriptase transcribes DNA from an RNA template; genetic researchers use reverse transcriptase to make complementary DNA (cDNA). 4. Scientists used synthetic nucleic acids to elucidate the genetic code, and they now use them to create genes for specific proteins and to synthesize DNA and RNA probes labeled with radioactive or fluorescent markers. o to id V e Tu r 6. In recombinant DNA technology, a vector is a small DNA molecule (such as a viral genome, transposon, or plasmid) that carries a particular gene and a recognizable genetic marker into a cell. 7. A gene library is a collection of bacterial or phage clones, each of which carries a fragment (typically a single gene) of an organism’s genome. ­ Techniques of Recombinant DNA Technology (pp. 242–247) 1. The polymerase chain reaction (PCR) allows researchers to replicate molecules of DNA rapidly. Real-time PCR can be used to measure the number of specific DNA or mRNA sequences in a sample. ▶ ANIMATIONS: Polymerase Chain Reaction (PCR): Overview, Components, The Process 2. Gel electrophoresis is a technique for separating molecules (including fragments of nucleic acids) by size, shape, and electrical charge. M08_BAUM7206_05_SE_C08.indd 256 4. DNA microarrays, containing nucleotide sequences of thousands of genes, are used to monitor gene activity and the presence of microbes in patients and the environment. 5. Geneticists artificially insert DNA into cells by electroporation, protoplast fusion, or injection. Applications of Recombinant DNA Technology (pp. 247–254) 1. The tools of recombinant DNA technology include mutagens, reverse transcriptase, synthetic nucleic acids, restriction enzymes, vectors, and gene libraries. 5. Restriction enzymes cut DNA at specific (usually palindromic) nucleotide sequences and are used to produce recombinant DNA molecules. ▶ ANIMATIONS: Recombinant DNA Technology 3. The Southern blot technique allows researchers to stabilize DNA sequences from an electrophoresis gel and then localize them using DNA dyes or probes. 1. Genomics is the sequencing (genetic mapping), analysis, and comparison of genomes. 2. Fluorescent in situ hybridization (FISH) uses fluorescent nucleic acid probes to localize specific genetic sequences. 3. Next-generation sequencing (NGS) has sped up determination of nucleotide sequences. Unique DNA sequences reveal the presence of microbes that have never been cultured in a laboratory. 4. Functional genomics involves determination of the function of a gene. This may be through the use of knockouts, which remove a single gene from a genome, or by overexpressing a gene so that its product builds up in a cell, allowing scientists to analyze the effect. 5. Determination of DNA sequences isolated in a particular environment allows scientists to deduce the presence of microbes that have been cultured or isolated by other methods. Complete sequences and relative abundance can be determined for the members of a microbiome, all the microorganisms in a specific environment. 6. Scientists synthesize subunit vaccines by introducing genes for a pathogen’s polypeptides into cells or viruses. When the cells, the viruses, or the polypeptides they produce are injected into a human, the body’s immune system is exposed to and reacts against relatively harmless antigens instead of the potentially harmful pathogen. 7. Genetic screening can detect infections and inherited diseases before a patient shows any sign of disease. 8. Gene therapy cures various diseases by replacing defective genes with normal genes. 9. Clinicians can use PCR, genetic probes, and DNA microarrays to rapidly diagnose diseases. 10. In xenotransplants involving recombinant DNA technology, human genes would be inserted into animals to produce cells, tissues, or organs for introduction into the human body. 11/23/16 3:02 AM 11. Transgenic organisms, also called genetically modified organisms (GMOs), are plants and animals that have been genetically altered by the inclusion of genes from other organisms. The Ethics and Safety of Recombinant DNA Technology (pp. 254–255) 1. Among the ethical and safety issues surrounding recombinant DNA technology are concerns over the accidental release of altered organisms into the environment and the potential for creating genetically modified biological weapons. VI W E RE STIONS FO R Q UE 12. Agricultural uses of recombinant DNA technology include advances in herbicide tolerance; salt tolerance; freeze resistance; pest resistance, such as insertion of the gene for Bt toxin (Bt) into plants; and improvements in nutritional value, yield, and shelf life.       R Questions for eview Answers to the Questions for Review (except Short Answer questions) begin on p. A-1. Multiple Choice 1. Which of the following statements is true concerning recombinant DNA technology? a. It will replace biotechnology in the future. b. It is a single technique for genetic manipulation. c. It is useful in manipulating genotypes but not phenotypes. d. It involves modification of an organism’s genome. 2. A DNA gene synthesized from an RNA template is __________________. a. reverse transcriptase c. recombinant DNA b. complementary DNA d. probe DNA 3. After scientists exposed cultures of Penicillium to agents X, Y, and Z, they examined the type and amount of penicillin produced by the altered fungi to find the one that is most effective. Agents X, Y, and Z were probably __________________. a. recombinant cells c. mutagens b. competent d. phages 4. Which of the following is false concerning vectors in recombinant DNA technology? a. Vectors are small enough to manipulate outside a cell. b. Vectors contain a recognizable genetic marker. c. Vectors survive inside cells. d. Vectors must contain genes for self-replication. 5. Which recombinant DNA technique is used to replicate copies of a DNA molecule? a. PCR c. electroporation b. gel electrophoresis d. reverse transcription 6. Which of the following would be most useful in following gene expression in a yeast cell? a. Southern blot c. DNA microarray b. reverse transcription d. restriction enzymes 7. Which of the following techniques is used regularly in the study of genomics? a. Clones are selected using a vector with two genetic markers. b. Genes are inserted to produce an antigenic protein from a pathogen. M08_BAUM7206_05_SE_C08.indd 257 c. Fluorescent nucleotide bases are sequenced. d. Defective organs are replaced with those made in animal hosts. 8. Restriction enzyme HhaI __________________. a. recombines DNA b. cuts DNA at a specific nucleotide sequence c. is likely derived from Haemophilus influenzae d. All of the above are correct. 9. Which application of recombinant DNA technology involves replacing a nonfunctional, mutated gene with a functional gene? a. gene therapy b. functional genomics c. genetic screening d. protein synthesis 10. A DNA microarray consists of __________________. a. a series of clones containing the entire genome of a microbe b. recombinant microbial cells c. restriction enzyme fragments of DNA molecules d. single-stranded DNA localized on a substrate Modified True/False Indicate which of the following are true and which are false. Rewrite any false statements to make them true by changing the underlined words. 1. __________________ Restriction enzymes inhibit the movement of DNA at specific sites. 2. __________________ Restriction enzymes act at specific nucleotide sequences within a double-stranded DNA molecule. 3. __________________ A thermocycler separates molecules based on their size, shape, and electrical charge. 4. __________________ Protoplast fusion is often used in the genetic modification of plants. 5. __________________ Gel electrophoresis is used in DNA microarrays. 11/23/16 3:02 AM t a       Ch p er 8   Recombinant DNA Technology Short Answer VISUALIZE IT! 1. Label the reagents and steps of PCR on the figure below. Indicate the temperature of the reaction at each numbered step. 1. Describe three artificial methods of introducing DNA into cells. 2. Why is cloning a practical technique for medical researchers? 3. Describe three ways scientists use synthetic nucleic acids. 4. Describe a gene library and its usefulness. 1 5. List three potential problems of recombinant DNA technology. 4 2 5¿ 3¿ 3¿ 5¿ 3 H R C ITICAL T INKING 1. Examine the restriction sites listed in Table 8.1 on p. 240. Which restriction enzymes produce restriction fragments with sticky ends? Which produce fragments with blunt ends? 2. A cancer-inducing virus, HTLV-1, inserts itself into a human chromosome, where it remains. How can a laboratory technician prove that a patient is infected with HTLV-1 even when there is no sign of cancer? 3. A thermocycler uses DNA polymerase from hyperthermophilic prokaryotes, but it cannot use DNA polymerase derived from E. coli. Why not? 4. How is the result of a Southern blot similar to the result of surveillance using a DNA microarray? 5. HhaI recognizes and cuts this DNA sequence at the sites indicated: G-C-G-C C-G-C-G 6. PCR replication of DNA is similar to bacterial population growth. If a scientist starts PCR with 15 DNA helices and runs the reaction for 15 cycles, how many DNA molecules will be present at the end? Show your calculations. 7. If a gene contains the sequence TACAATCGCATTGAA, what antisense RNA could be used to stop translation directly? 8. Suppose researchers learn that a particular congenital disease is caused by synthesis of a protein coded by a mutated gene. Describe a way in which recombinant DNA technology might be used to prevent translation of the protein. 9. Even though some students correctly synthesize a fluorescent cDNA probe complementary to mRNA for a particular yeast protein, they find that the probe does not attach to any portion of the yeast’s genome. Explain why the students’ probe does not work. Describe the fragments resulting from the use of this enzyme. M08_BAUM7206_05_SE_C08.indd 258 11/23/16 3:02 AM       Questions for Review CONCEPT MAPPING Using the following terms, fill in the following concept map that describes recombinant DNA technology. Southern blot Transposons Radioactive Restriction enzymes Reverse transcriptase Injection Mutagens Plasmids Protoplast fusion Blunt ends DNA probes Electroporation Fluorescent Recombinant DNA technology involves Tools Techniques include 1 2 3 Vectors DNA insertion methods uses can be Chemical 7 Polymerase chain reaction cut DNA into fragments with can be 8 Electrophoresis Physical which can be 4 Sticky ends 9 Viruses 5 6 include 11 M08_BAUM7206_05_SE_C08.indd 259 10 12 13 06/03/18 4:47 PM