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Chapter 17

BISC300 Chapter 17: BISC 300 - Chapter 17 Textbook Summary

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Biological Sciences
Cooper Carlton

Chapter 17: Recombinant DNA Technology 17.1: Key Discoveries Led to the Development of Recombinant DNA Technology Recombinant DNA is DNA with a new nucleotide sequence. Such DNA is formed by joining fragments from two or more different sources. These enzymes, known as restriction enzymes or restriction endonucleases, recognize and cleave specific sequences about four to eight base pairs long (figure 17.2). Restriction enzymes identify specific DNA sequences called recognition sites. Each restriction enzyme has its own recognition site. Hundreds of different restriction enzymes have been purified and are commercially available. When EcoRI cleaves between the G and A residues, unpaired 5′-AATTC-3′ remains at the end of each strand. They isolated the enzyme reverse transcriptase (RT) from retroviruses. These viruses have an RNA genome that is copied into DNA prior to replication. The mechanism by which reverse transcriptase accomplishes this is outlined in figure 17.5. Processed mRNA can be used as a template for complementary DNA (cDNA) synthesis in vitro. The resulting cDNA can then be cloned, without the need for RNA processing Agarose and polyacrylamide gel electrophoresis is now routinely used to separate and visualize DNA fragments. An oligonucleotide (Greek oligo, few or scant) probe, is a fragment of ssDNA complementary to the DNA of interest. • Streptavidin-Biotin Binding and Biotechnology - Egg white contains many proteins and glycoproteins with unique properties. One of the most interesting, which binds tenaciously to biotin, was isolated in 1963. This glycoprotein, called avidin due to its “avid” binding of biotin, was suggested to play an important role: making egg white antimicrobial by “tying up” the biotin needed by many microorganisms. Avidin, which functions best under alkaline conditions, has the highest known binding affinity between a protein and a ligand. Several years later, scientists at Merck & Co., Inc., discovered a similar protein produced by the actinomycete Streptomyces avidini, which binds biotin at a neutral pH and does not contain carbohydrates. These characteristics make this protein, called streptavidin, an ideal binding agent for biotin, and it has been used in an almost unlimited range of biotechnology applications. The streptavidin protein is joined to a probe. When a sample is incubated with the biotinylated binder, the binder attaches to any available target molecules. The presence and location of target molecules can be determined by treating the sample with a streptavidin probe because the streptavidin binds to the biotin on the biotinylated binder, and the probe is then visualized. This detection system is employed in a wide variety of biotechnological applications, including use as a nonradioactive probe in hybridization studies and as a critical component in biosensors for a wide range of environmental monitoring and clinical applications. 17.2: Polymerase Chain Reaction Amplifies Targeted DNA polymerase chain reaction (PCR): An in vitro technique used to synthesize large quantities of specific nucleotide sequences from small amounts of DNA. It employs oligonucleotide primers complementary to specific sequences in the target gene and special heat-stable DNA polymerases (e.g., Taq polymerase). Quite simply, it enables the rapid synthesis of billions of copies of a specific DNA fragment from a complex mixture of DNA molecules. Suppose that one wishes to make large quantities of a particular gene or other DNA sequence, a process known as gene or DNA amplification. 17.3: Cloning Vectors Are Needed to Create Recombinant DNA Plasmids make excellent cloning vectors because they replicate autonomously (i.e., independently of the chromosome) and are easy to purify. These plasmids are called shuttle vectors because they can be transferred, or “shuttle,” from one host to another. Alternatively, two different, unique sites may be cleaved and the DNA sequence between the two sites replaced with cloned DNA. Plasmids used for cloning have been designed with many restriction sites clustered in a single region called the mu
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