Cleaving and Rejoining DNA
► Knowledge of DNA transcription, translation, and replication has been used to create recombinant DNA molecules, made up of sequences from different organisms.
► Restriction enzymes, which are made by bacteria as a defense against viruses, bind to DNA at specific recognition sequences and cut it. Review Figure 16.1
► DNA fragments generated from cleavage by restriction enzymes can be separated by size using gel electrophoresis. The sequences of these fragments can be further identified by hybridization with a probe. Review Figures 16.2, 16.3. See Web/CD Tutorial 16.1
► Many restriction enzymes make staggered cuts in the two strands of DNA, creating "sticky ends" with unpaired bases. These sticky ends can be used to create recombinant DNA if DNA molecules from different species are cut with the same restriction enzyme. Review Figure 16.4
Getting New Genes into Cells
► Bacteria, yeasts, and cultured plant cells are commonly used as hosts for recombinant DNA procedures.
► Newly introduced DNA must be part of a replication unit if it is to be propagated in host cells. One way to make sure that the transfected DNA is part of such a unit is to insert it into a vector.
► There are specialized vectors for transfecting bacteria, yeasts, and plant cells. These vectors must contain an origin of replication, recognition sequences for restriction enzymes, and reporter genes to identify their presence in host cells. Review Figure 16.5
► Reporter genes conferring nutritional, antibiotic resistance, or fluorescent phenotypes can be used to identify which host cells have taken up the recombinant vector. Review Figure 16.6
► The cutting of DNA by a restriction enzyme produces many fragments that can be individually and randomly combined with a vector and inserted into a host to create a gene library. Review Figure 16.7
► The mRNAs produced in a certain tissue at a certain time can be extracted and used to create complementary DNA (cDNA) by reverse transcription. Review Figure 16.8
► A third source of DNA is synthetic DNA made by chemists in the laboratory. The methods of organic chemistry can be used to create or mutate DNA sequences.
Some Additional Tools for DNA Manipulation
► Homologous recombination can be used to "knock out" a gene in an organism. Review Figure 16.9
► DNA chip technology permits the screening of thousands of sequences at the same time. Review Figure 16.10. See Web/CD Tutorial 16.2
► An antisense or interfering RNA complementary to a specific mRNA can prevent translation of the mRNA by hybridizing with it. Review Figure 16.11
► A two-hybrid system allows scientists to determine which proteins interact in cells. Review Figure 16.12
Biotechnology: Applications of DNA Manipulation
► Recombinant DNA techniques have made possible many new applications of biotechnology, such as the large-scale production of eukaryotic gene products.
► Expression vectors carry sequences such as promoters and transcription terminators that allow a gene of interest to be expressed in a host cell. Review Figure 16.13. See Web/CD Activity 16.1
► Recombinant DNA techniques have been used to make medically useful proteins that would otherwise have been difficult to obtain in necessary quantities. Review Figure 16.14, Table 16.1
► Because recombinant DNA technology has several advantages over traditional agricultural biotechnology, it is being extensively applied to agriculture. Review Table 16.2
► Because plant cells can be cloned to produce adult plants, the introduction of new genes into crop plants has been advancing rapidly. Transgenic crop plants can be adapted to their environment, instead of vice versa.
► "Pharming" uses transgenic animals that produce useful products in their milk.
► There is public concern about the application of recombinant DNA technology to food production.
► Because the DNA of an individual is unique, the polymerase chain reaction can be used to identify an organism from a small sample of its cells—that is, to create a DNA fingerprint. Review Figures 16.17, 16.18
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This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.