16.1 Molecular biologists can manipulate DNA to clone genes.
The DNA Manipulator's Toolbox
• Restriction endonucleases are able to cleave DNA at specific restriction sites. (p. 320)
• DNA ligase joins DNA fragments by catalyzing the formation of phosphodiester bonds between DNA nucleotides. (p. 320)
Host/Vector Systems
• The ability to propagate DNA in a host cell requires a vector that can enter and replicate in the host. The two most commonly used vectors are plasmids and phages. (p. 321, p. 324)
• Using these vectors, the gene or genes of interest are introduced into the target organisms, where they infect cells and replicate the altered vector DNA. (p. 322)
Using Vectors to Transfer Genes
• Viruses and artificial chromosomes can also be used as vectors to insert foreign DNA into host cells, creating recombinant DNA and recombinant genomes. (p. 323)
DNA Libraries
• A DNA library is a collection of DNA fragments from a specific source in a form that can be propagated in a host. (p. 324)
• A genomic library is a representation of the entire genome in a vector. (p. 324)
16.2 Genetic engineering involves easily understood procedures.
The Four Stages of a Genetic Engineering Experiment
• The four stages of a genetic engineering experiment are (1) cleaving the source DNA, (2) producing recombinant DNA, (3) cloning copies of the recombinants, and (4) screening the cloned copies for the desired genes. (pp. 325-328)
Working with Gene Clones
• One approach to making enough DNA to work with is to use DNA polymerase to copy the gene of interest through the polymerase chain reaction (PCR). (p. 329)
• The three steps in PCR are denaturation, primer annealing, and primer extension. (p. 329)
• After 20 cycles, a single fragment produces more than one million copies of the target sequence. (p. 329)
• In the Southern Blotting process, DNA is cleaved into restriction fragments, and the fragments are then separated by gel electrophoresis. Fragments that contain the gene of interest can then be identified when a radioactively labeled probe is washed over paper that contains a copy of the gel's pattern (p. 330)
• Restriction fragment length polymorphisms (RFLP) analysis and DNA fingerprinting are both used to identify individuals and specific unknown gene sequences. (pp. 331-332)
16.3 Biotechnology is producing a scientific revolution.
Medical Applications
• Pharmaceuticals, gene therapy, and piggyback vaccines are all medical applications that have been developed from biotechnology advances. (pp. 333-334)
Agricultural Applications
• Increased nitrogen fixation, herbicide resistance, insect resistance, and dietary deficiencies are all agricultural problems that are currently being addressed via gene technology and transgenic species. (pp. 335-338)
Risk and Regulation
• Genetic engineering has the potential to create great medical and agricultural advances, although many people are concerned with possible risks of genetic engineering, especially those associated with transgenic species. (p. 340)
• In short, the potential benefits of genetic engineering appear to outweigh the potential risks. (p. 340)