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Solomon IM Ch 15

Solomon IM Ch 15 - ,Berg,andMartinsBiology,9thEdition 15...

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Instructor’s Manual for Solomon, Berg, and Martin’s Biology, 9 th Edition 148 15 DNA Technology and Genomics Lecture Outline I. DNA cloning A. Research on bacteriophages laid the foundation for recombinant DNA methods. B. Restriction enzymes are “molecular scissors.” 1. Restriction enzymes cut DNA in areas of specific base pair sequences that are palindromic. 2. Staggered cuts in palindromic regions leave strands with complementary (“sticky”) ends. 3. Segments of DNA with “sticky” ends can be joined with DNA ligases. 4. Restriction enzymes vary in the number of DNA bases they recognize. a) A restriction enzyme that recognizes a large number of nucleotides has a low probability of having the enzyme recognition sequence in a given DNA and is useful in research on entire chromosomes. C. Recombinant DNA is formed when DNA is spliced into a vector. 1. Bacteriophages or plasmids are common vectors. a) Foreign DNA and vector DNA are cut with the same restriction enzyme, making them useful tools. b) Using antibiotics, the transformed colonies can be identified. 2. The vector can be introduced into the host cell by transformation. a) Transformation involves making the bacterial cell wall permeable to the plasmid. b) Plasmids often carry genes for resistance to antibiotics. c) Plasmids can carry a DNA segment of smaller than 10 kb. d) Bacteriophage vectors can carry segments of up to 23 kb. 3. Another type of vector is a cosmid cloning vector, which is a combination vector with characteristics of both bacteriophages and plasmids. 4. Bacterial artificial chromosomes (BACs) accommodate larger fragments of DNA (up to 200 kg of extra DNA). a) These are particularly of importance in the processes used in the Human Genome Project. 5. Engineered viruses can also be used as vectors in mammalian cells. D. DNA can be cloned inside cells. 1. A genomic library contains fragments of all of the DNA in the genome. a) A chromosome library contains all of the DNA fragments isolated from the individual chromosomes of the organism of interest. b) It is easier to isolate a gene of interest from a chromosome library than a genomic library. 2. Cloning techniques provide the means for replicating and isolating many copies of a specific recombinant DNA molecule.
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Chapter 15: DNA Technology and Genomics 149 a) After the restriction enzyme cuts the DNA to be cloned, the fragments, all with complementary ends, are of different lengths. b) The restriction enzyme cuts both the plasmid and the DNA of the organism with the gene of interest. c) The two types of DNA are mixed, which allows pairing of the complementary ends. d) The gene is then cloned in E. coli. e) The cells are incubated on a medium with antibiotics. f) The selection scheme allows only the cells with the recombinant genes to survive, as plasmids often confer resistance to antibiotics.
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