Bio97_09+L18 - 1 Cloning a gene creating a transgenic...

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©2003 C. Brachmann 1 Cloning a gene, creating a transgenic & cloning a sheep Bio Sci 97 Lecture 18 Dr. Carrie Brachmann Office hours today 12-2 Friday 12:30-2:30 McGaugh Hall 4213
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©2003 C. Brachmann 2 Outline cloning a gene restriction enzymes vector making recombinant DNA making a transgenic plant transgenic animals gene targeting cloning a mammal pedigree analysis and human gene mapping
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©2003 C. Brachmann 3 Cloning: definition copying biological stuff human identical twins are biological copies (naturally occurring) artificially creating an organism that is genetically identical to another organism (later in this lecture)= reproductive cloning making many copies of a gene= recombinant DNA cloning
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©2003 C. Brachmann 4 Why clone a gene? Greatly aids further study of that gene, and its use in diagnosis or treatment A particular gene can be isolated and sequenced Control sequences of DNA can be identified and analyzed Protein/RNA function can be investigated Mutations can be identified (e.g. gene defects related to specific diseases) Organisms can be engineered for specific purposes (e.g. insulin production, insect resistance, etc.) Copyright © 2008 Dr. Salme Taagepera, All rights reserved.
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Gene cloning: an overview 5 In gene cloning, DNA fragments (genes) are: isolated inserted into suitable vector molecules introduced into a host cell The host cell replicates, thereby producing copies (clones) of the gene Copyright © 2008 Dr. Salme Taagepera, All rights reserved. Re c o m binant DNA
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©2003 C. Brachmann 6 A vector Serves as a carrier to transfer foreign genetic material into a cell Three types: Plasmid, circular, 3-10 kb in size Phage, packages 12-20 kb recomb DNA into phage head Cosmid, 40-45 kb, either plasmid or phage Can be introduced easily into host cells to be replicated (almost exclusively bacteria- E. coli)
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©2003 C. Brachmann 7 A vector Is not a chromosome and does not become part of (integrate into) the host chromosome Propagates independently as an extra- chromosomal element--contains a bacterial replication origin
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©2003 C. Brachmann 8 Components of a vector Bacterial replication origin Gene to make the bacteria resistant to ampicillin (or other antibiotic) Restriction sites into which the DNA of interest can be introduced. The restriction site region is called a polylinker or multiple cloning site
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9 R ORI GAATTC CTTAAG Antibiotic resistance gene Replication origin Useful Restriction site A plasmid vector ©1999 Lee Bardwell
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©2003 C. Brachmann 10 DNA cloning utilizes restriction endonucleases Cleave insert and vector DNA with the same restriction enzyme -”sticky ends” Complementary sticky ends base pair and are “glued together” or ligated using an enzyme called DNA ligase .
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