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Chapter 13

Chapter 13. Recombinant DNA: Cloning and Creation of Chimeric Genes to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham.

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Chapter 13

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  1. Chapter 13 Recombinant DNA: Cloning and Creation of Chimeric Genes to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777

  2. Outline • 13.1 Cloning • 13.2 DNA Libraries • 13.3 Polymerase Chain Reaction (PCR) • 13.4 Recombinant DNA Technology

  3. 13.1 Cloning Clone: a collection of molecules or cells, all identical to an original molecule or cell • To "clone a gene" is to make many copies of it - for example, in a population of bacteria • Gene can be an exact copy of a natural gene • Gene can be an altered version of a natural gene • Recombinant DNA technology makes it possible

  4. Plasmids Naturally occurring extrachromosomal DNA • Plasmids are circular dsDNA • Plasmids can be cleaved by restriction enzymes, leaving sticky ends • Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid

  5. Cloning Vectors Plasmids that can be modified to carry new genes • Plasmids useful as cloning vectors must have • a replicator (origin of replication) • a selectable marker (antibiotic resistance gene) • a cloning site (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers

  6. Chimeric Plasmids Named for mythological beasts with body parts from several creatures • After cleavage of a plasmid with a restriction enzyme, a foreign DNA fragment can be inserted • Ends of the plasmid/fragment are closed to form a "recombinant plasmid" • Plasmid can replicate when placed in a suitable bacterial host • See Figure 13.3

  7. Directional Cloning Often one desires to insert foreign DNA in a particular orientation • This can be done by making two cleavages with two different restriction enzymes • Construct foreign DNA with same two restriction enzymes • Foreign DNA can only be inserted in one direction • See Figure 13.6

  8. 13.2 DNA Libraries Sets of cloned DNA fragments that together represent the genes of a particular organism • Any particular gene may represent a tiny, tiny fraction of the DNA in a given cell • Can't isolate it directly • Trick is to find the fragment or fragments in the library that contain the desired gene

  9. DNA Libraries - II The probabilities are staggering! • Consider the formula on page 406 for probability of finding a particular fragment in N clones • Suppose you seek a 99% probability of finding a given fragment in N clones of 10 kbp fragments • If your library is from the human genome, you would need 1,400,000 clones to reach 99% probability of finding the fragment of interest!

  10. Colony Hybridization A way to screen plasmid-based genome libraries for a DNA fragment of interest • Host bacteria containing a plasmid-based library of DNA fragments are plated on a petri dish and allowed to grow overnight to form colonies • Replica of dish made with a nitrocellulose disk

  11. Colony Hybridization • Disk is treated with base or heated to convert dsDNA to ssDNA and incubated with probes • Colonies that bind probe (with P-32) hold the fragment of interest

  12. Southern Blots Another way to find desired fragments • Subject the DNA library to agarose gel electrophoresis • Soak gel in NaOH to convert dsDNA to ssDNA • Neutralize and blot gel with nitrocellulose sheet • Nitrocellulose immobilizes ssDNA • Incubate sheet with labelled oligonucleotide probes • Autoradiography should show location of desired fragment(s)

  13. The Polymerase Chain Reaction What if you don't have enough DNA for colony hybridization or Southern blots? • The small sample of DNA serves as template for DNA polymerase • Make complementary primers • Add primers in more than 1000-fold excess • Heat to make ssDNA, then cool • Run DNA polymerase (usually Taq) • Repeat heating, cooling, polymerase cycle

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