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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 13 Recombinant DNA: Cloning and Creation of Chimeric Genes to.

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Presentation on theme: "Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 13 Recombinant DNA: Cloning and Creation of Chimeric Genes to."— Presentation transcript:

1 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

2 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Outline 13.1 Cloning 13.2 DNA Libraries 13.3 Polymerase Chain Reaction (PCR) 13.4 Recombinant DNA Technology

3 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

6 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

7 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

8 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

9 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

10 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

11 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

12 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

13 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

14 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

15 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

16 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

17 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

18 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

19 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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!

20 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

21 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

22 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

23 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

24 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

25 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

26 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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)

27 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

28 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

29 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

30 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

31 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

32 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

33 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

34 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

35 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

36 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

37 Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company


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