Biochemistry 2e Garrett Grisham Chapter 13 Recombinant DNA

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

Biochemistry 2/e - Garrett & Grisham Outline • • 13. 1 Cloning 13. 2

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

Biochemistry 2/e - Garrett & Grisham Plasmids Naturally occurring extrachromosomal DNA • Plasmids are circular ds. DNA • 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 Copyright © 1999 by Harcourt Brace & Company

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

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

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

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

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

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

Biochemistry 2/e - Garrett & Grisham Colony Hybridization A way to screen plasmid-based genome libraries for a DNA fragment of interest • Host bacteria containing a plasmidbased 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 Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Colony Hybridization • Disk is treated with base or heated to convert ds. DNA to ss. DNA and incubated with probes • Colonies that bind probe (with P-32) hold the fragment of interest Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Southern Blots • • • Another way to find desired fragments Subject the DNA library to agarose gel electrophoresis Soak gel in Na. OH to convert ds. DNA to ss. DNA Neutralize and blot gel with nitrocellulose sheet Nitrocellulose immobilizes ss. DNA Incubate sheet with labelled oligonucleotide probes Autoradiography should show location of desired fragment(s) Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham 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 ss. DNA, then cool • Run DNA polymerase (usually Taq) • Repeat heating, cooling, polymerase cycle Copyright © 1999 by Harcourt Brace & Company