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

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 32887-6777



Outline

• 13.1 Cloning

• 13.2 DNA Libraries

• 13.3 Polymerase Chain Reaction (PCR)

• 13.4 Recombinant DNA Technology



13.1 CloningClone: 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



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





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





Chimeric PlasmidsNamed 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









Directional CloningOften 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











13.2 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





DNA Libraries - IIThe 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!



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





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









Southern BlotsAnother 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)















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







