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, Sea Harbor Drive, Orlando, Florida

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. Naturally occurring extrachromosomal DNA
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

6. Plasmids that can be modified to carry new genes
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

8. Named for mythological beasts with body parts from several creatures
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

12. Often one desires to insert foreign DNA in a particular orientation
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

17. 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

19. The probabilities are staggering!
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. 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

22. 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

26. Another way to find desired fragments
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)

33. 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