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Chapter 13
Recombinant DNA: Cloning and Creation of Chimeric Genes
Outline
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13.1 Cloning
13.2 DNA Libraries
13.3 Polymerase Chain Reaction (PCR)
13.4 Recombinant DNA Technology
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
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 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
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
13.2 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 - 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!
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 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)
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