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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
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Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Outline
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13.1 Cloning
13.2 DNA Libraries
13.3 Polymerase Chain Reaction (PCR)
13.4 Recombinant DNA Technology
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
13.1 Cloning
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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 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
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
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
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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
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
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Southern Blots
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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)
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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 ssDNA, then cool
• Run DNA polymerase (usually Taq)
• Repeat heating, cooling, polymerase cycle
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company