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Chapter 12: Recombinant DNA:
Cloning and Creation of Chimeric Genes
The Chimera of Arezzo, of
Etruscan origin, probably
from the 5th century B.C.
Chimeric DNA molecules
have opened up a new field
of scientific investigation.
“..how many vain
chimeras have you
created?...Go and take your
place with the seekers after
gold.”
Leonardo da Vinci
Essential Questions
• What are the methods that scientists use to
create recombinant DNA molecules?
• Can scientists create genes from recombinant
DNA molecules?
• Can scientists modify the heredity of an
organism using recombinant DNA?
Outline
• What Does It Mean: “To Clone”?
• What Is a DNA Library?
• Can the Cloned Genes in Libraries Be Expressed?
• What Is the Polymerase Chain Reaction (PCR)?
• How is RNA Interference Used to Reveal the Function
of Genes?
• Is It Possible to Make Directed Changes in the
Heredity of an Organism?
12.1 What Does It Mean “To Clone”?
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 Are Very Useful in Cloning Genes
• Plamids are 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
• These recombinant molecules can be
autonomously replicated, and hence propagated
Cloning Vectors
Cloning vectors are 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)
Plasmids as Cloning Vectors
Figure 12.1 One of
the first widely used
cloning vectors was
the plasmid pBR322.
Note the antibiotic
resistance genes
(ampr and tetr).
Virtually Any DNA Sequence Can Be
Cloned
Nuclease cleavage at a restriction site
linearizes the circular plasmid so that a
foreign DNA fragment can be inserted.
Recombinant plasmids are hybrid
DNA molecules consisting of plasmid
DNA sequences plus inserted DNA
elements (pink here).
Such hybrid molecules are called
chimeric plasmids.
Figure 12.2 An EcoRI restriction
fragment of foreign DNA can be
inserted into a plasmid.
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 12.2
Short DNA Duplexes With Restriction Sites
Can Be Used as Linkers
Figure 12.3 The use of
linkers to create tailormade ends on cloning
fragments.
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 12.4
Directional Cloning
Figure 12.4 Directional cloning.
DNA molecules whose ends
have different overhangs can
be used to form chimeric
constructs in which the foreign
DNA can enter the plasmid in
only one orientation.
Biologically Functional Chimeric Plasmids
• Plasmids can be used to transform recipient E. coli
cells
• (“Transformation” means the uptake and replication
of exogenous DNA by a recipient cell.)
• To facilitate transformation, the bacterial cells are
rendered somewhat permeable to DNA by Ca2+
treatment and a brief 42°C heat shock
• The useful upper limit on cloned inserts in plasmids
is about 10 kbp. Many eukaryotic genes exceed this
size.
Biologically Functional Chimeric Plasmids
Figure 12.5 A typical
bacterial transformation
experiment. Here
pBR322 is the cloning
vector.
Shuttle Vectors Are Plasmids That Can
Propagate in Two Different Organisms
Shuttle vectors are plasmids capable of
propagating and transferring (“shuttling”)
genes between two different organisms.
Figure 12.6 A typical shuttle vector. LEU2+ is a gene in the
yeast pathway for leucine biosynthesis.
12.2 What Is a DNA Library?
A DNA library is a set 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
12.2 What is a DNA Library?
The probabilities are daunting
• Consider the formula on page 361 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 disc
• Disc 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
What is a DNA Library?
Figure 12.7 Screening a genomic
library by colony hybridization. Host
bacteria transformed with a plasmidbased genomic library are plated on a
petri plate and incubated overnight to
allow bacterial colonies to form.
A replica of the colonies is obtained
by overlaying the plate with a flexible
disc composed of absorbent material
(such as nitrocellulose or nylon).
Probes for Southern Hybridization Can Be
Prepared in a Variety of Ways
Figure 12.8 Cloning genes using
oligonuceotide probes from a known amino
acid sequence. A radioactively labeled set
of DNA (degenerate) oligonucleotides
representing all possible mRNA coding
sequences is synthesized and is used to
probe the genomic library by colony
hybridization (see Figure 12.7).
Labeling methodologies other than
radioactivity are also available.
Identifying Specific DNA Sequences by
Southern Blotting
• Finding one particular DNA segment among a vast
population of different DNA fragments (e.g., in a
genomic DNA preparation) is to exploit its sequence
specificity to identify it.
• Southern blots (invented by E.M. Southern) do this
• DNA fragments (the “library”) are fractionated by size
with agarose gel electrophoresis
• Gel is blotted to an absorbent support and then
incubated with radioactively labeled oligonucleotide
probes
• An autoradiograph shows the hybridized DNA
fragments
Identifying Specific DNA Sequences by
Southern Blotting
The Southern blotting
technique involves the
transfer of
electrophoretically
separated DNA
fragments to an
absorbent sheet and
subsequent detection of
the specific DNA
sequences.
cDNA Libraries Are DNA Libraries
Prepared from mRNA
• cDNAs are DNAs copied from mRNA templates.
• cDNA libraries are constructed by synthesizing cDNA
from purified cellular mRNA.
• Because most eukaryotic mRNAs carry 3'-poly(A)
tails, mRNA can be selectively isolated from
preparations of total cellular RNA by oligo(dT)cellulose chromatography (Figure 12.9)
• DNA copies of the purified mRNAs are synthesized by
first annealing short oligo(dT) chains to the poly(A)
tails.
• These serve as primers for reverse transcriptasedriven synthesis of DNA (Figure 12.10)
cDNA Libraries Are DNA Libraries
Prepared from mRNA
Figure 12.9 Isolation of eukaryotic mRNA via
oligo(dT)-cellulose chromatography.
cDNA Libraries Are DNA Libraries
Prepared from mRNA
• Reverse transcriptase is an enzyme that synthesizes
a DNA strand, copying RNA as the template
• DNA polymerase is then used to copy the DNA
strand and form a double-stranded duplex DNA
• Linkers are then added to the DNA duplexes
rendered from the mRNA templates
• The cDNA is then cloned into a suitable vector
• Once a cDNA derived from a particular gene has
been identified, the cDNA becomes an effective
probe for screening genomic libraries for isolation of
the gene itself
cDNA Libraries Are DNA Libraries
Prepared from mRNA
Figure 12.10 Reverse
transcriptase-driven
synthesis of cDNA from
oligo(dT) primers annealed
to the poly(A) tails of
purified eukaryotic mRNA.
DNA Microarrays Are Arrays of Different
Oligonucleotides Immobilized on a Chip
• Robotic methods can be used to synthesize
combinatorial libraries of DNA oligonucleotides
directly on a solid support.
• The completed library is a 2-D array of different
oligonucleotides
• The final products of such procedures are referred to
as “gene chips” because the sequences synthesized
upon the chip represent the sequences of chosen
genes
• The oligonucleotides on such gene chips are used
as probes in hybridization experiments to reveal
gene expression patterns
DNA Microarrays Are Arrays of Different
Oligonucleotides Immobilized on a Chip
Figure 12.11 Gene chips (DNA
microarrays) in the analysis of
gene expression.
The Human Genome Project
• A working draft of the
human genome was
completed in June, 2000
and published in February
2001.
• The genomes of many
other organisms have now
been sequenced as well.
• Information about whole
genome sequences has
created a new branch of
science called
bioinformatics.
12.3 Can the Cloned Genes in Libraries
Be Expressed?
Expression vectors are engineered
so that the RNA or protein products
of cloned genes can be expressed.
Figure 12.12 Expression vectors
carrying the promoter recognized by
the RNA polymerase of bacteriophage
SP6 are useful for the production of
multiple RNA copies of any DNA
inserted at the polylinker.
12.3 Can the Cloned Genes in Libraries
Be Expressed?
To express a eukaryotic protein in E. coli, the eukaryotic
cDNA must be cloned in an expression vector that contains
regulatory signals for transcription and translation.
Figure 12.13 A typical expression-cloning vector.
12.3 Can the Cloned Genes in Libraries
Be Expressed?
Strong promoters have
been constructed to
drive synthesis of
foreign proteins to
levels of 30% of total E.
coli protein.
Figure 12.14 A ptac
protein expression
vector contains the
hybrid promoter ptac
derived from fusion
of the lac and trp
promoters.
12.3 Can the Cloned Genes in Libraries
Be Expressed?
Some expression vectors carry
cDNA inserts cloned directly into
the coding sequence of a
protein-coding gene.
Figure 12.15 A typical
expression vector for
the synthesis of a
hybrid protein.
12.3 Can the Cloned Genes in Libraries
Be Expressed?
Reporter Gene Constructs
Reporter gene constructs are
chimeric DNA molecules
composed of gene regulatory
sequences next to an easily
expressible gene product.
Figure 12.16 Green
fluorescent protein
(GFP) as a reporter
gene.
Specific Protein-Protein Interactions Can Be
Identified Using the Two-Hybrid System
Figure 12.17 The yeast twohybrid system for identifying
protein-protein interactions.
If proteins X and Y interact,
the lacZ reporter gene is
expressed. Cells
expressing lacZ exhibit βgalactosidase activity.
12.4 What Is the Polymerase Chain
Reaction (PCR)?
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
12.4 What Is the Polymerase Chain
Reaction (PCR)?
Figure 12.18 Polymerase chain
reaction (PCR).
In Vitro Mutagenesis
Figure 12.19 One method of PCRbased site-directed mutagenesis.
(1) Template DNA strands are
separate and amplified by PCR.
(2) Following many cycles of PCR,
the DNA product can be used to
transform E. coli cells.
(3) The plasmid DNA can be
isolated and screened for the
presence of the unique
restriction site (by restriction
endonuclease cleavage.
12.5 How Is RNA Interference Used to
Reveal the Function of Genes?
• RNA interference (RNAi) has emerged as a
method of choice in eukaryotic gene
inactivation
• RNAi leads to targeted destruction of a selected
gene’s transcript
• The consequences following loss of gene
function reveal the role of the gene product in
cell metabolism
12.6 Is It Possible to Make Directed
Changes in the Heredity of an Organism?
Figure 12.20 Gene knockdown by
RNAi.
The dsRNA is processed by
DICER. Following unwinding by
DICER, the guide strand is
delivered to the RISC complex.
The guide strand and a
complementary nucleotide are
brought together by Ago. Rnase
on Ago cleaves the gene transcript,
rendering it incapable of
translation by ribosomes.
Human Gene Therapy Can Repair Genetic
Deficiencies
A basic strategy of human gene therapy
involves incorporation of a functional
gene into target cells.
Retroviruses (RNA viruses that make
DNA from RNA) provide a route for
permanent modification of host cells ex
vivo.
Figure 12.21 Retrovirus-mediated
gene delivery ex vivo using MMLV.
Human Gene Therapy Can Repair Genetic
Deficiencies
Adenovirus vectors are a possible in
vivo approach to human gene therapy.
Figure 12.22 Adenovirus-mediated
gene delivery in vivo.
Adenoviruses are DNA viruses.
The Biochemical Defects in Cystic Fibrosis
and ADA- SCID