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INTRODUCTION
•  Recombinant DNA technology is the use of in vitro molecular
techniques to isolate and manipulate fragments of DNA
•  In the early 1970s, researchers at Stanford University were
able to construct chimeric molecules called recombinant DNA
molecules
–  Shortly thereafter, it became possible to introduce such molecules into
living cells where they are replicated to make many identical copies
–  This achievement ushered in the era of gene cloning
•  Recombinant DNA technology and gene cloning have been
fundamental to our understanding of gene structure and
function
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18-2
18.1 GENE CLONING
•  The term gene cloning refers to the technique of
isolating and making many copies of a gene
•  The laboratory methods that are necessary to clone
a gene were devised during the early 1970s
–  Since then, many technical advances have enabled gene
cloning to become a widely used procedure in science
•  Table 18.1 summarizes some of the more common
uses of gene cloning
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18-3
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18-4
Cloning Experiments Involve
Chromosomal and Vector DNA
n
Cloning experiments usually involve two kinds of
DNA molecules
n
Chromosomal DNA
n
n
Vector DNA
n
n
n
Serves as the source of the DNA segment of interest
Serves as the carrier for the DNA segment that is to be cloned
Can replicate independently of the host chromosomal DNA
To prepare chromosomal DNA, the scientist has to
n
n
n
Obtain cellular tissue from the organism of interest
Break open the cells
Extract and purify DNA using a variety of biochemical techniques
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18-5
n
The cell that harbors the vector is called the host cell
n
n
n
When a vector is replicated inside a host cell, the DNA that
it carries is also replicated
The sequence of the origin of replication determines whether a vector can replicate in a
particular host cell
The vectors commonly used in gene cloning were
originally derived from two natural sources
n
n
n
1. Plasmids
2. Viruses
Many naturally occurring plasmids have selectable
markers
n
n
Typically, genes conferring antibiotic resistance to the host cell
Table 18.2 provides a general description of several
vectors used to clone small segments of DNA
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18-6
18-7
Cloning Experiments Involve
Enzymes that Cut and Join DNA
n
n
Insertion of chromosomal DNA into a vector
requires the cutting and joining of DNA fragments
The enzymes used to cut DNA are known as
restriction endonucleases or restriction enzymes
n
n
These bind to specific DNA sequences and then cleave
the DNA at two defined locations, one on each strand
Figure 18.1 shows the action of a restriction
endonuclease
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18-8
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DNA from 2 different sources
5′
3′
T T C
G A A
A A G
C T T
3′
T T C
G A A
A A G
C T T
5′
EcoRI recognition"
sequence"
A A
T T C
G
3′
T T C
G A A
A A G
C T T
3′
5′
Incubate both DNAs with EcoRI,"
which cuts the DNA backbone"
between G and A.
A sticky"
3′end "
5′
3′
5′
T T C
G A A
A A G
C T T
5′
G
A A
C T T
3′
T T C
G
A sticky"
3′
end
A A
5′
G
A A
C T T
5′
Cleavage by restriction enzymes is the first step to making
recombinant DNA. In this case, the ends are sticky in that
they are short, single-stranded regions of DNA that can basepair with another piece of DNA with complementary sequence
(e.g. other DNA cut with the same enzyme)
Figure 18.1 (partial)
18-9
n
n
Restriction enzymes were discovered in the 1960s
and 1970s by Werner Arber, Hamilton Smith and
Daniel Nathans
Restriction enzymes are made naturally by many
species of bacteria
n
n
They protect bacterial cells from invasion by foreign DNA,
particularly that of bacteriophage
Currently, several hundred different restriction
enzymes are available commercially
n
Table 18.3 gives a few examples
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18-10
18-11
n
Restriction enzymes bind to specific DNA sequences
n
These are typically palindromic
n
n
The sequence is identical when read in the opposite direction in the
complementary strand
For example, the EcoRI recognition sequence is
5
3
n
Some restriction enzymes digest DNA into
fragments with sticky ends (see figure 18.1)
n
n
GAATTC 3
CTTAAG 5
These DNA fragments will hydrogen bond to each other
due to their complementary sequences
Other restriction enzymes generate blunt ends
n
The enzyme NaeI (Refer to Table 18.3)
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18-12
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DNA from 2 different sources
5′
3′
T T C
G A A
A A G
C T T
T T C
G A A
A A G
C T T
3′
T T C
G A A
A A G
C T T
5′
T T C
G A A
A A G
C T T
3′
EcoRI recognition"
sequence"
5′
Incubate both DNAs with EcoRI,"
which cuts the DNA backbone"
between G and A.
A sticky"
3′end "
5′
A A
3′
5′
T T C
G
5′
G
A A
C T T
3′
3′
T T C
A A
G
A sticky"
3′
end
5′
This interaction is not stable because
it involves only a few hydrogen bonds
G
A A
C T T
5′
Incubate the DNAs"
together, allowing sticky"
ends to hydrogen bond.
5′
3′
A A
T T C
G
G A A T T C
C T T A A G
G
C T T A A
3′
5′
To establish a permanent connection, the
sugar-phosphate backbones of the two
DNA fragments must be covalently linked
Add DNA ligase, which"
covalently links the"
DNA backbones.
Covalent bond
5′
A recombinant
DNA molecule
3′
A A
T T C
G
T T C
G A A
A A G
C T T
3′
G
A A
C T T
5′
Covalent bond
Figure 18.1
A recombinant DNA molecule
18-13
The Steps in Gene Cloning
n
n
The general strategy followed in a typical cloning
experiment is outlined in Figure 18.2
The procedure shown seeks to clone the human
β-globin gene into a plasmid vector
n
The vector carries two important genes
n
ampR à Confers antibiotic resistance to the host cell
n
n
Identifies cells that have taken up the vector
lacZ à Encodes β-galactosidase
n
n
Provides a means by which bacteria that have picked up the cloned
gene can be identified
More on that later
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18-14
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ampR gene
lacZ gene
Plasmid DNA
Origin of"
replication
Gene of interest
Unique"
restriction"
site
Chromosomal DNA"
from human cells
Cut the DNAs with the"
same restriction enzyme.
Digestion of DNA
from a human cell
would actually
produce tens of
thousands of
fragments.
Mix the DNAs together. Allow time for"
sticky ends to base-pair. Add DNA ligase"
to covalently link the DNA backbones.
Vector with the"
gene of interest
Recircularized"
vector
or
Vector with"
another fragment"
of chromosomal DNA
or
Recombinant"
vectors
This is termed
a hybrid vector
Figure 18.2
18-15
This step of the procedure is
termed transformation when
plasmid vectors are used, and
transfection when a viral vector is
introduced into a host cell
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Vector with the"
gene of interest
Recircularized"
vector
or
Vector with"
another fragment"
of chromosomal DNA
or
Recombinant"
vectors
Mix DNA with many E.coli!
cells that have been treated"
with agents that make them"
permeable to DNA.
Cells that are able to take
up DNA are called
competent cells
E. coli cell without a plasmid
Recircularized vector"
without an insert
Blue colony
Note: This shows a bacterial cell with"
the plasmid carrying the gene of"
interest. Other bacterial cells"
would have other recombinant"
vectors or a recircularized vector.
Plate cells on media"
containing X-Gal, IPTG,"
and ampicillin."
Incubate overnight.
White colony
Recombinant"
vector"
with an"
insert
Each bacterial colony is derived from a single cell;"
so all the cells in a colony are genetically identical.
Figure 18.2
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18-16
n
All bacterial colonies growing on the plate had to
have picked up the vector and its ampR gene
n
n
n
In the hybrid vector, the chromosomal DNA inserts
into the lacZ gene, thereby disrupting it
n
n
Now need to differentiate between the colonies that have a
recircularized vector from those with a hybrid vector
This is where the lacZ gene comes into play
By comparison, the recircularized vector has a functional
lacZ gene
But how is the functionality of the lacZ gene
determined?
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18-17
n
The growth media contains two relevant compounds:
n
IPTG (isopropyl-β-D-thiogalactopyranoside)
n
n
X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)
n
n
A colorless compound that is cleaved by β-galactosidase into a
blue dye
The color of bacterial colonies will therefore depend on
whether or not the β-galactosidase enzyme is functional
n
n
n
A lactose analogue that can induce lacZ gene expression
If it is, the colonies will be blue
If not, the colonies will be white
In this experiment
n
n
Bacterial colonies with recircularized vectors form blue colonies
While those with hybrid vectors form white colonies
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18-18
n
The net result of gene cloning is to produce an
enormous amount of copies of a gene
n
n
During transformation, a single bacterial cell usually
takes up a single copy of a vector
Amplification of a cloned gene occurs in two ways:
n
1. The vector gets replicated by the host cell many times
n
n
2. The bacterial cell divides approximately every 20 minutes
n
n
This will generate a lot of copies per cell (25-50 for plasmids)
This will generate a population of many millions of cells overnight
Recombinant DNA technology is not only used to
clone genes
n
Sequences such as telomeres, centromeres and highly
repetitive sequences can be cloned as well
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18-19
cDNA
n
To clone DNA, one can start with a sample of RNA
n
The enzyme reverse transcriptase is used
n
n
n
DNA that is made from RNA is called complementary
DNA (cDNA)
n
n
Uses RNA as a template to make a complementary strand of DNA
Used by retroviruses to copy their RNA genome to DNA
It could be single- or double-stranded
Synthesis of cDNA is presented in Figure 18.3
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18-20
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A A A A A A
5′
polyA tail
3′
mRNA
Add a poly-dT primer that binds"
to the polyA tail of mRNA.
3′
5′
T T T T T T
A A A A A A
3′
5′
Add reverse transcriptase"
+ dNTPs to synthesize a"
complementary DNA strand.
3′
5′
T T T T T T
A A A A A A
3′
5′
Add RNaseH to"
cut up the RNA"
and generate"
RNA primers.
5′
3′
T T T T T T
Add DNA polymerase and"
DNA ligase to synthesize"
the second DNA strand.
3′
5′
T T T T T T
A A A A A A
Figure 18.3
3′
5′
Double-stranded cDNA
18-21
n
n
From a research perspective, an important
advantage of cDNA is that it lacks introns
This has two ramifications
n
n
1. It allows researchers to focus their attention on the
coding sequence of a gene
2. It allows the expression of the encoded protein
Especially, in cells that would not splice out the introns properly
(e.g., a bacterial cell)
n
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18-22
Restriction Mapping
n
Sometimes, it is necessary to obtain smaller clones
from a large chromosomal DNA insert
n
n
n
This process is termed subcloning
Cloning and subcloning require knowledge of the
locations of restriction enzyme sites in vectors and
hybrid vectors
A common approach to examine the locations of
restriction sites is known as restriction mapping
n
Figure 18.4 outlines the restriction mapping of a bacterial
plasmid, pBR322
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18-23
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Bacterial"
host cell"
(all cells"
carry the"
same"
plasmid)"
Plasmid"
DNA"
(pBR322)"
Isolate plasmid DNA"
from host cells."
Plasmid DNA
Place samples in"
separate tubes."
Figure 18.4
Cut the DNA with different"
restriction enzymes.
18-24
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EcoRI
BamHI
PstI
EcoRI"
BamHI"
EcoRI"
PstI"
EcoRI"
BamHI"
PstI"
BamHI"
PstI"
Separate the DNA fragments"
by gel electrophoresis."
Lane
Restriction"
enzyme(s)"
added"
1
2
3
4
5
6
EcoRI
BamHI
PstI
EcoRI"
BamHI"
EcoRI"
PstI"
7
8
EcoRI"
BamHI" BamHI"
PstI"
PstI"
Markers
Used for
fragment
size
comparison
(bp)
4360
4000
3600
3200
2300
1600
1100
750
400
200
Figure 18.4
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18-25
n
The restriction map can be deduced by comparing the sizes
of DNA fragments obtained from the single, double and triple
digestions
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EcoRI
PstI
~770 bp
~380 bp
BamHI
4,363 bp
Figure 18.4
~3210 bp
n
Another way to obtain a restriction map is via DNA
sequencing
n
Once the DNA sequence of a vector has been determined, computer
programs can scan the sequence and identify restriction enzyme sites
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18-26
18.2 Polymerase Chain Reaction
n
Another way to copy DNA is a technique called
polymerase chain reaction (PCR)
n
n
n
It was developed by Kary Mullis in 1985
Unlike gene cloning, PCR can copy DNA without
the aid of vectors and host cells
The PCR method is outlined in Figure 18.5
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18-27
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Chromosomal DNA
Gene of
interest
Primer binding"
near one end"
of the gene
A different primer"
binding near the other"
end of the gene
Many PCR cycles
Many copies"
of the gene of"
interest, flanked"
by the regions"
where the"
primers bind.
(a) The outcome of a PCR experiment
Template"
DNA
Site where reverse primer binds
5′
3′
3′
5′
Site where forward primer binds
Denaturation: Separate DNA"
strands with high temperature.
5′
3′
3′
5′
Primer annealing: Lower"
temperature, which allows primers"
to bind to template DNA.
5′
3′
Forward primer
5′
3′
3′
5′
Reverse primer
3′
Primer extension: Incubate at a"
temperature that allows DNA"
synthesis to occur.
5′
3′
T
C
C C
C
T C C
T C
G
A
A G
A
G A
A C G T G G T C G T A G G C T A G
3′
5′
Reverse primer
3′
3′
5′
5′
3′
Figure 18.5
5′
5′
3′
5′
(b) The 3 steps of a PCR cycle
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18-28
n
The starting material for PCR includes
n
1. Template DNA
n
n
2. Oligonucleotide primers
n
n
n
Provide the precursors for DNA synthesis
4. Taq polymerase
n
n
n
Complementary to sequences at the ends of the DNA fragment to
be amplified
These are synthetic and about 15-20 nucleotides long
3. Deoxynucleoside triphosphates (dNTPs)
n
n
Contains the region that needs to be amplified
DNA polymerase isolated from the bacterium Thermus aquaticus
This thermostable enzyme is necessary because PCR involves
heating steps that inactivate most other DNA polymerases
Refer to Figure 18.6
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18-29
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Template DNA
Region of interest that will be copied
During each cycle, the DNA"
strands are separated via heating."
The temperature is then lowered"
to allow the primers to bind, and"
a complementary strand is made.
Mix together template DNA,"
present in low amounts,"
Cycle 1
with dNTPs, Taq!
polymerase, and 2 primers"
present in high amounts.
n
+
n
Cycle 2
n
+
+
PCR is carried out in a thermocycler,
which automates the timing of
each cycle
All the ingredients are placed in
one tube
The experimenter sets the
machine to operate within a
defined temperature range and
number of cycles
+
Cycle 3
Figure 18.6
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18-30
Figure 18.6
n
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The sequential process of
denaturing-annealingsynthesis is then repeated for
many cycles
+
+
+
+
+
With each successive"
cycle, the relative amount"
of this type of DNA fragment"
increases. Therefore, after"
many cycles, the vast"
majority of DNA fragments"
contain only the region that"
is flanked by the 2 primers.
+
+
n
n
n
A typical PCR run is likely to involve 20 to 30 cycles of replication
n  This takes a few hours to complete
After 20 cycles, a target DNA sequence will increase 220-fold (~ 1 million-fold)
After 30 cycles, a target DNA sequence will increase 230-fold (~ 1 billion-fold)
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18-31
n
The PCR reaction shown in Figure 18.5 seeks to
amplify a specific DNA segment
n
For this type of experiment, a researcher must have prior
knowledge about the sequence of the template DNA
n
n
Required to construct the synthetic primers
PCR can also be used to amplify chromosomal DNA
semispecifically or nonspecifically
n
1. Semispecific approach
n
Primers recognize a repetitive DNA sequence found at several
sites within the genome
n
n
Therefore, many different DNA fragments will be amplified
2. Nonspecific approach
n
A mixture of primers with many different random sequences is used
n
These will anneal randomly throughout the genome and amplify most
of the chromosomal DNA
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18-32
n
PCR is also used to detect and quantitate the
amount of an RNA in living cells
n
n
RT-PCR is carried out in the following manner
n
n
n
n
The method is called reverse transcriptase PCR (RT-PCR)
RNA is isolated from a sample
It is mixed with reverse transcriptase and a primer that will
anneal to the 3 end of the RNA of interest
This generates a single-stranded cDNA which can be used
as template DNA in conventional PCR
Refer to Figure 18.7
n
RT-PCR is extraordinarily sensitive
n
It can detect the expression of small amounts of RNA in a single
cell
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18-33
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3′
3′
5′
3′
5′
5 ′
3′
5′
RNA isolated"
from a sample"
of cells
5′
3′
RNA of interest
Add reverse transcriptase, a primer"
that binds near the 3′ of the RNA of"
interest, and deoxyribonucleotides
3′
3′
5′
3′
5′
5′
3′
3′
5′
5′
3′
5′
Primer
Subject to PCR as described"
in Figures 18.5 and 18.6
Double-stranded"
cDNAs derived"
from the RNA"
of interest
Figure 18.7
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18-34
Real-Time PCR is used to Quantitate the
Amount of a Specific Gene or mRNA
n
n
Real-time PCR is carried out in a thermocycler that can
measure changes in fluorescence emitted by detector
molecules in the PCR reaction mix
The TaqMan system uses a detector oligonucleotide that has
a fluorescent reporter molecule at one end and a quencher
molecule at the other end
n
n
n
n
Due to their proximity, the quencher molecule blocks the fluorescence
of the reporter molecule on the oligonucleotide
During primer extension, Taq polymerase 5 -3 exonuclease activity
digests the detector oligonucleotide, separating reporter and quencher
Fluorescence will increase in proportion to the amount of PCR product
produced
See Figure 18.8 and 18.9
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18-35
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Reporter
Quencher
Oligonucleotide that is complementary"
to one strand of the PCR product
(a) TaqMan detector
During the primer annealing step, both a primer and TaqMan"
detector bind to the template DNA.
Forward"
primer
5′
3′
TaqMan detector
3′
5′
Template DNA"
that is being amplified
During the primer extension step,"
the detector is digested by Taq!
polymerase, which separates"
the reporter from the quencher.
Reporter is"
not quenched
5′
3′
5′
Taq polymerase
Figure 18.8
(b) Use of a TaqMan detector in real-time PCR
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18-36
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PCR product
Fluorescence
Plateau
Linear
Ct
Cycle threshold
Exponential
Cycle
(a) Phases of PCR
Fluorescence
High
Medium
Ct
Ct
Low
Ct
Cycle threshold
0
5
10
15
20
25
30
35
40
45
Cycle number
(b) Real-time PCR at high, medium, and low concentrations of the!
starting template DNA
Unknown"
sample
Fluorescence
Standard at"
a known high"
concentration
Ct Ct
Standard at"
a known lower"
concentration
Ct
Cycle threshold
0
5
10
15
20
25
30
35
40
45
Cycle number
(c) A comparison between an unknown sample and standards of!
known concentrations
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18-37
18.3 DNA LIBRARIES AND
BLOTTING METHODS
•  Molecular geneticists usually want to study particular
genes within the chromosomes of living species
–  This presents a problem, because chromosomal DNA
contains thousands of different genes
–  The term gene detection refers to methods that distinguish
one particular gene from a mixture of thousands of genes
•  Scientists have also developed techniques to identify
gene products
–  RNA that is transcribed from a particular gene
–  Protein that is encoded in an mRNA
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18-44
DNA Libraries
n
A DNA library is a collection of thousands of
different fragments of DNA, each of which is
inserted into a vector
n
n
When the starting material is chromosomal DNA, the
library is called a genomic library
A cDNA library contains hybrid vectors with cDNA inserts
n
n
Should represent the genes expressed in the cells from which the
RNA was isolated
The construction of a DNA library is shown in
Figure 18.11
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18-45
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Chromosomal DNA with"
many restriction sites
Plasmid vectors"
with a single"
restriction site
Cleave DNA"
with restriction"
enzyme.
Fragment with"
gene of interest
Different fragments"
of chromosomal DNA
Opened"
vectors
Figure 18.11
Mix vectors and DNA"
fragments under"
conditions that favor"
base pairing.
18-46
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Treat with DNA ligase to"
covalently join pieces together.
Each hybrid vector contains a different"
fragment of chromosomal DNA.
Transform bacteria.
Select for bacteria that"
have taken up a plasmid."
(Note: In this experiment,"
only 1 plasmid is taken"
up by a bacterium.)
Plate on petri plates"
containing the selected"
antibiotic.
Each bacterial colony"
contains millions of cells"
that were derived from a"
single transformed cell."
A collection of many"
colonies is a DNA library.
(a) Making a genomic library
Figure 18.11
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-47
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Isolate mRNAs from a sample of cells.
mRNA
cDNA
Make cDNAs"
as described"
in Figure 18.3."
Attach oligonucleotide linkers"
to ends of cDNAs using DNA ligase.
Linker DNA, which has a"
sequence that is recognized"
by a particular restriction enzyme
Cut cDNAs and plasmid DNA with"
a restriction enzyme and ligate the"
cDNAs into vectors.
Recombinant plasmid"
with a cDNA insert
Transform bacteria. Place"
on petri plates containing"
the selected antibiotic.
(b) Making a cDNA library!
A cDNA library can be made from mRNA
Figure 18.11
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-48
n
n
In most cloning experiments, the ultimate goal is to
clone a specific gene
For example, suppose that a geneticist wishes to
clone the rat β-globin gene
n
n
n
Only a small percentage of the hybrid vectors in a DNA
library would actually contain the gene
Therefore, geneticists must have a way to distinguish
those rare colonies from all the others
This can be accomplished by using a DNA probe in
a procedure called colony hybridization
n
Refer to Figure 18.12
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-49
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Master plate
A nylon membrane is"
gently laid onto the"
master plate and"
lifted, yielding a replica"
of the master plate.
Nylon membrane
The membrane is treated with detergent"
to permeabilize the bacteria, and the"
DNA is fixed to the membrane. NaOH is"
added to denature the DNA. The membrane"
is submerged in a solution containing a"
radiolabeled probe that is complementary"
to the β-globin gene.
Radiolabeled"
probe
The membrane is washed to remove"
unbound probe and then placed"
next to X-ray film.
β-globin gene in"
a bacterial colony
X-ray film
Based on the orientation of the membrane"
and X-ray film (see X), the colonies"
containing the β-globin gene are"
identified on the master plate.
Figure 18.12
Colonies"
containing"
the cloned"
β-globin gene
Master plate (see above)
18-50
n
But how does one obtain the probe?
n
n
If the gene of interest has been already cloned, a piece of
it can be used as the probe
If not, one strategy is to use a probe that likely has a
sequence similar to the gene of interest
n
n
For example, use the rat β-globin gene to probe for the β-globin
gene from another rodent
What if a scientist is looking for a novel gene that no one
has ever cloned from any species?
n
n
If the protein of interest has been previously isolated, amino acid
sequence is obtained from it
The researcher can use the amino acid sequence to design short
DNA probes that can bind to the protein s DNA coding sequence
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-51
Southern Blotting
n
n
Southern blotting can detect the presence of a particular
gene sequence within a complex genetic background
n  It was developed by E. M. Southern in 1975
Southern blotting has several uses
n  1. It can determine copy number of a gene in a genome
n  2. It can detect small gene deletions that cannot be
detected by light microscopy
n  3. It can identify gene families
n  4. It can identify homologous genes among different
species
n  5. It can determine if a transgenic organism is carrying a
new or modified gene
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-52
n
Prior to a Southern blotting experiment, the gene of
interest, or a fragment of the gene, has been cloned
n
n
n
This cloned DNA is labeled (e.g., radiolabeled) and used
as a probe
The probe will be able to detect the gene of interest
within a mixture of many DNA fragments
The technique of Southern Blotting is shown in
Figure 18.13
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-53
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Weight
Glass"
plate
Dry paper"
towels
A sample of chromosomal"
DNA is digested into"
small fragments with"
a restriction enzyme.
Blotting"
paper
Nylon"
membrane
Gel
Support for"
blotting paper"
and gel
The fragments are"
separated by gel"
electrophoresis,"
and then denatured.
Transfer"
solution
(b) Transfer step (traditional method)
Gel
Nylon"
membrane
An alternative type
of transfer uses a
vaccuum
As shown in parts b"
and c, the DNA bands"
are transferred (blotted)"
to a nylon membrane. "
After transfer, the"
DNA is permanently"
attached to the"
membrane.
Lid
–
Cathode"
plate
Blotting"
paper
Gel
The membrane is placed in a solution"
containing a radiolabeled probe."
The binding can be done under"
conditions of low or high stringency."
Excess probe is washed away, and"
the membrane is exposed to X-ray film.
High"
stringency
Nylon"
membrane
Blotting"
paper
Low"
stringency
+
Anode"
plate
X-ray film
Base
Figure 18.13
(a) The steps in Southern blotting
(c) The transfer step via electrophoresis
18-54
a) The steps in
Southern blotting
Nylon"
membrane
Conditions of high temperature
and/or low salt concentration
Probe DNA and chromosomal
fragment must be nearly
identical to hybridize
The membrane is placed in a solution"
containing a radiolabeled probe."
The binding can be done under"
conditions of low or high stringency."
Excess probe is washed away, and"
the membrane is exposed to X-ray film.
High"
stringency
A common labeling method is
the use of the radioisotope 32P
Low"
stringency
Conditions of low temperature
and/or high salt concentration
X-ray film
Gene of interest is
found only in single
copy in the genome
Figure 18.13
Probe DNA and chromosomal
fragment must be similar but not
necessarily identical to hybridize
Gene is member of a gene
family composed of three
distinct members
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-55
Northern Blotting
n
n
Northern blotting is used to identify a specific RNA
within a mixture of many RNA molecules
n
It was not named after anyone called Northern!
n
Originally known as Reverse-Southern which became Northern.
Northern blotting has several uses
n
1. It can determine if a specific gene is transcribed in a
particular cell type
n
n
2. It can determine if a specific gene is transcribed at a
particular stage of development
n
n
Nerve vs. muscle cells
Fetal vs. adult cells
3. It can reveal if a pre-mRNA is alternatively spliced
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-56
n
n
Northern blotting is rather similar to Southern blotting
It is carried out in the following manner
n
n
n
n
n
RNA is extracted from the cells and purified
It is separated by gel electrophoresis
It is then blotted onto nitrocellulose or nylon filters
The filters are placed into a solution containing a
radioactive probe
The filters are then exposed to an X-ray film
n
n
RNAs that are complementary to the radiolabeled probe are
detected as dark bands on the X-ray film
Figure 18.14 shows the results of a Northern blot for
mRNA encoding a protein called tropomyosin
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-57
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Figure 18.14
Lane 1: Smooth muscle cells
Lane 2: Striated muscle cells
Lane 3: Brain cells
1
n
3
Smooth and striated muscles produce a larger amount of
tropomyosin mRNA than do brain cells
n
n
2
This is expected because tropomyosin plays a role in muscle
contraction
The three mRNAs have different molecular weights
n
This indicates that the pre-mRNA is alternatively spliced
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-58
Western Blotting
n
Western blotting is used to identify a specific
protein within a mixture of many protein molecules
n
n
Again, it was not named after anyone called Western!
Western blotting has several uses
n
1. It can determine if a specific protein is made in a
particular cell type
n
n
Red blood cells vs. brain cells
2. It can determine if a specific protein is made at a
particular stage of development
n
Fetal vs. adult cells
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-59
n
Western blotting is carried out as follows:
n
n
Proteins are extracted from the cells
They are then separated by SDS-PAGE
n
They are first dissolved in the detergent sodium dodecyl sulfate
n
n
n
n
n
The secondary antibody is also conjugated to alkaline phosphatase
The colorless dye XP is added
n
n
The negatively charged proteins are then separated by
polyacrylamide gel electrophoresis
They are then blotted onto nitrocellulose or nylon filters
The filters are placed into a solution containing a primary
antibody (recognizes the protein of interest)
A secondary antibody, which recognizes the constant
region of the primary antibody, is then added
n
n
This denatures proteins and coats them with negative charges
Alkaline phosphatase converts the dye to a black compound
Thus proteins of interest are indicated by dark bands
18-60
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display."
XP"
(colorless)
X"
(black)
+
Pi
Alkaline phosphatase
Secondary antibody
Primary antibody
Protein of interest
(a) Interactions between the protein of interest and antibodies
Lane 1: Red blood cells
Lane 2: Brain cells
Lane 3: Intestinal cells
n
1
2
3
n
(b) Results from a Western blotting experiment
The results of a Western blot for the
β-globin polypeptide
The experiment indicates that βglobin is made in red blood cells but
not in brain or intestinal cells
18-61
Techniques that Detect the Binding
of Proteins to DNA or RNA
n
Researchers often want to study the binding of
proteins to specific sites on a DNA or RNA molecule
n
n
For example, the binding to DNA of transcription factors
To study protein-DNA interactions, the following two
methods are used
n
1. Gel retardation assay
n
n
Also termed gel mobility shift assay
2. DNA footprinting
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-62
n
The technical basis for a gel retardation assay is this:
n
The binding of a protein to a fragment of DNA retards its rate of
movement through a gel
Lower mass and
therefore fast migration
Higher mass and
therefore slow migration
Figure 18.16
n
Gel retardation assays must be performed under
nondenaturing conditions
n
Buffer and gel should not cause the unfolding of the proteins nor the
separation of the DNA double helix
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-63
n
DNA footprinting was described originally by David
Galas and Albert Schmitz in 1978
n
They identified a DNA site in the lac operon that is bound
by the lac repressor
n
n
The technical basis for DNA footprinting is this:
n
n
This DNA site is, of course, the operator
A segment of DNA that is bound by a protein will be
protected from digestion by the enzyme DNase I
Figure 18.17 shows a DNA footprinting experiment
involving RNA polymerase holoenzyme
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-64
Did not contain RNA pol
holoenzyme
Tube A
Tube B
Labeled end
RNA"
polymerase"
holoenzyme
150-bp"
fragment
A site where"
DNase I randomly"
cuts the fragment
A single cut can"
occur anywhere in"
the DNA fragment.
A single cut can only"
occur where the protein"
is not bound.
Load onto a gel.
Expose the gel to X-ray film."
Only the pieces of DNA with"
a labeled end are detected.
Figure 18.17
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-65
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
In the absence of RNA
pol holoenzyme, a
continuous range of
fragment sizes occurs
Tube A
150 bases
Tube B
Promoter"
numbering
+75
+50
105 bases
Fragment"
size
RNA pol holoenzyme is
bound to this DNA
region, and thus
protects it from DNase I
Figure 18.17
Region"
where"
RNA"
polymerase"
binds
No bands in
this range
+30
+1 Transcription"
start site
–30
25 bases
–50
1 base
–75
Thus RNA pol
holoenzyme binds to an
80-nucleotide region
(from -50 to +30)
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-66
18.5 DNA SEQUENCING AND SITEDIRECTED MUTAGENESIS
•  Analyzing and altering DNA sequences is a powerful
approach to understanding genetics
–  A technique called DNA sequencing enables researchers
to determine the base sequence of DNA
•  It is one of the most important tools for exploring genetics at the
molecular level
–  Another technique known as site-directed mutagenesis
allows scientists to change the sequence of DNA
•  This too provides information regarding the function of genes
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-67
DNA Sequencing
n
During the 1970s two DNA sequencing methods
were devised
n
n
n
One method, developed by Allan Maxam and Walter
Gilbert, involves the base-specific cleavage of DNA
The other method, developed by Frederick Sanger, is
known as dideoxy sequencing
The dideoxy method has become the more popular
and will therefore be discussed here
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-68
n
The dideoxy method is based on our knowledge of DNA
replication but uses a clever twist
n
n
DNA polymerase connects adjacent deoxynucleotides by covalently
linking the 5 –P of one to the 3 –OH another (Refer to Fig. 11.12)
Nucleotides missing that 3 –OH can be synthesized
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
O
O–
P
–
O
O
O
P
O
O
–
O
Adenine
P
O
–
O
CH2
5′
4′
H
3′
Figure 18.18
n
H
O
H
1′
H
2′
H
H
2′, 3′-Dideoxyadenosine triphosphate (ddA)
Sanger reasoned that if a dideoxynucleotide is added to a
growing DNA strand, the strand can no longer grow
n
n
This is referred to as chain termination
If ddATP is used, termination will always be at an A in the DNA
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-69
n
Prior to DNA sequencing, the DNA to be sequenced must be
obtained in large amounts
n
n
n
This is accomplished using cloning or PCR techniques
In many sequencing experiments, the target DNA is cloned
into the vector at a site adjacent to a primer annealing site
In the experiment shown in Figure 18.19, the recombinant
vector DNA is heat denatured into single strands
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-70
Figure 18.19
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
C
Sequence to"
be analyzed"
(target DNA)
Primer
C
The newly-made DNA fragments can be
separated according to their length by
running them on an acrylamide gel
A T
T
Annealing"
site
5′
Recombinant vector
They can then be visualized as
fluorescence peaks as the bands run
off the bottom of the gel
Many copies of the recombinant vector, primer,"
dNTPs, fluorescently labeled dideoxynucleotides,"
and DNA polymerase are mixed together."
Incubate to allow the synthesis of DNA.
CACCGTAAGGACTddG"
CACCGTAAGGACddT"
CACCGTAAGGAddC"
CACCGTAAGGddA"
CACCGTAAGddG"
CACCGTAAddG"
CACCGTAddA"
CACCGTddA"
CACCGddT"
CACCddG"
CACddC"
CAddC"
CddA"
ddC
Nucleotides added to primer
Separate newly made strands by"
gel electrophoresis.
Sequence"
deduced"
from gel
Laser"
beam
(a) Automated DNA sequencing
G
T
C
A
G
G
A
A
T
G
C
C
A
C
CA C C G T A A G G A C T G
Fluorescence"
detector
(b) Output from automated sequencing
18-71
n
An important innovation in the
method of dideoxy sequencing
is automated sequencing
n
n
n
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
G
T
C
A
G
G
A
A
T
G
C
C
A
C
It uses a single tube containing all
four dideoxyribonucleotides
However, each type (ddA, ddT,
ddG, and ddC) has a differentcolored fluorescent label attached
After incubation and
polymerization, the sample is
loaded into a single lane of a gel
(a) Automated DNA!
sequencing
Figure 18.19
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-72
n
n
The procedure is automated using a laser and fluorescence
detector
The fragments are separated by gel electrophoresis
n
n
Indeed, the mixture of DNA fragments are electrophoresed off the end
of the gel
As each band comes off the bottom of the gel, the fluorescent
dye is excited by the laser
n
The fluorescence emission is recorded by the fluorescence detector
n
The detector reads the level of fluorescence at four wavelengths
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
CA C C G T A A G G A C T G
Figure 18.19
(b) Output from automated sequencing
18-73
Site-Directed Mutagenesis
n
Analysis of mutations can provide important
information about normal genetic processes
n
n
n
Therefore, researchers are constantly looking for mutant
organisms
Mutations can arise spontaneously, or be induced
by mutagens
Researchers have recently developed techniques
to make mutations within cloned DNA
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-74
n
One widely-used method is known as site-directed
mutagenesis
n
n
It allows the alteration of a DNA sequence in a specific way
The site-directed mutant can then be introduced into a
living organism
n
This will allow the researchers to see how the mutation affects
n
n
n
n
The expression of a gene
The function of a protein
The phenotype of an organism
Mark Zoller and Michael Smith developed a protocol
for the site-directed mutagenesis of DNA cloned in a
viral vector
n
Refer to Figure 18.20
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
18-75
Figure 18.20
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Oligonucleotide"
primer with"
a mismatch
Gene in"
a vector"
(template"
DNA)
AC
G
G
CC
Mismatch
Vector
The vector and insert are
denatured into singlestranded DNA prior to the
experiment
Add dNTPs,"
DNA polymerase,"
and DNA ligase.
AC
G
G
CC
Can be identified by
DNA sequencing and
used for further studies
Mismatch
The DNA is introduced"
into a living cell, where"
the mismatch is repaired.
C
G AG
CT
A site-directed mutant is made.
or
Depending on which
base is replaced,
the mutant or original
sequence is produced
C
G GG
CC
The DNA is repaired back"
to the original sequence.
18-76