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Recombinant DNA Technology
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 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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Cloning Experiments Involve Chromosomal and
Vector DNA

Cloning experiments usually involve two kinds of DNA
molecules
 Chromosomal DNA


Vector DNA



Serves as the source of the DNA segment of interest
Serves as the carrier of the DNA segment that is to be cloned
Can replicate independently of the host chromosomal DNA
To prepare chromosomal DNA, the scientist has to

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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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

The cell that harbors the vector is called the host cell
 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 that vector can replicate in a particular host cell
The vectors commonly used in gene cloning were originally
derived from two natural sources
 1. Plasmids
 2. Viruses

Many naturally occurring plasmids have selectable
markers
 Typically, genes conferring antibiotic resistance to the
host cell
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Cloning Experiments Involve Enzymes that Cut and
Paste DNA

Insertion of chromosomal DNA into a vector
requires the cutting and pasting of DNA fragments

The enzymes used to cut DNA are known as
restriction endonucleases or restriction enzymes


These bind to specific DNA sequences and then cleave
the DNA at two defined locations, one on each strand
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
Restriction enzymes bind to specific DNA sequences

These are typically palindromic

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The sequence is identical when read in the opposite direction in the
complementary strand
For example, the EcoRI recognition sequence is
5’ GAATTC 3’
3’ CTTAAG 5’

Some restriction enzymes digest DNA into
fragments with “sticky ends” These DNA fragments
will hydrogen bond to each other due to their
complementary sequences

Other restriction enzymes generate blunt ends

The enzyme NaeI
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Cleavage by restriction enzymes is the first step to making
recombinant DNA. In this case, the ends are ‘sticky’ in that
they have a short, single-stranded end that can base-pair with
another piece of DNA cut with the same enzyme.

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


They protect bacterial cells from invasion by foreign DNA,
particularly that of bacteriophage
Currently, several hundred different restriction
enzymes are available commercially
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This interaction is not stable because
it involves only a few hydrogen bonds
To establish a permanent connection, the
sugar-phosphate backbones of the two DNA
fragments must be covalently linked
A recombinant
DNA molecule
The Steps in Gene Cloning
The procedure shown seeks to clone the human b-globin gene
into a plasmid vector

The vector carries two important genes


ampR  Confers antibiotic resistance to the host cell
 Identifies cells that have taken up the vector
lacZ  Encodes b-galactosidase
 Provides a means by which bacteria that have
picked up the cloned gene can be identified
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Digestion of a
human cell would
actually produce
tens of thousands
of fragments.
This is termed
a hybrid vector
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
Cells that are able to take
up DNA are called
competent cells
X-Gal is an analog of lactose
IPTG – molecular mimic of allolactose
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
All bacterial colonies growing on the plate had to
have picked up the vector and its ampR gene

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But how to differentiate between the colonies that have a
circularized vector from those with a hybrid vector?
In the hybrid vector, the chromosomal DNA inserts
into the lacZ gene, thereby disrupting it

By comparison, the recircularized vector has a functional
lacZ gene
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
The growth media contains two relevant compounds:

IPTG (isopropyl-b-D-thiogalactopyranoside)
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X-Gal (5-bromo-4-chloro-3-indoyl-b-D-galactoside)
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A colorless compound that is cleaved by b-galactosidase into a
blue dye
The color of bacterial colonies will therefore depend on
whether or not the b-galactosidase is functional
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A lactose analogue that can induce the lacZ gene
If it is, the colonies will be blue
If not, the colonies will be white
In this experiment
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Bacterial colonies with recircularized vectors form blue colonies
While those with hybrid vectors form white colonies
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
The net result of gene cloning is to produce an
enormous amount of copies of a gene
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During transformation, a single bacterial cell usually
takes up a single copy of the hybrid vector

Amplification of the gene occurs in two ways:

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1. The vector gets replicated by the host cell many times
 This will generate a lot of copies per cell (25-50 for plasmids)
2. The bacterial cell divides approximately every 20 minutes
 This will generate a population of many million overnight
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cDNA

To clone DNA, one can start with a sample of RNA

The enzyme reverse transcriptase is used
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
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Uses RNA as a template to make a complementary strand of DNA
From retroviruses to copy their RNA genome to DNA
DNA that is made from RNA is called complementary
DNA (cDNA)

It could be single- or double-stranded
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polyA tail

From a research perspective, an important
advantage of cDNA is that it lacks introns

This has two ramifications

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)

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Polymerase Chain Reaction

Another way to copy DNA is a technique called
polymerase chain reaction (PCR)


It was developed by Kary Mullis in 1985
Unlike gene cloning, PCR can copy DNA without
the aid of vectors and host cells
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Chromosomal DNA
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′
5′
3′
5′
Primer extension: Incubate at a
temperature that allows DNA
synthesis to occur.
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
5′
3′
3′
5′
5′
3′
3′
5′
(b) The 3 steps of a PCR cycle
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
The starting material for PCR includes

1. Template DNA
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2. Oligonucleotide primers
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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)
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Contains the region that needs to be amplified
Provide the precursors for DNA synthesis
4. Taq polymerase


DNA polymerase isolated from the bacterium Thermus aquaticus
This thermostable enzyme is necessary because PCR involves
heating steps that inactivate most other DNA polymerases
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Binding of the primers to the
DNA is called annealing

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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
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

A typical PCR run is likely to involve 20 to 30 cycles of replication

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The sequential process of
denaturing-annealingsynthesis is then repeated for
many cycles
This takes a few hours to complete
After 20 cycles, a DNA sample will increase 220-fold (~ 1 million-fold)
After 30 cycles, a DNA sample will increase 230-fold (~ 1 billion-fold)
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

PCR is also used to detect and quantitate the
amount of RNA in living cells


The method is called reverse transcriptase PCR (RT-PCR)
RT-PCR is carried out in the following manner

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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
RT-PCR is extraordinarily sensitive

It can detect the expression of small amounts of RNA in a single
cell
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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′
3′
5′
5′
3′
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
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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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DNA Libraries

A DNA library is a collection of thousands of
different cloned fragments of DNA

When the starting material is chromosomal DNA, the
library is called a genomic library

A cDNA library contains hybrid vectors with cDNA inserts
 Should represent the genes expressed in the cells the
RNA was isolated from
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Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

In most cloning experiments, the ultimate goal is to
clone a specific gene

For example, suppose that a geneticist wishes to
clone the rat b-globin gene


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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
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
But how does one obtain the probe?


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
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
For example, use the rat b-globin gene to probe for the b-globin
gene from another rodent
What if a scientist is looking for a novel type of gene that
no one else has ever cloned from any species?


If the protein of interest has been previously isolated, amino acid
sequences are obtained from it
The researcher can use these amino sequences to design short
DNA probes that can bind to the protein’s coding sequence
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Stained DNA gel under UV
Southern Blotting

Southern blotting can detect the presence of a
particular gene sequence within a mixture of many

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It was developed by E. M. Southern in 1975
Southern blotting has several uses
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1. It can determine copy number of a gene in a genome
2. It can detect small gene deletions that cannot be
detected by light microscopy
3. It can identify gene families
4. It can identify homologous genes among different
species
5. It can determine if a transgenic organism is carrying a
new or modified gene
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
Prior to a Southern blotting experiment, the gene of
interest, or a fragment of a gene, has been cloned

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
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Gel electrophoresis of total DNA digested with restriction
enzyme and subjected to Southern blot hybridization
Northern Blotting


Northern blotting is used to identify a specific RNA
within a mixture of many RNA molecules

It was not named after anyone called Northern!

Originally known as ‘Reverse-Southern’ which became Northern.
Northern blotting has several uses

1. It can determine if a specific gene is transcribed in a
particular cell type


2. It can determine if a specific gene is transcribed at a
particular stage of development


Nerve vs. muscle cells
Fetal vs. adult cells
3. It can reveal if a pre-mRNA is alternatively spliced
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

Northern blotting is rather similar to Southern blotting
It is carried out in the following manner
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RNA is extracted from the cell(s) 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
 RNAs that are complementary to the radiolabeled probe
are detected as dark bands on the X-ray film
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
Smooth and striated muscles produce a larger amount of
tropomyosin mRNA than do brain cells


This is expected because tropomyosin plays a role in muscle
contraction
The three mRNAs have different molecular weights

This indicates that the pre-mRNA is alternatively spliced
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Western Blotting

Western blotting is used to identify a specific
protein within a mixture of many protein molecules


Again, it was not named after anyone called Western!
Western blotting has several uses

1. It can determine if a specific protein is made in a
particular cell type


Red blood cells vs. brain cells
2. It can determine if a specific protein is made at a
particular stage of development

Fetal vs. adult cells
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
Western blotting is carried out as follows:


Proteins are extracted from the cell(s) and purified
They are then separated by SDS-PAGE

They are first dissolved in the detergent sodium dodecyl sulfate
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The secondary antibody is also conjugated to alkaline phosphatase
The colorless dye XP is added


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


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
Western blot analysis
Western blot analysis

This experiment indicates that b-globin is made in
red blood cells but not in brain or intestinal cells
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Techniques that Detect the Binding of Proteins
to DNA or RNA

Researchers often want to study the binding of
proteins to specific sites on a DNA or RNA
molecule


For example, the binding to DNA of transcription factors
To study protein-DNA interactions, the following two
methods are used

1. Gel retardation assay


Also termed band shift assay
2. DNA footprinting
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
The technical basis for a gel retardation assay is this:

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
Gel retardation assays must be performed under
nondenaturing conditions

Buffer and gel should not cause the unfolding of the proteins nor the
separation of the double helix
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DNA Sequencing

During the 1970s two DNA sequencing methods
were devised



One method, developed by Alan 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
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
The dideoxy method is based on our knowledge of DNA
replication but uses a clever twist


DNA polymerase connects adjacent deoxynucleotides by covalently
linking the 5’–P of one and the 3’–OH of the other. Nucleotides
missing that 3’–OH can be synthesized
Sanger reasoned that if a dideoxynucleotide is added to a
growing DNA strand, the strand can no longer grow


This is referred to as chain termination
If ddATP is used, termination will always be at an A in the DNA
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
Prior to DNA sequencing, the DNA to be sequenced must be
obtained in large amounts
 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
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Sequence to
be analyzed
(target DNA)
Primer
The newly-made DNA fragments can be
separated according to their length by
running them on an acrylamide gel
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

An important innovation in the
method of dideoxy sequencing
is automated sequencing

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
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

The procedure is automated using a laser and fluorescent
detector
The fragments are separated by gel electrophoresis


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

The fluorescence emission is recorded by the fluorescence detector

The detector reads the level of fluorescence at four wavelengths