Recombinant DNA
Technology
Deqiao Sheng PhD
Dept. Of Biochemistry and Molecular
Biology
Reference Books
1. Hortons’ Principles of Biochemistry 4th
edition
2. Molecular Biology, Robert Weaver 2nd
edition
3. Harpers’ Biochemistry 26th edition
4. Styers’ Biochemistry
Recombinant DNA and
Gene Cloning


Recombinant DNA (rDNA) is a form of artificial
DNA that is created by combining two or more
sequences that would not normally occur together
through the process of gene splicing.
Recombinant DNA technology is a technology
which allows DNA to be produced via artificial
means. The procedure has been used to change
DNA in living organisms and may have even more
practical uses in the future.
Chinese Dragon
龙
The dragon is a mythical creature that can
fly and walk. Dragon can change its form
and has divine powers to summon wind and
rain.
 The dragons are said to be made up of many
different types of animals of the Earth.

The Nine parts of A Chinese Dragon

Dragon is an imagination creature, which has
deer's antlers, camel's head, hare's eye, snake's
neck, carp's scales, eagle's claws, tiger's paws;
and ox's ears.
Recombinant DNA:
Cloning and Creation of Chimeric Genes
Recombinant DNA technology is
one of the recent advances in
biotechnology, which was
developed by two scientists named
Boyer and Cohen in 1973.
Stanley N. Cohen , who
received the Nobel Prize in
Medicine in 1986 for his
work on discoveries of
growth factors.
Stanley N. Cohen (1935–) (top)
and Herbert Boyer (1936–)
(bottom), who constructed the
first recombinant DNA using
bacterial DNA and plasmids.
What is Recombinant DNA Technology?
Recombinant DNA technology is a
technology which allows DNA to be
produced via artificial means.
 The procedure has been used to change
DNA in living organisms and may have even
more practical uses in the future.
 It is an area of medical science that is just
beginning to be researched in a concerted
effort.

Recombinant DNA technology works by
taking DNA from two different sources and
combining that DNA into a single molecule.
That alone, however, will not do much.
 Recombinant DNA technology only becomes
useful when that artificially-created DNA is
reproduced. This is known as DNA cloning.

Brief Introduction
Recombinant DNA Technology
1. The basic concepts for recombinant
DNA technology
2. The basic procedures of recombinant
DNA technology
3. Application of recombinant DNA
technology
The basic concepts for
recombinant DNA technology

In the early 1970s, technologies for the
laboratory manipulation of nucleic acids
emerged. In turn, these technologies led to
the construction of DNA molecules
composed of nucleotide sequences taken
from different sources. The products of
these innovations, recombinant DNA
molecules, opened exciting new avenues of
investigation in molecular biology and
genetics, and a new field was born—
recombinant DNA technology.
Concept of Recombinant DNA


Recombinant DNA is a molecule that combines
DNA from two sources . Also known as gene
cloning.
Creates a new combination of genetic material
–
–
–

Human gene for insulin was placed in bacteria
The bacteria are recombinant organisms and
produce insulin in large quantities for diabetics
Genetically engineered drug in 1986
Genetically modified organisms are possible
because of the universal nature of the genetic
code!

Genetic engineering is the application of
this technology to the manipulation of
genes. These advances were made
possible by methods for amplification of
any particular DNA segment( how? ),
regardless of source, within bacterial
host cells. Or, in the language of
recombinant DNA technology, the
cloning of virtually any DNA sequence
became feasible.

Recombinant technology begins with the
isolation of a gene of interest (target gene).
The target gene is then inserted into the
plasmid or phage (vector) to form replicon.
 The replicon is then introduced into host cells
to cloned and either express the protein or not.
 The cloned replicon is referred to as
recombinant DNA. The procedure is called
recombinant DNA technology. Cloning is
necessary to produce numerous copies of the
DNA since the initial supply is inadequate to
insert into host cells.

Some other terms are also in common use to
describe genetic engineering.
 Gene manipulation
 Recombinant DNA technology
 Gene cloning (Molecular cloning)
 Genetic modification

Cloning——In classical biology, a clone is a
population of identical organisms derived
from a single parental organism.

For example, the members of a colony of
bacterial cells that arise from a single cell on a
petri plate are clones. Molecular biology has
borrowed the term to mean a collection of
molecules or cells all identical to an original
molecule or cell.

Recombinant DNA technology——A series
of procedures used to join together
(recombine) DNA segments. A recombinant
DNA molecule is constructed (recombined)
from segments from 2 or more different
DNA molecules. Under certain conditions, a
recombinant DNA molecule can enter a cell
and replicate there, autonomously (on its
own) or after it has become integrated into a
chromosome.
How recombinant technology works

These steps include isolating of the target
gene and the vector, specific cutting of
DNA at defined sites, joining or splicing of
DNA fragments, transforming of replicon
to host cell, cloning, selecting of the positive
cells containing recombinant DNA, and
either express or not in the end.
Six steps of Recombinant DNA
1.
2.
3.
4.
5.
6.
Isolating (vector and target gene)
Cutting (Cleavage)
Joining (Ligation)
Transforming
Cloning
Selecting (Screening)
Recombinant DNA Technology
1. The basic concepts for recombinant
DNA technology
2. The basic procedures of recombinant
DNA technology
3. Application of recombinant DNA
technology
The basic procedures of
recombinant DNA technology


DNA molecules that are constructed with DNA
from different sources are called recombinant
DNA molecules.
Recombinant DNA molecules are created in
nature more often than in the laboratory;
– for example, every time a bacteria phage or
eukaryotic virus infects its host cell and
integrates its DNA into the host genome, a
recombinant is created.
– Occasionally, these viruses pick up a fragment
of host DNA when they excise from their host’s
genome; these naturally occurring
recombinant DNA molecules have been used to
study some genes.
Six basic steps are common to most
recombinant DNA experiments
1. Isolation and purification of DNA.
Both vector and target DNA molecules
can be prepared by a variety of
routine methods, which are not
discussed here. In some cases, the
target DNA is synthesized in vitro.
2. Cleavage of DNA at particular sequences. As
we will see, cleaving DNA to generate
fragments of defined length, or with specific
endpoints, is crucial to recombinant DNA
technology. The DNA fragment of interest is
called insert DNA. In the laboratory, DNA is
usually cleaved by treating it with
commercially produced nucleases and
restriction endonucleases.
3. Ligation of DNA fragments.
A recombinant DNA molecule is usually
formed by cleaving the DNA of interest to
yield insert DNA and then ligating the insert
DNA to vector DNA (recombinant DNA or
chimeric DNA). DNA fragments are
typically joined using DNA ligase (also
commercially produced).
– T4 DNA Ligase
4. Introduction of recombinant DNA into
compatible host cells. In order to be
propagated, the recombinant DNA
molecule (insert DNA joined to vector DNA)
must be introduced into a compatible host
cell where it can replicate. The direct
uptake of foreign DNA by a host cell is
called genetic transformation (or
transformation). Recombinant DNA can
also be packaged into virus particles and
transferred to host cells by transfection.
5. Replication and expression of
recombinant DNA in host cells.
Cloning vectors allow insert DNA to be
replicated and, in some cases, expressed
in a host cell. The ability to clone and
express DNA efficiently depends on the
choice of appropriate vectors and hosts.
6. Identification of host cells that contain
recombinant DNA of interest. Vectors
usually contain easily scored genetic
markers, or genes, that allow the
selection of host cells that have taken up
foreign DNA. The identification of a
particular DNA fragment usually
involves an additional step—screening a
large number of recombinant DNA
clones. This is almost always the most
difficult step.
DNA cloning in a plasmid
vector permits amplification
of a DNA fragment.
First step:
Isolating DNA
1. Vector
2. Target gene
How to get a target genes?
1.
2.
3.
4.
Genomic DNA
Artificial synthesis
PCR amplification
RT-PCR
Polymerase chain reaction (PCR)

A technique called the polymerase chain
reaction (PCR) has revolutionized
recombinant DNA technology. It can
amplify DNA from as little material as a
single cell and from very old tissue such
as that isolated from Egyptian mummies,
a frozen mammoth, and insects trapped
in ancient amber.
 method is used to
amplify DNA
sequences
 The polymerase chain
reaction (PCR) can
quickly clone a small
sample of DNA in a
test tube
Initial
DNA
segment
Number of DNA
molecules
PCR primers
RT-PCR


Reverse transcription polymerase chain reaction
(RT-PCR) is a variant of polymerase chain
reaction (PCR.
In RT-PCR, however, an RNA strand is first
reverse transcribed into its DNA complement
(complementary DNA, or cDNA) using the enzyme
reverse transcriptase, and the resulting cDNA is
amplified using traditional.
– Template:RNA
– Products: cDNA
Vectors- Cloning Vehicles
 Cloning
vectors can be plasmids,
bacteriophage, viruses, or even small
artificial chromosomes. Most vectors
contain sequences that allow them to be
replicated autonomously within a
compatible host cell, whereas a minority
carry sequences that facilitate integration
into the host genome.

All cloning vectors have in common at least
one unique cloning site, a sequence that can
be cut by a restriction endonuclease to allow
site-specific insertion of foreign DNA. The
most useful vectors have several restriction
sites grouped together in a multiple cloning
site (MCS) called a polylinker.
Types of vector
1.
2.
3.
4.
Plasmid Vectors
Bacteriophage Vectors
Virus vectors
Shuttle Vectors--can replicate in either
prokaryotic or eukaryotic cells.
5. Yeast Artificial Chromosomes as
Vectors
Plasmid Vectors
Plasmids are circular, double-stranded
DNA (dsDNA) molecules that are separate
from a cell’s chromosomal DNA.
 These extra chromosomal DNAs, which
occur naturally in bacteria and in lower
eukaryotic cells (e.g., yeast), exist in a
parasitic or symbiotic relationship with
their host cell.

Plasmid

Plasmids can replicate autonomously within
a host, and they frequently carry genes
conferring resistance to antibiotics such as
tetracycline, ampicillin, or kanamycin. The
expression of these marker genes can be
used to distinguish between host cells that
carry the vectors and those that do not
pBR322


pBR322 was one of the first versatile plasmid
vectors developed; it is the ancestor of many of the
common plasmid vectors used in biochemistry
laboratories.
pBR322 contains an origin of replication (ori) and
a gene (rop) that helps regulate the number of
copies of plasmid DNA in the cell. There are two
marker genes: confers resistance to ampicillin,
and confers resistance to tetracycline. pBR322
contains a number of unique restriction sites that
are useful for constructing recombinant DNA.
pBR322
1. Origin of
replication
2. Selectable
marker
3. unique
restriction
sites
Enzymes
1.
2.
3.
4.
5.
6.
Restriction endonuclease, RE
DNA ligase
Reverse transcriptase
DNA polymerase, DNA pol
Nuclease
Terminal transferase
Restriction Enzymes and DNA Ligases Allow
Insertion of DNA Fragments into Cloning Vectors
Restriction enzymes cleave DNA

The same sequence of bases is
found on both DNA strands, but
in opposite orders. GAATTC
CTTAAG

This arrangement is called a
palindrome. Palindromes are
words or sentences that read the
same forward and backward.

form sticky ends: single
stranded ends that have a
tendency to join with each
other ( the key to
recombinant DNA)
Restriction Enzymes Cut DNA Chains at
Specific Locations
Restriction enzymes are endonucleases
produced by bacteria that typically
recognize specific 4 to 8bp sequences,
called restriction sites, and then cleave both
DNA strands at this site.
 Restriction sites commonly are short
palindromic sequences; that is, the
restriction-site sequence is the same on
each DNA strand when read in the 5′ → 3′
direction.

Cut out the gene
Restriction enzymes
Restriction enzymes

Restriction enzymes are named after the
bacterium from which they are isolated
–
For example, Eco RI is from Escherichia coli,
and Bam HI is from Bacillus amyloliquefaciens .
The first three letters in the restriction enzyme
name consist of the first letter of the genus (E)
and the first two letters of the species (co). These
may be followed by a strain designation (R) and
a roman numeral (I) to indicate the order of
discovery (eg, EcoRI, EcoRII).
Blunt ends or sticky ends
Each enzyme recognizes and cleaves a
specific double-stranded DNA sequence that
is 4–7 bp long. These DNA cuts result in
blunt ends (eg, Hpa I) or overlapping (sticky)
ends (eg, BamH I) , depending on the
mechanism used by the enzyme.
 Sticky ends are particularly useful in
constructing hybrid or chimeric DNA
molecules .

Results of restriction endonuclease digestion.
Digestion with a restriction endonuclease can result
in the formation of DNA fragments with sticky, or
cohesive ends (A) or blunt ends (B). This is an
important consideration in devising cloning
strategies.
Inserting DNA Fragments into Vectors



DNA fragments with either sticky ends or blunt
ends can be inserted into vector DNA with the
aid of DNA ligases.
For purposes of DNA cloning, purified DNA
ligase is used to covalently join the ends of a
restriction fragment and vector DNA that have
complementary ends . The vector DNA and
restriction fragment are covalently ligated
together through the standard 3 → 5
phosphodiester bonds of DNA.
DNA ligase “pastes” the DNA fragments
together
Ligation of restriction fragments
with complementary sticky ends.
Identification of Host Cells
Containing Recombinant DNA


Once a cloning vector and insert DNA have
been joined in vitro, the recombinant DNA
molecule can be introduced into a host cell,
most often a bacterial cell such as E. coli.
In general, transformation is not a very
efficient way of getting DNA into a cell
because only a very small percentage of cells
take up recombinant DNA. Consequently,
those cells that have been successfully
transformed must be distinguished from the
vast majority of untransformed cells.




Identification of host cells containing
recombinant DNA requires genetic selection or
screening or both.
In a selection, cells are grown under conditions in
which only transformed cells can survive; all the
other cells die.
In contrast, in a screen, transformed cells have to
be individually tested for the presence of the
desired recombinant DNA.
Normally, a number of colonies of cells are first
selected and then screened for colonies carrying
the desired insert.
Selection Strategies Use Marker Genes
(Primary screening)
Many selection strategies involve selectable
marker genes— genes whose presence can
easily be detected or demonstrated. ampR
 Selection or screening can also be achieved
using insertional inactivation.

insertional inactivation
A method of screening recombinants for inserted DNA fragments.
Using the plasmid pBR322, a piece of DNA is inserted into the unique
PstI site. This insertion disrupts the gene coding for a protein that
provides ampicillin resistance to the host bacterium. Hence, the
chimeric plasmid will no longer survive when plated on a substrate
medium that contains this antibiotic. The differential sensitivity to
tetracycline and ampicillin can therefore be used to distinguish clones
of plasmid that contain an insert.
Screening (Strategies)
1. Gel Electrophoresis Allows Separation of
Vector DNA from Cloned Fragments
2. Cloned DNA Molecules Are Sequenced
Rapidly by the Dideoxy Chain-Termination
Method
3. The Polymerase Chain Reaction Amplifies a
Specific DNA Sequence from a Complex
Mixture
4. Blotting Techniques Permit Detection of
Specific DNA Fragments and mRNAs with
DNA Probes
A
B
C
M
bp
—1534
— 994
— 695
— 515
— 377
— 237
Gel Electrophoresis
negative charged DNA run to the anode
Sequencing
results
Southern blot technique can detect a specific DNA
fragment in a complex mixture of restriction fragments.
Hybridization
Radioactive isotope
Types of blotting techniques

Southern blotting



Northern blotting


Southern blotting techniques is the first nucleic acid
blotting procedure developed in 1975 by Southern.
Southern blotting is the techniques for the specific
identification of DNA molecules.
Northern blotting is the techniques for the specific
identification of RNA molecules.
Western blotting


Western blotting involves the identification of proteins.
Antigen + antibody
Expression of Proteins Using
Recombinant DNA Technology


Cloned or amplified DNA can be purified and
sequenced, used to produce RNA and protein, or
introduced into organisms with the goal of
changing their phenotype.
One of the reasons recombinant DNA technology
has had such a large impact on biochemistry is
that it has overcome many of the difficulties
inherent in purifying low-abundance proteins and
determining their amino acid sequences.

Recombinant DNA technology allows the
protein to be purified without further
characterization. Purification begins with
overproduction of the protein in a cell
containing an expression vector.
– Prokaryotic Expression Vectors
– Eukaryotic Expression Vectors
Prokaryotic Expression Vectors

Expression vectors for bacterial hosts are
generally plasmids that have been
engineered to contain appropriate
regulatory sequences for transcription and
translation such as strong promoters,
ribosome-binding sites, and transcription
terminators.


Eukaryotic proteins can be made in bacteria by
inserting a cDNA fragment into an expression
vector . Large amounts of a desired protein can be
purified from the transformed cells.
In some cases, the proteins can be used to treat
patients with genetic disorders.

For example, human growth hormone, insulin, and
several blood coagulation factors have been produced
using recombinant DNA technology and expression
vectors.
Expression of Proteins in Eukaryotes

Prokaryotic cells may be unable to produce
functional proteins from eukaryotic genes
even when all the signals necessary for gene
expression are present because many
eukaryotic proteins must be posttranslationally modified.
Several expression vectors that function in
eukaryotes have been developed.
 These vectors contain eukaryotic origins of
replication, marker genes for selection in
eukaryotes, transcription and translation
control regions, and additional features
required for efficient translation of
eukaryotic mRNA, such as polyadenylation
signals and capping sites.

商业化的载体
Recombinant DNA Technology
1. The basic concepts for recombinant
DNA technology
2. The basic procedures of recombinant
DNA technology
3. Application of recombinant DNA
technology
Applications of Recombinant
DNA Technology
1. Analysis of Gene Structure and
Expression
2. Pharmaceutical Products
–
–
Drugs
Vaccines
3. Genetically modified organisms (GMO)
–
–
Transgenic plants
Transgenic animal
4. Application in medicine
5. 
Analysis of Gene Structure and
Expression

Using specialized recombinant DNA techniques,
researchers have determined vast amounts of DNA
sequence including the entire genomic sequence of
humans and many key experimental organisms.
This enormous volume of data, which is growing at
a rapid pace, has been stored and organized in two
primary data banks:


the GenBank at the National Institutes of Health,
Bethesda, Maryland,
and the EMBL Sequence Data Base at the European
Molecular Biology Laboratory in Heidelberg, Germany.
Pharmaceutical Products

Some pharmaceutical applications of DNA
technology:
Large-scale production of human hormones
and other proteins with therapeutic uses
 Production of safer vaccines


A number of therapeutic gene products —
insulin, the interleukins, interferons, growth
hormones, erythropoietin, and coagulation
factor VIII—are now produced
commercially from cloned genes
 Pharmaceutical
companies already are
producing molecules
made by recombinant
DNA to treat human
diseases.
 Recombinant bacteria
are used in the
production of human
growth hormone and
human insulin
Use recombinant cells to mass produce proteins
– Bacteria
– Yeast
– Mammalian
• Insulin
– Hormone required to
properly process sugars
and fats
– Treat diabetes
– Now easily produced by
bacteria
• Growth hormone
deficiency
– Faulty pituitary and
regulation
– Had to rely on cadaver
source
– Now easily produced by
bacteria
Subunit Herpes Vaccine
Not always used for good...
• High doses of HGH can
cause permanent side
effects
– As adults normal growth
has stopped so excessive GH
can thicken bones and
enlarge organs
Genetically modified organisms (GMO)
Use of recombinant plasmids in
agriculture
– plants with genetically desirable
traits
• herbicide or pesticide resistant corn
& soybean
– Decreases chemical insecticide use
– Increases production
• “Golden rice” with beta-carotene
– Required to make vitamin A, which in
deficiency causes blindness

Crops have been
developed that are
better tasting, stay
fresh longer, and are
protected from disease
and insect infestations.
“Golden rice” has been
genetically modified to
contain beta-carotene
Genetic Engineering of Plants

Plants have been bred for millennia to
enhance certain desirable characteristics in
important food crops.

Transgenic plants.
The luciferase gene from a
firefly is transformed into
tobacco plant using the Ti
plasmid. Watering the plant
with a solution of luciferin
(the substrate for firefly
luciferase) results in the
generation of light by all
plant tissues.
Insect-resistant tomato plants
The plant on the left contains a gene that encodes a
bacterial protein that is toxic to certain insects that
feed on tomato plants. The plant on the right is a
wild-type plant. Only the plant on the left is able to
grow when exposed to the insects.
Transgenic animals
Green fluorescence
Red fluorescence
Transgenic animals
A transgenic
mouse
Mouse on right is
normal; mouse on
left is transgenic
animal expressing
rat growth hormone
Farm Animals and “Pharm”
Animals


Trangenic plants and animals
have genes from other
organisms.
These transgenic sheep
carry a gene for a
human blood protein
– This protein may help in
the treatment of cystic
fibrosis
just a joke
Other benefits of GMOs

Disease resistance



Cold tolerance


There are many viruses, fungi, bacteria that cause plant
diseases
“Super-shrimp”
Antifreeze gene from cold water fish introduced to
tobacco and potato plants
Drought tolerance & Salinity tolerance

As populations expand, potential to grow crops in
otherwise inhospitable environments
Where in the world?
Downsides???


Introduce allergens?
Pass trans-genes to
wild populations?
–

Pollinator transfer
R&D is costly
– Patents to insure
profits
• Patent infringements
• Lawsuits
• potential for capitalism
to overshadow
humanitarian efforts
Application in medicine
 Human
Gene Therapy
 Diagnosis of genetic disorders
 Forensic Evidence
Human Gene Therapy

Human gene therapy seeks to repair the damage
caused by a genetic deficiency through
introduction of a functional version of the
defective gene. To achieve this end, a cloned
variant of the gene must be incorporated into the
organism in such a manner that it is expressed
only at the proper time and only in appropriate
cell types. At this time, these conditions impose
serious technical and clinical difficulties.




Gene therapy is the alteration of an afflicted
individual’s genes
Gene therapy holds great potential for treating
disorders traceable to a single defective gene
Vectors are used for delivery of genes into cells
Gene therapy raises ethical questions, such as
whether human germ-line cells should be treated to
correct the defect in future generations

Many gene therapies have received approval from
the National Institutes of Health for trials in
human patients, including the introduction of gene
constructs into patients. Among these are
constructs designed to cure ADA- SCID (severe
combined immunodeficiency due to adenosine
deaminase [ADA] deficiency), neuroblastoma, or
cystic fibrosis, or to treat cancer through
expression of the E1A and p53 tumor suppressor
genes.
Cloned gene
Insert RNA version of normal allele
into retrovirus.
Viral RNA
Retrovirus
capsid
Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
Somatic cells
Only!
Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient
Inject engineered
cells into patient.
Bone
marrow
Not for
reproductive
cells !!
However, there are some challenging
issues that need to be considered:
1. In mammalian cells, mRNA is processed
before it is translated into a protein:
–
–
Introns are cut out and exons are spliced
together
Bacteria can not process mRNA
2. Post-translational modifications
–
–
Enzymatic modifications of protein
molecules after they are synthesized in cells
Post-translational modifications include:
•
•
•
•
Disulfide bond formation (catalyzed by disulfide
isomerases) and protein folding
Glycosylation (addition of sugar molecules to
protein backbone, catalyzed by glycosyl
transferases)
Proteolysis (clipping of protein molecule, e.g.,
processing of proinsulin to insulin)
Sulfation, phosphorylation (addition of sulfate,
phosphate groups)
3. Recombinant proteins are particularly
susceptible to proteolytic degradation in
bacteria
4. Recombinant protein may accumulate in
bacteria as refractile inclusion bodies
So how can these problems be tackled?
Problem:
1. mRNA processing in mammalian cells but not in
bacteria
Solution:
• Synthesize chemically gene containing only
exons and insert that into vector;
• or, Make cDNA by reverse-transcription of
processed mRNA (using the enzyme reverse
transcriptase)
Problem:
2. Bacteria cannot perform posttranslational modifications
Solution:
• This is a tough one! Only proteins that do
not undergo extensive post-translational
processing can be synthesized in bacteria
Problem:
3. Recombinant proteins particularly susceptible to
proteolysis
Solution:
• Design fusion protein consisting of an
endogenous bacterial protein connected to the
recombinant protein through a specific amino
acid sequence. Fusion protein is then specifically
cleaved at the fusion site
The Hope
Summary
1. Recombinant DNA technology builds on a
few basic techniques: isolation of DNA,
cleavage of DNA at particular sequences,
ligation of DNA fragments, introduction of
DNA into host cells, replication and
expression of DNA, and identification of
host cells that contain recombinants.
2. DNA Fragments generated by
restriction endonucleases can be
ligated into a wide range of cloning
vectors, including: plasmids,
bacteriophage, viruses, or artificial
chromosomes.
3. Cells containing recombinant DNA
molecules can be selected, often by
the activity of a marker gene. Cells
containing the desired recombinant
are identified by screening.
4. The product of a gene that has been
incorporated into an appropriate
expression vector can be generated in
prokaryotic or eukaryotic cells.
Foreign genes can also be stably
incorporated into the genomes of
animals and plants.
5. Recombinant DNA methods allow the
production of proteins for therapeutic
use and the identification of
individuals with genetic defects.
REVIEW QUESTIONS
Choose the ONE BEST answer or completion.
Plasmids used as cloning vectors
A. are circular molecules.
B. have an origin of replication.
C. carry antibiotic resistance genes.
D. have unique restriction endonuclease cutting sites.
E. all of the above.