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Recombinant DNA
Technology
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.
 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.
2.
3.
The basic concepts for recombinant DNA technology
The basic procedures of recombinant DNA technology
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.
2.
3.
The basic concepts for recombinant DNA technology
The basic procedures of recombinant DNA technology
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.
2.
Vector
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.
5.
Plasmid Vectors
Bacteriophage Vectors
Virus vectors
Shuttle Vectors--can replicate in either prokaryotic or
eukaryotic cells.
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 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
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
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 post-translationally 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.
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.
2.
Analysis of Gene Structure and Expression
Pharmaceutical Products
•
•
3.
Genetically modified organisms
•
•
4.
5.
Drugs
Vaccines
Transgenic plants
Transgenic animal
Application in medicine

(GMO)
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
of therapeutic gene products —insulin, the interleukins,
interferons, growth hormones, erythropoietin, and coagulation factor
VIII—are now produced commercially from cloned genes
 A number
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
There
are many viruses, fungi, bacteria that cause
plant diseases
“Super-shrimp”
Cold
tolerance
Antifreeze
gene from cold water fish introduced to
tobacco and potato plants
Drought
As
tolerance & Salinity tolerance
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.
4.
Recombinant proteins are particularly susceptible to
proteolytic degradation in bacteria
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 post-translational 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.
THANK YOU
-PHARMA STREET