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Recombinant DNA Technology
 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.
 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.
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
 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 cuttingof
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 ofRecombinant DNA
1. Isolating (vector and target gene)
2. Cutting (Cleavage)
3. Joining (Ligation)
4. Transforming
5. Cloning
6. Selecting (Screening)
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.
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
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 vectorscan 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. Plasmid Vectors
2. Bacteriophage Vectors
3. Virus vectors
4. 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.
 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. Restriction endonuclease, RE
2. DNA ligase
3. Reverse transcriptase
4. DNA polymerase, DNA pol
5. Nuclease
6. Terminal transferase
Restriction Enzymes and DNA Ligases Allow Insertion of DNA Fragments into
Cloning Vectors
 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.
Restriction enzymes
 Restriction enzymes are named after the bacterium from which they are isolated
–
For example, Eco RIis from Escherichia coli, and Bam HIis 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
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 fromthe 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.
Screening (Strategies)
1. Gel Electrophoresis Allows Separation of Vector DNA from Cloned Fragments
2. Cloned DNA Molecules Are Sequenced Rapidly by the Dideoxy ChainTermination 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
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.
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
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 factorVIII—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
•
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.
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 tolerance & Salinity tolerance
 As populations expand, potential to grow crops in otherwise inhospitable
environments
Disadvantage
 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 therapyseeks 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 theNational 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.
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:
1. Disulfide bond formation (catalyzed by disulfide isomerases) and
protein folding
2. Glycosylation (addition of sugar molecules to protein backbone,
catalyzed by glycosyl transferases)
3. Proteolysis (clipping of protein molecule, e.g., processing of
proinsulin to insulin)
4. 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 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
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.