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RECOMBINANT DNA
What is genetic recombinant?
 Genetic recombination refers to the exchange of
segment of DNA.
 It also refers to the ability to manipulate the structure
of DNA.
 Recombinant DNA technology is not a single
procedure or technique, but rather a collection of
tools and technique use to manipulate the genome of
organism.
an overview of
recombinant
DNA technology
The Tools of Recombinant DNA
Technology
 Tools of recombinant DNA technology include
 mutagens,
 reverse transcriptase,
 synthetic nucleic acids,
 restriction enzymes, and
 vectors.
 To create gene libraries, which are a time-saving tool
for genetic researchers.
Restriction enzymes
 Restriction enzymes cut DNA molecules and are restricted in their action
 They cut DNA only at locations with specific and usually palindromic
nucleotide sequences called restriction sites.
 In nature, bacterial cells use restriction enzymes to protect themselves
from phages by cutting phage DNA into nonfunctional pieces.
 Restriction enzymes were name with three letters
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denoting the genus and specific epithet of the source bacterium
and Roman numerals (to indicate the order in which enzymes from
the same bacterium were discovered).
 In some cases, a fourth letter denotes the strain of the bacterium. Thus
Escherichia coli strain R produces the restriction enzymes EcoRI and
EcoRII. HindIII is the third restriction enzyme isolated from Haemophilus
influenzae strain Rd.
 Restriction enzymes can
categorize in two groups
based on the types of cuts
they make.
 The first type,
 As exemplified by EcoRI,
makes staggered cuts of the
two strands of DNA,
producing fragments that
terminate in mortise-like
sticky ends.
 Each sticky end is composed
of up to four nucleotides that
form hydrogen bonds with its
complementary sticky end.
 These bits of single stranded
DNA can use to combine
pieces of DNA from different
organisms into a single
recombinant DNA molecule
(the enzyme ligase unites the
sugar-phosphate backbones
of the pieces)
 Other restriction enzymes, such as
HindII and Smal, cut both strands
of DNA at the same point, resulting
in blunt ends (Figure c).
 It is more difficult to make
recombinant DNA from bluntended fragments because they are
not sticky, but they have a potential
advantage blunt ends are
nonspecific.
 This enables any blunt-ended
fragments, even those produced
by different restriction enzymes, to
be combined easily (Figure d).
 In contrast, sticky-ended fragments
bind only to complementary, stickyended fragments produced by the
same restriction enzyme.
 Table 8.1 identifies several restriction
enzymes and their target DNA sequences.
Vector
 Vectors are use to deliver a gene into a cell
 Vectors are nucleic acid molecules such as
viral genomes, transposons and plasmids.
Genetic vectors share several useful
properties:
 Vectors are small enough to manipulate in a
laboratory
 Vectors survive inside cells
 Vectors contain a recognizable genetic
marker
 Vectors can ensure genetic expression
 An example of
the process used
to produce a
vector containing
a specific gene is
depicted as
shown.
 After a given restriction enzyme cuts both the DNA
molecule containing the gene of interest (in this example,
the human growth hormone gene) and the vector DNA
(here a plasmid containing a gene for antibiotic
resistance as a marker) into fragments with sticky ends
 1) ligase anneals the fragments to produce a
recombinant plasmid.
 2) After the recombinant plasmid has been inserted into
a bacterial cell
 3) The bacteria are grown on a medium containing the
antibiotic
 4) only those cells that contain the recombinant plasmid
(and thus the human growth hormone gene as well) can
grow on the medium.
Techniques of Recombinant DNA
Technology
 The tools of recombinant DNA technology were use
in a number of basic techniques to
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Multiply
Identify
Manipulate
Isolate
map, and
sequence
the nucleotides of genes
Multiplying DNA in vitro: The
Polymerase Chain Reaction (PCR)
 The polymerase chain reaction (PCR) is a technique
to produce a large number of identical molecules of
DNA in vitro.
 Using PCR, we start with a single molecule of DNA
and generate billions of exact replicate within hours.
 Such rapid amplification of DNA is critical in a variety
of situations.
 PCR is a repetitive process that alternately
separates and replicates the two strands of
DNA. Each cycle of PCR consists of the
following three steps:
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Denaturation.
Priming.
Extension.
 Denaturation.
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Exposure to heat (about 94°C)
separates the two strands of the target DNA
by breaking the hydrogen bonds between
base pairs but otherwise leaves the two
strands unaltered.
 Priming.
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A mixture containing an excess of
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DNA primers (synthesized such that they are complementary to
nucleotide sequences near the ends of the target DNA),
DNA polymerase, and
an abundance of the four deoxyribonucleotide triphosphates (A, T,
G, and C)
is added to the target DNA
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This mixture is then cooled to about 65°C, enabling doublestranded DNA to reform.
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Because there is an excess of primers, single strands are more
likely to bind to a primer than to one another.
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The primers provide DNA polymerase with the 3' hydroxyl group
it requires for DNA synthesis.
 Extension.
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Raising the temperature to about 72°C
increases the rate at which DNA polymerase
replicates each strand to produce more DNA
 These steps are repeated over and over, so the
number of DNA molecules increases
exponentially (Figure 8.6b).
 After only 30 cycles - which requires only a few
hours to complete
 PCR produces over 1 billion identical copies of
the original DNA molecule.
 The process can be automated using a thermocycler,
 a device that automatically performs PCR by
continuously cycling all the necessary reagents
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DNA polymerase,
primers, and
triphosphate deoxynucleotides-
 through the three temperature regimes.
 A thermocycler uses DNA polymerase derived from
hyperthermophilic archaea such as Thermus aquaticus.
 This enzyme is not denatured at 94°C, so the machine
need not be replenished with DNA polymerase after
each cycle.
Genetic recombination and transfer
 Horizontal gene transfer
 A donor cell contributes part of its genom to a recipient
cell which may be of a different species or even a
different genus from the donor.
 3 types
 Transformation
 Transduction
 Bacterial conjugation
(animation)
Separating DNA Molecules:
Gel Electrophoresis and the Southern Blot

Electrophoresis is a technique that involves separating molecules based on their
electrical charge, size, and shape. In recombinant DNA technology, gel
electrophoresis were use to isolate fragments of DNA molecules that can then
be inserted into vectors, multiplied by PCR, or preserved in a gene library.
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In gel electrophoresis, DNA molecules, which have an overall negative charge,
are drawn through a semisolid gel by an electric current toward the positive
electrode within an electrophoresis chamber.
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The gel is typically composed of a purified sugar component of agar, called
agarose, which in addition to making a more uniform gel than agar, acts as a
molecular sieve that retards the movement of DNA fragments down the chamber
and separates the fragments by size. Smaller DNA fragments move faster and
farther than larger ones. The size of a fragment can determine by comparing the
distance it travels to the distances traveled by standard DNA fragments of
known sizes.
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DNA probes allow us to find specific DNA sequences such as genes in a cell.
We could also use probes to localize specific sequences in electrophoresis gels,
but because gels are flimsy, easily broken, and deform as they dry, it is difficult
to probe gels.
 In 1975, Ed Southern (1938) devised a method, called the Southern blot, to
transfer DNA from agarose gels to nitrocellulose membranes, which are less
delicate.
 The Southern blot technique begins with the procedures of gel
electrophoresis just described.
 Once the DNA fragments have been separated by size, the liquid in the
electrophoresis gel is blotted out, the DNA is denatured with NaOH, and its
single strands are transferred and bonded to a nitrocellulose membrane.
Radioactive probes are used to localize DNA sequences of interest in the
membrane.
 A northern blot is a similar technique used to detect specific RNA molecules.
Southern blots were use for a variety of purposes, including genetic
‘fingerprinting’ and diagnosis of infectious diseases. For example, we can
detect the presence of genetic sequences unique to hepatitis B virus in a
blood sample of an infected patient even before the patient shows
symptoms or an immune response.
 Southern blotting also were use to demonstrate the incidence and
prevalence in an environmental sample of archaea, bacteria, and viruses,
particularly those that cannot be cultured. Most microorganisms have never
been grown in a laboratory; indeed, scientists know them only by unique
DNA patterns in electrophoresis gels and Southern blot membranes, called
their DNA fingerprints or ‘signatures’.
DNA Microarrays
 A recent advance in biotechnology is the development of DNA
micro arrays (DNA chips). An array consists of molecules of
single-stranded DNA either genetic DNA or cDNA immobilized
on glass slides, silicon chips, or nylon membranes.
 Robots, similar to those that construct computer chips, deposit
PCR-derived copies of hundreds of thousands of different DNA
sequences in precise locations on the array.
 An array may consist of DNA from a single species (for
example, DNA micro arrays containing sequences from all the
genes of E. coli are available commercially), or a DNA array
may contain sequences from numerous species. In any case,
single strands of fluorescently labeled DNA in a sample washed
over an array adhere only to locations on the array where there
are complementary DNA sequences.
DNA micro arrays were used in a
number of ways, including:
 Monitoring gene expression
 Diagnosis of infection.
 Identification of organisms in an
environmental sample.
Inserting DNA into Cells
 A goal of recombinant DNA technology is the
insertion of a gene into a cell, a process also known
as transformation.
 In addition to using vectors and the natural methods
of transformation of competent cells, transduction,
and conjugation, there are also several artificial
methods that have been developed to introduce DNA
into cells, including:
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Electroporation
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Protoplast fusion
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Injection
Electroporation
 Electroporation involves using an electrical current to
puncture microscopic holes through a cell's
membrane so that DNA can enter the cell from the
environment.
 Electroporation can be used on all types of cells,
though the thick-walled cells of fungi and algae must
first be converted to protoplasts, which are cells
whose cell walls have been enzymatically removed.
 Cells treated by electroporation repair their
membranes and cell walls after a time.
Protoplast fusion
 When protoplasts encounter one another, their
cytoplasmic membranes may fuse to form a single
cell that contains the genomes of both "parent" cells.
 Exposure to polyethylene glycol increases the rate of
fusion. The DNA from the two fused cells recombines
to form a recombinant molecule.
 Scientists often use protoplast fusion for the genetic
modification of plants.
Injection
 Two types of injection are commonly used with larger eukaryotic
cells.
 Researchers use a gene gun powered by a blank .22-caliber
cartridge or compressed gas to fire tiny tungsten or gold beads
coated with DNA into a target cell.
 The cell eventually eliminates the inert metal beads.
 In microinjection, a geneticist inserts DNA into a target cell with
a glass micropipette having a tip diameter smaller than that of
the cell or nucleus.
 Unlike electroporation and protoplast fusion, injection can be
used on intact tissues such as in plant seeds.
 In every case, foreign DNA that enters a cell
remains in a cell's progeny only if the DNA is
self-replicating, as in the case of plasmid and
viral vectors, or if the DNA integrates into a
cellular chromosome by recombination.
Applications of Recombinant DNA
Technology
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Genomics is the sequencing (genetic mapping), analysis, and
comparison of genomes. Genetic sequencing has been speeded up
by an automated machine that distinguishes among fluorescent dyes
attached to each type of nucleotide base.
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Scientists synthesize subunit vaccines by introducing genes for a
pathogen's polypeptides into cells or viruses. When the cells, the
viruses, or the polypeptides they produce are injected into a human,
the body's immune system is exposed to and reacts against
relatively harmless antigens instead of the potentially harmful
pathogen.
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Genetic screening can detect infections and inherited diseases
before a patient shows any sign of disease.
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Genetic fingerprinting (DNA fingerprinting), which identifies unique
sequences of DNA, is used in paternity investigations, crime scene
forensics, diagnostic microbiology, and epidemiology.
 Gene therapy cures various diseases by replacing
defective genes with normal genes.
 In xenotransplants involving recombinant DNA
technology, human genes would be inserted into animals
to produce cells, tissues, or organs for introduction into
the human body.
 Transgenic plants and animals have been genetically
altered by the inclusion of genes from other organisms.
 Agricultural uses of recombinant DNA technology include
advances in herbicide resistance, salt tolerance, freeze
resistance, and pest resistance, as well as
improvements in nutritional value and yield.
The Ethics and Safety of
Recombinant DNA Technology
 The procedures of recombinant DNA technology provide the
opportunity to transfer genes among unrelated organisms, even
among organisms in different kingdoms, but how safe and ethical
are they? "Super-broccoli," "frankenfood," "biological Russian
roulette," and" designer humans" are some of the terms opponents
use to denigrate gene therapy and transgenic agricultural products.
 Some opponents question the ethics of raising genetically altered
animals solely for creating products for human use. They contend
that this exemplifies a supremacist view-the view that humans are of
greater intrinsic value than animals.
 Other critics of transgenic crops and animals correctly state that the
long-term effects of transgenic manipulations are unknown, and that
unforeseen problems arise from every new technology and
procedure. Recombinant DNA technology may burden society with
complex and as yet unforeseen regulatory, administrative, financial,
legal, social, and environmental problems.
 Critics also argue that natural genetic transfer through
sexual reproduction and processes such as
transformation and transduction could deliver genes from
transgenic plants and animals into other organisms.
 For example, if a herbicideresistant plant cross-pollinates
with a related weed species, we might be cursed with a
weed that is nearly impossible to kill.
 Opponents further express concern that transgenic
organisms could trigger allergies or cause harmless
organisms to become pathogenic.
 Therefore, some opponents of recombinant DNA
technology desire a ban on all genetically modified
agricultural products.
 The U.S. National Academy of Sciences, the
U.S. National Research Council, and 81
research projects conducted between 1985 and
2001 by the European Union have not revealed
any risks to human health or the environment
from genetically modified agricultural products
beyond the usual uncertainties inherent in
conventional plant breeding.
 In fact, the European Union concluded in 2001
that "the use of more precise technology and the
greater regulatory scrutiny probably make them
[genetically modified foods] even safer than
conventional plants and foods."
 As the debate continues, governments continue
to impose standards on laboratories involved in
recombinant DNA technology. These are intended
to prevent the accidental release of altered
organisms or exposure of laboratory workers to
potential dangers.
 Additionally, genetic researchers often design
organisms to lack a vital gene so that they cannot
survive for long outside of a laboratory.
 Unfortunately, biologists can apply the
procedures used to create beneficial crops and
animals to create biological weapons that are
more infective and more resistant to treatment
than their natural counterparts are, though
international treaties prohibit the development of
biological weapons.
 Nevertheless, B. anthracis spores were used in
bioterrorist attacks in the United States in 2001,
though, thankfully, the strain utilized was not
genetically altered to realize its deadliest
potential.
 Emergent recombinant DNA technologies raise numerous other
ethical issues. Should people be routinely screened for diseases
that are untreatable or fatal? Who should pay for these procedures:
individuals, employers, prospective employers, insurance
companies, HMOs, government agencies? What rights do
individuals have to genetic privacy?
 If entities other than individuals pay the costs involved in genetic
screening, should those entities have access to all the genetic
information that results? Should businesses be allowed to have
patents on and make profits from any living organisms they have
genetically altered? Should governments be allowed to require
genetic screening and then force genetic manipulations on
individuals to correct so-called genetic abnormalities that some
claim are the bases of criminality, manic depression, risk-taking
behavior, and alcoholism? Should HMOs, physicians, or the
government demand genetic screening and then refuse to provide
services related to the birth or care of supposedly" defective"
children?
 We as a society will have to confront these and other ethical
considerations as the genomic revolution continues to affect
people's lives in many unpredictable ways.