Detail description of rDNA technology

1. Recombinant DNA
Technology
Prepared by Arfa Shazad
Dated 12.8.2020

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Basics
1
Recombination also known
as gene cloning i.e.
manipulation of gene
makeup
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Joining together of DNA
molecules from two different
species named as
recombinant DNA
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The DNA from a donor organism
is extracted, enzymatically cleaved
and joined to another DNA entity
to form a new, recombined DNA
molecule (cloning vector– insert
DNA construct, or DNA
construct)
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This cloning vector–insert DNA construct is
transferred into and maintained within a host
cell.The protein product encoded by the
cloned DNA sequence is produced in the
host cell
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Recombinant DNA technology
would not be possible without the
availability of enzymes.
 Endonuclease
 Exonucleases
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discovery of restriction
endonucleases byWernerArber,
Daniel Nathans and Hamilton Smith

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Cloning vectors
1.
They accept different sizes of
source DNA fragments known
as insert DNA to carry them
as recombinant.
2.
 They must have circular DNA.
 They must have origin of replication
 They must contain selectable marker
gene sequences.
 They must have multiple cloning site
3.
Plasmids are the most studied type
of cloning vector.They have
features that are required for a
high-quality cloning vector
4.
• A choice of unique (single) restriction
endonuclease recognition sites into
which the insert DNA can be cloned.
• One or more selectable genetic markers
for identifying recipient cells that carry
the cloning vector–insert DNA
construct.
5.
Closed circular plasmid vector DNA
molecules are cut with a restriction
enzyme that lies within either of the
antibiotic resistance genes and cleaves
the plasmid DNA only once to create
single, linear, sticky-ended DNA
molecules
6.
Discovered in the 1980s. the work of
the researchers F. Bolivar and R.
Rodriguez. Plasmid pBR322 contains
4,361 bp.

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Protocol of Recombinant DNA Technology
Gene
Isolation
Restriction
Digestion.
Ligation
Transformation
Selection and
screening of
genes.

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Isolation
• A gene or any DNA fragment to be used in cloning is
isolated.
• Isolated done from both the source DNA containing
the gene of interest and the vector i.e. bacterial
plasmids that are self replicating circular DNA.
• The isolation can be done by different methods such as
PCR based on sequence specific primers, amplification
of DNA fragment from extracted genomic DNA and
via genomic libraries.
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• bacterial enzymes that cut DNA molecules internally at
specific base pair sequences thus making molecular cloning
feasible
• Major role plays by enzyme type II restriction
endonucleases that are of many different types.
• EcoRI.
From the bacterium Escherichia coli, EcoRI is a homodimeric
protein (i.e. it is made up of two identical proteins) that binds
to a DNA region with a specific recognition/ binding site. The
EcoRI recognition sequence consists of 6 base pairs (bp) and is
cut between the guanine and adenine residues on each strand.
EcoRI carried out symmetrical staggered cleavage forming
staggered or sticky ends by specifically cleaving the
internucleotide bond between the oxygen of the 3′ carbon of
the sugar of one nucleotide (3-OH overhangs or 3` protruding
end) and the phosphate group attached to the 5′ carbon of the
sugar of the adjacent nucleotide (5` phosphate overhang or 5`
protruding end). Thus, EcoRI cleaves DNA and produces two
single-stranded, complementary cut ends, each with extensions
of 4 nucleotides, known as sticky ends
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Restriction Digestion.

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• HindIII:
• HindIII is another type II restriction endonucleases
isolated from Haemophilus influenzae that cleaves
a DNA molecule are within the recognition sites.
This restriction enzyme cut the backbones of both
strands within a recognition site to produce blunt-
ended (flush-ended) DNA molecules. The lengths
of the recognition sites for this enzyme can be four,
five, six, eight, or more nucleotide pairs.
• FokI, XhoI, PaeR7I, BamHI and Sau3AI etc. from
different organisms are have different restriction
sites within source DNA.
• Similar to the restriction digestion of source DNA
molecule the purified, closed circular plasmid
vector DNA molecules are cut with a restriction
enzyme. These linear molecules are combined with
prepared target DNA from a source organism in
next step called ligation.
Continued…
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Ligation
• When two different DNA samples (a source/target DNA and
vector DNA) are digested with the same restriction
endonuclease that produces sticky ends are then mixed
together, new DNA combinations can be formed as a result of
base pairing between them.
• The enzyme DNA ligase, obtained from bacteriophage T4 is
used. This enzyme catalyzes the formation of phosphodiester
bonds at the ends of DNA strands that are already held
together by the base pairing of two extensions (sticky ends).
The DNA mixture is treated with T4 DNA ligase in the
presence of ATP. To reduce the amount of unwanted ligation
product, the cleaved plasmid DNA preparation is treated with
the enzyme alkaline phosphatase to remove the 5′ phosphate
groups from the linearized plasmid DNA. As a consequence,
T4 DNA ligase cannot join the ends of the dephosphorylated
linear plasmid DNA. However, the two phosphodiester bonds
that are formed by T4 DNA ligase after the ligation and
circularization of alkaline phosphatase-treated plasmid DNA
with restriction endonuclease-digested source DNA, which
provides the phosphate groups, are sufficient to hold the two
molecules together, despite the presence of two nicks. After
transformation, these nicks are sealed by the host cell DNA
ligase system. 8

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• The uptake of the cloned plasmid DNA by a bacterial cell,
usually E. coli is called transformation, and a cell that is capable
of taking up DNA is said to be competent.
• In different bacterial species, usually when cell density is high
or starvation is impending, a set of proteins is produced that
facilitates the uptake of DNA molecules.
• the binding of double-stranded DNA to components of the cell
wall.
• Entry of the DNA into an inner compartment (periplasm), where
it is protected from enzymes that degrade nucleic acids
(nucleases).
• Transmission of one strand into the cytoplasm while the other
one is degraded.
• Maintenance of DNA in the cytoplasm after the second strand
is synthesized.
• The recombinant plasmid-cloned DNA construct will replicate
within bacterial cell using the enzymatic machinery of bacterial
host cell. A transformed cell with recombinant DNA will then
divide and multiply to form a colony with millions of cells,
each of which carries a recombinant DNA.
Transformation
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Selection:
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• It is necessary to identify, those cells that contain plasmids with
cloned DNA. Bacterial colonies containing a recombinant plasmid-
cloned DNA construct are selected on the basis of markers present in
the vector.
• the cells are incubated in medium without antibiotics to allow the
antibiotic resistance genes to be expressed, and then the
transformation mixture is plated onto medium that contains the
antibiotic ampicillin. Cells that carry plasmid with or without insert
DNA can grow under these conditions because the “Amp” gene on
vector plasmid is intact. The nontransformed cells are sensitive to
ampicillin. Therefore, cells with these plasmid–cloned DNA
constructs are resistant to ampicillin and sensitive to tetracycline (i.e.
the source DNA site in plasmid is within the “Tet” gene, so the
insertion of DNA into this gene disrupts the coding sequence and
tetracycline resistance is lost).
• Cells that grow on the ampicillin-containing medium are transferred
to a tetracycline-containing medium. The relative positions of the cells
transferred to the tetracycline–agar plate are the same. Cells that form
colonies on the tetracycline–agar plates carry recircularized plasmid
without insert DNA, because these cells are resistant to both
ampicillin and tetracycline. Those cells that do not grow on the
tetracycline–agar plates, however, are sensitive to tetracycline and
carry recombinant plasmid–cloned DNA constructs.

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Screening
• For the determination of plasmid-cloned DNA carries
specific genes coding for protein, the specific clone that
carries the target DNA sequence must be identified,
isolated, and characterized and this can be done via
screening of recombinants selected via replica plate.
• DNA hybridization with a labeled DNA:
• In general, for a DNA hybridization assay, the target
DNA is denatured and the single strands are irreversibly
bound to a matrix, e.g., nitrocellulose or nylon. Then, the
single strands of a DNA probe, which are labeled with
either a radioisotope or another tagging system, are
incubated with the bound DNA sample. If the sequence
of nucleotides in the DNA probe is complementary to a
nucleotide sequence in the sample, then base pairing, i.e.,
hybridization, occurs. The hybridization can be detected
by autoradiography or other visualization procedures
depending on the nature of the probe label. If the
nucleotide sequence of the probe does not base pair with
a DNA sequence in the sample, then no hybridization
occurs and the assay gives a negative result.

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• Immunological screening for the protein product
• If a cloned DNA sequence is transcribed and translated,
the presence of the protein, or even part of it, can be
determined by an immunological assay. All the clones of
the library are grown on several master plates. A sample
of each colony is transferred to a known position on a
matrix, where the cells are lysed and the released proteins
are attached to the matrix. The matrix with the bound
proteins is treated with primary antibody that specifically
binds to the protein encoded by the target gene. After that
the matrix is treated with a secondary antibody that is
specific for the primary antibody. In many assay systems,
the secondary antibody has an enzyme, such as alkaline
phosphatase, attached to it. After the matrix is washed, a
colorless substrate is added. If the secondary antibody has
bound to the primary antibody, the colorless substrate is
hydrolyzed by the attached enzyme and produces a
colored compound that accumulates at the site of the
reaction hence, gives a positive result for the presence of
protein
Continued…
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Pros and cons
• Recombinant DNA is widely used in biotechnology
and medicine research with its importance to most
recent works in biomedical sciences.
• Recombinant DNA technology improved disease
resistant livestock.
• In agriculture, better crops with improved shelf life,
nutritional values and resistance to parasites has
been developed.
• Recombinant DNA technology made possible to
treat pre-existing conditions like cancer and many
genetic disorders via gene therapy.
• Recombinant DNA technology is also now a days,
used to treat HIV.
• Many additional practical applications of
recombinant DNA are found in industry, food
production, human and veterinary medicine
Cons
• The recombinant organisms are population of
clones, vulnerable in exact same ways. A single
disease or pest can wipe out the entire population
quickly.
• Development of many drug resistant bacteria and
viruses that have resistance against many antibiotics
use to treat severe infections
• There are many ethical dilemmas over using
recombinant DNA technology for human treatment.
• As men tries to play GOD, germ line treatment,
instead of treating diseases may turn into a method
of creating customized babies which will raise
serious ethical and medical issues.
• Genetically modified crops may cause health
hazards like Bt. Brinjal which have 15 % less
calories and many antiparasitic toxins in it
Pros

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References
• Bibliographic reference:
• Bernard R. Glick., Jack J. Pasternak., Cheryl L. Patten. Molecular Biotechnology
Principles and Applications of Recombinant DNA. 4th Edition. Page 49-90.
• Chaudhary. Keya. 2013. Recombinant DNA Technology. Delhi. TERA. Page 1-26. Ijaz.
Iqra., Huq. Imran. 2019. Recombinant DNA Technology. Page 2-7.
• Webiogaphic reference:
• https://www.pdfdrive.com/campbell-biology-10th-edition-d127216552.html
• https://microbenotes.com/recombinant-dna-technology-steps-applications-
andlimitations/#:~:text=Recombinant%20DNA%20technology%20refers%20to,medic
ine%2C%20agriculture%2C%20and%20industry.
• https://www.britannica.com/science/recombinant-DNA-technology
References