Recombinant DNA Technology (Key-Notes)

Nimish Shirish Khandekar
Roll no. 45, Div-1
T.Y. B. Pharm,
ADCBP, Ashta
RECOMBINANT DNA (rDNA)
TECHNOLOGY

Introduction
Recombinant DNA Technology
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 Also known as cloning or molecular cloning.
 Recombinant DNA is a technology in
which molecules of DNA from two
different species that are inserted into a host
organism to produce new genetic combinations
that are of value to science, medicine, agriculture,
and industry.
 In short, the process of rDNA is:

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From
Donor
• Cloned DNA, insert DNA,
target DNA or foreign DNA
Extration
• Enzymatically cleavage
(cut/digested)
Ligated
• Joined to another DNA
molecule

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Transformation
• DNA construct
transferred into and
maintained in host cell
Selection
• The host cells which take
or do not take the DNA
construct are identified.

History
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YEAR EVENT
1953 Discovery of DNA Structure by Watson & Crick (Double helix model)
1967 Isolation of DNA Ligase
1970 Isolation REase
1972 Generation of DNA Technology by Paul Berg
1973 Production of First Plasmid Vector which was capable of being
replicated within a bacterial host.

Restriction Endonuclease (RE)
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 rDNA does not exist without availability of
enzymes that recognize specific double stranded
DNA and cleave the DNA in both strands at these
sequences.
 Nuclease which internally cleave →
endonucleases
 Nuclease which externally cleave →
exonucleases

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 More than 3,700 type II restriction endonucleases
with about 250 different recognition sites have been
isolated from various bacteria.
 Target sequence and cloning vector → cut into
discrete and reproducible fragments.
 REs are bacterial enzymes.
 Formally designated as type II endonucleases.
 First → type II endonucleases → from Escherichia
coli → named as EcoRI
 EcoRI → Homodimeric protein

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 EcoRI recognition sequence consist → 6 base pairs
→ cut between guanine and adenine.
 EcoRI cleave → internucleotide bond → oxygen of
the 3’ carbon of the sugar of one nucleotide & the
phosphate group attached to the 5’ carbon of the
sugar of the adjacent nucleotide.
 Symmetrical cleavage of DNA by EcoRI → produce
two single stranded complementary cuts with
extension of 4 nucleotides known as sticky ends.

Plasmid Cloning Vectors
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 Plasmids have the basic attributes to make them
potential vectors for carrying closed DNA
 Plasmids → self replicating, double stranded,
circular DNA molecules → maintained in bacteria
as independent extrachromosomal entities.
 Plasmid → carry genes that are advantageous
under particular condition

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Number of
Copies
High copy
number
plasmids
Low copy
number
plasmids
Specificity of
their origin of
replication
Replicate in
only one
species
Replicate in
number of
bacterial
species
Plasmid sequence → functions as
an origin of DNA replication.

Plasmid Cloning Vector pBR322
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 pBR322 → carries two antibiotic resistance genes
→ amphicillin and tetracycin resistant.
 Has unique recognition sites → BmHI, Hind III &
Sa II.
 Stepwise procedure:

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pBR322 molecules cut with
restriction enzyme
Sticky ended DNA molecules
are formed
Combined with prepared target
DNA from source organism

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Treated with T4 DNA ligase in
the presence of ATP
Number of different ligated
combinations are produced,
including original circular plasmid
DNA
Treatment with alkaline
phosphate to remove
unwanted ligation mainly 5’
groups.

Transformation & Selection
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 Uptake of the cloned plasmid DNA by a bacterial
cell, usually E.Coli.
 Process of introducing purified DNA into a
bacterial cell is called transformation.
 Cell capable of taking DNA is said to be
competent.

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 Phenomenon of transformation:

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1. Binding of double stranded DNA to component of
cell wall.
2. Entry of DNA into inner compartment (periplasm),
where it is protected from enzymes that degrade
nucleic acids (nucleases).
3. Transmission of one strand into cytoplasm and
other one is degraded.
4. If DNA is linear, then integration into the host
chromosomes.

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 Competence can be induced in E.Coli by → cold,
calcium cloride → enchance acquisition of DNA by
the cell.
 Success of DNA transformation depends upon:
Success
Transformation
frequency
Transformation
efficiency

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 After transformation → it is necessary to identify as
easily possible, those cells that contain plasmids
with cloned DNA.
E.g: pBR322 → ampicillin & tetracyclin medium

Overview
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Recombinant DNA Technology (Key-Notes)

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