in this topic we will discuss the following contents Introduction. Cloning. Discovery. Molecules need in rDNA technology. Enzymes. Vectors. Procedure or steps involves in rDNA technology. Application of rDNA technology. Advantages and disadvantages.

1. RECOMBINANT DNA
TECHNOLOGY
Muhammad Kamil Khan
1st Semister M.phil Microbiology
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2. Contents
• Introduction.
• Cloning.
• Discovery.
• Molecules need in rDNA technology.
• Enzymes.
• Vectors.
• Procedure or steps involves in rDNA technology.
• Application of rDNA technology.
• Advantages and disadvantages.
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3. Introduction
• The technique through which we join the DNA molecules
together from two different species in lab.
• Then these joining DNA molecules are inserted into a
host organism to produce new genetic combinations
that are of valuable to science, medicine, agriculture,
and industry.
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4. Cloning
• In recombinant DNA technology we usually perform the
cloning process.
• In biotechnology cloning is refers to the process of creating
clones of organisms or copies of cells or DNA fragments.
• In cloning we usually clone the DNA or genes.
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5. Discovery
• Paul Berg generated rDNA technology in 1972
• Cohen & Boyer in 1973 produced first plasmid
vector capable of being replicated within a
bacterial host
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6. Molecules need in cloning
• Target DNA having genes of interest
• Enzymes
• Vectors
• Host cell i.e E. coli
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7. Enzymes used for rDNA technology
• DNA ligase • Bind to DNA molecules.
• Type II restriction endonuclease • Cleaves DNA at
specific sites.
• Polynycleotide Kinase • Adds a phosephate to the 5'-OH
end of a polynucleotide.
• Terminal transferase • Adds homopolymer tails to the 3'-
OH ends.
• Alkaline phosphatase • Removes terminal phosphates.
• Polymerase enzymes that add nucleotides.
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8. Vectors
• Vectors are the vehicles or delivery system that can
deliver or transfer our target DNA having gene of
interest in to the host cell.
• Types of vectors
I. Expression vectors.
II. Cloning vectors.
1. Plasmid.
2. Cosmid.
3. Phage vector.
4. Bacterial artificial chromosomes (BAC).
5. Yeast artificial chromosomes (YAC). 8

9. Procedure or steps need in rDNA
• Isolating of DNA.
• Cutting of DNA.
• Joining of DNA.
• Transferring of the rDNA in to host.
• Amplification of DNA.
• Screening.
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11. 1. Isolation
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12. 2. Cutting of DNA molecules
• The same type 2 R.E are used to cut DNA
molecules having gene of interest as well as
vectors i.e EcoR1 or BamH1 etc restriction
endonuclease to produce sticky ends
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13. 3. Joining of DNA molecules
• Once the target DNA is uptake by the vectors
then the gaps is still remaining which is sealed
by ligase enzymes
• Finally the vector and target DNA are joined
and we called that rDNA vector
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14. 4. Transferring of the rDNA into the host
• The recombinant dna vector should be transfer into the
host cell i.e E.coli
• It can be possible by making the cell competent
1. Treat the host cell by cacl2 or Mgcl2.
2. Electric shock to host cell.
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15. 5. Amplification of rDNA vector
• Once the host cell uptake the rDNA vector then we allow
the cell inside the media to multiply and divided
• Thus our target DNA is also replicated because
transcription, translation and replication is always going
on inside host cell and we get cDNA or gene library
• Finally the target amplified.
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16. 6. Screening
• Now to identify that which cell have our rDNA vector
we perform screening technique.
• It may be
• Blue white screening.
• Using selectable marker genes.
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17. Application of rDNA technology
• Agriculture: growing crops of your choice (GM
food),pesticide resistant crops, fruits with attractive
colors, all being grown in artificial conditions.
• Pharmacology: artificial insulin production, drug
delivery to target sites.
• Medicine: gene therapy, antiviral therapy, vaccination,
synthesizing clotting factors.
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18. Advantages
• To isolate and characterize a gene.
• To make desired alterations in one or more isolated genes.
• To return altered genes to living cells.
• Artificially synthesize new gene.
• Understanding the hereditary diseases and their cure
• Improving human genome.
• Improve fertilization inside woman
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19. Disadvantages
• Expensive method
• Sometime the vector self anneal with each other
• Difficult process
• Effect on health
• Can take long time
• Can cause mutation
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