recombinant DNA, various enzymes, types of vectors, examples

1. RECOMBINANT
DNA TECHNOLOGY
Presented by
Pranav C. Joshi
M. Pharm (Pharmacology)
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2. Contents…
• Introduction
• Principle
• Enzymes
• Vectors
• Applications
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3. Basic in rDNA technology
Hybridized DNA molecule
created by cutting
when introduced into host cell,
rDNA multiply and code for desired proteins.
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4. Introduction
• DNA:DNA is Deoxyribose nucleic acid.
It is made up of a base consisting of sugar,
phosphate and one nitrogen base.
These nitrogen bases are adenine(A)
guanine(G) cytosine(C) and thymine(T).
The nitrogen bases are grouped as…
• PURINES : Adenine and guanine
• PYRAMIDINES: Cytosine and thymine.
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5. What is rDNA …?
• Recombinant DNA –
A DNA is form artificially by combining constituents
from different organism.
• Genetically engineered DNA prepared by
transplanting from one species into cells of host
organisms of different species.
• rDNA also called Chimeric DNA or Hybrid DNA
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6. Principle…
 Generation of DNA fragments and selection of the
desired piece of DNA
 Insertion of the selected DNA into a cloning vector
(ex: plasmid) to create a cloning vector or a chimeric
DNA.
 Introduction of the recombinant vectors into the host
cells(bacteria).
 Multiplication and selection of clones containing the
recombinant molecules.
 Expression of the gene to produce the desired product
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8. Enzymes…
• Enzymes are proteins that act as catalyst within living
cell.
Enzymes – genetically engineers toolkit.
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10. Restriction enzymes…
• These are the bacterial enzymes that can cut / split
DNA at specific sites.
Ex- E.coli R1, Hind 3.
• Enzyme that cleave the sugar-phosphate backbone of
DNA stands.
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11. History –
Restriction endonuclease
• Prior to 1968 the existence of restriction enzymes was
unknown.
• In 1968, Matthew Meselson and Robert Yuan
reported that they had identified an enzyme.
• They conclude that this enzymes could be
responsible to restriction they coined
Restriction endonuclease
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12. Mechanism of action…
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13. Examples of
restriction enzymes
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Enzyme Microorganism Recognition
Sequence
Alu I Arthrobacter luteus AG|CT
Apa I Acetobacter
pasteurianus
GGGCC|C
Bgl II Bacillus globigii A|GATCT
Cla I Caryophanon latum
L
AT|CGAT

14. Vectors…
• Vector is a DNA molecule used as a vehicle to
artificially carry foreign genetic material into another
cell.
• Purpose - transfers genetic information to another
cell is typically to isolate, multiply, or express the
insert in the target cell.
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15. Requirements for a cloning vector
• Should be capable of replicating in host cell.
• Should have convenient RE sites for inserting DNA of
interest.
• Should have a selectable marker to indicate which host
cells received recombinant DNA molecule.
• Should be small and easy to isolate.
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16. Types of vectors…
Vectors
Artificial
chromosome
vectors
Plasmid
Bacteriophages
Cosmids
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17. Vectors Insert size (kb)
Plasmid <10 kb
Bacteriophage 9-15 kb
Cosmid 33-50 kb
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18. Plasmid vector…
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• History - The word 'plasmid' was first coined by
Joshua Lederberg in 1952. He used it to describe 'any
extrachromosomal hereditary element’.
• Characteristics – small circular DNA
• Host – bacteria nothing but E.coli.
 pUC18 -
in host it replicates 500 copies per cell and
contains several RE sites.

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20. Bacteriophage lambda vector…
• It is a virus that infects
and replicates
within bacteria.
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22. Cosmid vector…
• Cosmids are hybrids of phages and plasmids.
• They can carry DNA fragments up to 45 kb.
• They can replicate like plasmids but can be packaged
like phage lambda.
• Cosmids can be used to build genomic libraries.
• Ex- pHV79
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24. Applications…
• Medical Research - Diagnostic techniques
for HIV.
• Pharmacology - Blood clotting factors (haemophilia),
artificial insulin production,
Hepatitis B.
• Agriculture technology –
growing crops of your choice,
pesticide resistant crops,
fruits with attractive colours,
all being grown in artificial conditions. 24

25. • Medicine - gene therapy,
vaccination,
synthesizing clotting factors.
• Food Industry – larger production
of cheese.(chymosin)
• Other uses - fluorescent fishes, glowing plants etc.
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26. References…
• Biochemistry, U. Satyanarayna, U. Chakrapani, third
edition, Books and allied publications, pg.no 578-
615.
• Molecular biology, David freifelder, second edition,
Narosa publishing house, pg.no 705-714.
• http://www.infoplease.com/cig/biology/dna-
technology-applications.html
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