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RECOMBINANT
DNA TECHNOLOGY
Presented by
Pranav C. Joshi
M. Pharm (Pharmacology)
1
Contents…
• Introduction
• Principle
• Enzymes
• Vectors
• Applications
2
Basic in rDNA technology
Hybridized DNA molecule
created by cutting
when introduced into host cell,
rDNA multiply and code for desired proteins.
3
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.
4
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
5
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
6
7
Enzymes…
• Enzymes are proteins that act as catalyst within living
cell.
Enzymes – genetically engineers toolkit.
8
9
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.
10
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
11
Mechanism of action…
12
Examples of
restriction enzymes
13
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
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.
14
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.
15
Types of vectors…
Vectors
Artificial
chromosome
vectors
Plasmid
Bacteriophages
Cosmids
16
Vectors Insert size (kb)
Plasmid <10 kb
Bacteriophage 9-15 kb
Cosmid 33-50 kb
17
Plasmid vector…
18
• 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.
19
Bacteriophage lambda vector…
• It is a virus that infects
and replicates
within bacteria.
20
21
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
22
23
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
• Medicine - gene therapy,
vaccination,
synthesizing clotting factors.
• Food Industry – larger production
of cheese.(chymosin)
• Other uses - fluorescent fishes, glowing plants etc.
25
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
26
27

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rDNA technology

  • 1. RECOMBINANT DNA TECHNOLOGY Presented by Pranav C. Joshi M. Pharm (Pharmacology) 1
  • 2. Contents… • Introduction • Principle • Enzymes • Vectors • Applications 2
  • 3. Basic in rDNA technology Hybridized DNA molecule created by cutting when introduced into host cell, rDNA multiply and code for desired proteins. 3
  • 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. 4
  • 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 5
  • 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 6
  • 7. 7
  • 8. Enzymes… • Enzymes are proteins that act as catalyst within living cell. Enzymes – genetically engineers toolkit. 8
  • 9. 9
  • 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. 10
  • 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 11
  • 13. Examples of restriction enzymes 13 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. 14
  • 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. 15
  • 17. Vectors Insert size (kb) Plasmid <10 kb Bacteriophage 9-15 kb Cosmid 33-50 kb 17
  • 18. Plasmid vector… 18 • 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.
  • 19. 19
  • 20. Bacteriophage lambda vector… • It is a virus that infects and replicates within bacteria. 20
  • 21. 21
  • 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 22
  • 23. 23
  • 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. 25
  • 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 26
  • 27. 27