recombinant dna technology is most important molecular technique..

1. RECOMBINANT
DNA TECHNOLOGY
PRESENTER : SARANYA . S
MODERATOR : DR.V.BALASUBRAMANIYAN
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2. DNA CLONING
 DNA cloning protocol
 Plasmid cloning
 Bacteriophage λ
cloning
 Yeast artificial
chromosome
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Recombinant DNA libraries
Genomic library
cDNA library
Summary

3. Molecular cloning / DNA cloning
Molecular
cloning refers to
process of
making multiple
copies of DNA
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Step 1 : Fragmentation – breaking apart a
strand of DNA
Step 2 : ligation – gluing together pieces of
DNA in a desired sequence
Step 3 : transfection – inserting the newly
formed DNA in to cells
Step 4: screening / selection – selecting
out the cells that were successfully
transfected with the new DNA

4. DNA Cloning Protocol
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PLASMID CLONING STRATEGY

5. Recombinant DNA Cloning Procedure 5
Foreign
DNA
rDNA Host cell Desired
products
Antibiotics
Antibodies
Blood
factors
Hormones
Enzymes
Vaccine
Fine
chemicals
Plasmid
DNA

6. STEP 1. RE DIGESTION OF DNA
SAMPLE
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7. STEP 2. RE DIGESTION OF PLASMID
DNA
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8. STEP 3. LIGATION OF DNA SAMPLE
AND PLASMID DNA
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9. STEP 4. TRANSFORMATION OF
LIGATION PRODUCTS
 The process of transferring exogenous DNA into cells is called
“transformation”
 Chemical method utilizing CaCl2 and heat shock to promote
DNA entry into cells.
 Electroporation based on a short pulse of electric charge to
facilitate DNA uptake.
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10. CHEMICAL TRANSFORMATION
WITH CALCIUM CHLORIDE
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11. TRANSFORMATION BY
ELECTROPORATION
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12. STEP 5. GROWTH ON AGAR PLATES 12

13. STEP 5 : Blue-white Screening
 Blue colonies represent Ampicillin-resistant bacteria
that contain vector and express a functional alpha
fragment from an intact LacZ alpha coding
sequence
 White colonies represent Ampicillin-resistant
bacteria that contain Insert and do not produce
LacZ alpha fragment
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14. β-Galactosidase Activity Can Be Used As An
Indicator Of The Presence Of A Foreign DNA Insert
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15. OVERVIEW 15

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17. Amplification and purification of
recombinant plasmid DNA
 Positive colony containing recombinant plasmid DNA is transferred
aseptically to liquid growth medium, the cells will continue to
multiply exponentially
 Within a day or two, a culture containing trillions of identical cells
can be harvested
 Plasmid DNA can be purified from crude cell lysates by
chromatography (using silica gel or anion exchange resins)
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18. Amplification and purification of
recombinant plasmid DNA
 The purified plasmid DNA can then
be eluted and recovered by
ethanol precipitation in the
presence of monovalent cations
 Ethanol precipitation of plasmid
DNA from aqueous solutions yields
a clear pellet that can be easily
dissolved in an appropriate
buffered solution
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19. Bacteriophage Lambda (λ)
Cohesive Sites
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20. Bacteriophage Lambda (λ) Cohesive
Sites
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21. Yeast Artificial
Chromosome
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22. Red white screening
 SUP4 encodes a tRNA that suppresses the Ade2-1 UAA
mutation
 ADE1 and ADE2 encode enzymes involved in the
synthesis of adenine (phosphoribosyl amino-imidazole-
succinocarbozamide synthetase and
phosphoribosylamino-imidazole carboxylase).
 In the absence of these critical enzymes, Ade2-1
mutant cells produce a red pigment
 But Ade2-1 mutant cells expressing SUP4 are white
because the Ade2-1 mutation is suppressed
 Red colonies contain recombinant YAC vector DNA
 White colonies contain non recombinant YAC vector
DNA.
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23. Recombinant DNA Libraries
1. Genomic library
2. cDNA library
3. Chromosomal library
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24. Creation of a Genomic DNA library
using the phage-λ vector EMBL3A
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# High-molecular-weight
genomic DNA is partially digested
with Sau3AI
# The vector is digested with
BamHI and EcoRI, which cut within
the polylinker sites
# The vector arms are then ligated
with the partially digested
genomic DNA

25. # The only package able
molecules are recombinant
phages. These are obtained as
plaques on a P2 lysogen of sup+ E.
coli
# The Spi− selection ensures
recovery of phage lacking red
and gam genes
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26. cDNA Libraries
 A cell’s mRNA molecules can be
copied to make complementary DNA
strands (cDNA)
 The cDNA derives only from mature
mRNA. Introns are not present
 The poly(A) tail at the 3’ end of the
mRNA is useful for:
1. Isolating mRNA from cell lysates by
passage over an oligo(dT) column
2. Priming the synthesis of cDNA, by
providing a known 5’ sequence
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27. An Early cDNA Cloning Strategy, Involving Hairpin
Primed Second-strand DNA Synthesis And Homopolymer
Tailing To Insert The cDNA Into The Vector
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28. An Early cDNA Cloning Strategy, Involving Hairpin Primed
Second-strand DNA Synthesis And Homopolymer Tailing To
Insert The cDNA Into The Vector
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29. Improved Method For cDNA Cloning. The First Strand
Is Tailed With Oligo(dc) Allowing The Second Strand
To Be Initiated Using An Oligo(dg) Primer
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Oligo-dG,
Reverse
transcriptase
+ 4 dNTPs
Insert in to vector
by either
homopolymer
tailing or linkers

30. Summary
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Gene cloning
vectors
Host cell
Preparation of
cDNA library
# Orgin of replication
# Suitable marker
# Single restriction
# Expression signal
Plasmids Phage
Prokaryote Eukaryote
# transformation
#transfection
# physical method
#chemical method
Genomic
library
cDNA
library
Can be cloned for
further use
Inserted in
to host cell

31. References
 Sambrook, J., Russell, D.W. (2001) Molecular Cloning: a Laboratory
Manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York
 Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith,
J.A., Struhl, K., eds (2002) Short Protocols in Molecular Biology, 5th edn.
John Wiley & Sons, New York
 Lodge j., lund P., Gene cloning principles and application 2nd edition
 Primrose SB., Twyman RM., Principles of gene manipulation and genomics
 Brown TA., Gene cloning and DNA analysis., 6th edition
 Recombinant DNA technology and DNA cloning., chapter 8
 Molecular cloning technical guide
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32. THANK YOU
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