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BIO20 RDNA Technology
BIO20 RDNA Technology
BIO20 RDNA Technology
RECOMBINANT DNA
TECHNOLOGY
BIO20 Intro. to
Biomimetics Prof. UREAH THEA A. SEVILLA
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Agriculture
Pest-
resistant
plants
Frost-resistant
plants
Medicine
recombinant blood
clotting factor Recombinant
recombinant human
vaccine
insulin
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Environment
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DNA Cloning
Amplification of rDNA using host organism’s
replication mechanism
DNA Cloning
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DNA Cloning
Procedure in Recombination
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Recombination
Gene of
interest
+
Enzymes involved
RESTRICTION ENDONUCLEASES / RESTRICTION
ENZYMES
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Restriction Endonucleases
Blunt ends – when both strands of the
molecule are cut at the same position that
leaves no unpaired nucleotides.
Staggered or “Sticky” ends – when each
strand of the molecules is cut at different
position so that one strand (either 5’ or 3’)
overhangs by several nucleotides, these single-
stranded ends can be spontaneously base pair
with each other –that is, they are sticky or
cohesive.
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Restriction Enzymes
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Enzymes involved
DNA LIGASE
DNA Ligase
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Restriction enzyme
recognition sequence
5’ GAAT TC 3’
3’ CT TAAG 5’
Restriction enzyme
cuts DNA
Sticky ends
5’ G AA T T C 3’
3’ CT TAA G 5’
5’ G A AT T C G A A T T C 3’
3’ C T T AA G C T T AA G 5’
Recombinant DNA
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Recombinant DNA
The enzyme that cuts both the vector and DNA of
interest internally is called
a) DNA ligase
b) DNA nuclease
c) Restriction endonuclease
d) DNA exonuclease
B.
C.
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Cloning Vectors
Cell Transformation
introduction of new DNAs into organisms or to a host for gene cloning.
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Electroporation
Cell Transformation
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Cell Transformation
Microinjection
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rDNA
Technology
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DNA Techniques
•Polymerase Chain Reaction
•Gel Electrophoresis
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PCR Components
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Steps in PCR
DENATURATION
Double-stranded DNA sample is unwound by heating to 92-94ºC. The
single strands will serve as templates for reaction.
ELONGATION
DNA molecules in the mixture become doubled.
The mixture is heated again which cause unwinding of the double-
strands and the cycle repeats producing new DNA strands.
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Gel Electrophoresis
A technique used to separate macromolecules
(nucleic acids or proteins) on the basis of size,
electrical charge, and other physical properties. It
sorts a mixture of DNA molecules into bands, each
consisting of DNA molecules of the same length.
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Southern Blot
Hybridization
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DNA Fingerprinting
Amplified DNAs
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DNA Fingerprinting
DNA Fingerprinting
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DNA Fingerprinting
Based on the results
shown for a single locus
probe, which of the
children could not be the
progeny of these
parents?
DNA Fingerprinting
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References:
Voet, Voet and Pratt, Principles of Biochemistry 3rd
ed. Wiley Publication (2008).
Lodish H, Berk A, Zipursky SL, et al. Molecular Cell
Biology, 5th edition. New York: W. H. Freeman;
(2004).
Barnum, S., Biotechnology, Wadsworth – Thomson
Publishing Co. (1998).
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