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DNA REPAIR MECHANISMS
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Photoreactivation
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b. Nucleotide Excision
Repair
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Enzymes involved in Neutralization
of Free Radicals
1. Superoxide dismutase (SOD) 2O 2• +
2H+ → O 2 + H2O22. Catalase
2H2O2 → O 2 + H2O
3. Glutathione Peroxidase 2OH• + 2GSH → 2H 2O + GSSG
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Vitamins
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Recombination Repair
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Demethylase
SH + G-CH 3 G+S-CH 3
Sulfitase
HSO 3- SO 4-2
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Nucleic Acid Biotechnology
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What is Recombinant DNA?
DNA molecule containing DNA fragments (fromdifferent sources) that are not naturally found.
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CHIMAERA A mythical creature with the head of a lion, the body of a goat, and the
tail of a serpent
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Gene Transfer
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Restriction Endonucleases?!
• Restriction endonucleasesrecognize and cut specificsequences in the double-stranded DNA.
• This specific base sequence isknown as the " recognitionsequence "
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“STICKY” Ends
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VECTORS
Vectors - gene carrier;also known ascloning vehicle
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VECTOR CHARACTERISTICS
It must be small , and unlikely todegrade during purificationIt has markers (such as antibioticresistance) that can indicate (inculture) whether transformationhas been successfulIt must have an origin ofreplication so that DNA can bereplicated by the host cell'smachineryIt has have several uniquerestriction sites so that the vectorDNA will be cut only in the desiredlocation, and that several suchlocations will be available forinsertion of foreign DNA
pbr3224361bp
(SMORES!)
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GeneticEngineering :Recombinant DNATechnology andGene Cloning
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Recombinant DNATechnology
Steps:
1. Isolation of DNA
2. Cleaving of DNA withrestriction enzymes
3. Formation of recombinantDNA
4. Introduce recombinantDNA to host cells
5. Propagation of clones
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Genetic Engineering
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Polymerase Chain Reaction
• It is first developed by KaryMullis in 1983
• A more convenient methodof amplifying specific DNAfragment.
• Allows investigation ofminute samples of DNA
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PCR: in vitro repliaction
• It allows you to carryout in vitro multiple
replications of targetDNA• Can produce millions of
copies DNA fragments
from a single template• PCR is very specific
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Components of PCR tubes• DNA template , which contains the
region of the DNA fragment to beamplified
• Two primers , which determine thebeginning and end of the region to beamplified
• DNA polymerase , which copies theregion to be amplified
• Nucleotides (dNTPs) , from which theDNA polymerase builds the new DNA
• Buffer , which provides a suitablechemical environment for the DNApolymerase
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Instrument for PCR• The PCR reaction is carried out in a thermocycler, an
instrument that automatically controls and alternates thetemperatures for programmed periods of time for the
appropriate number of PCR cycles (usually bet 30 - 40cycles)
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Three Steps in PCR
There are three majorsteps in a PCR, all carriedout in the same test tubebut at differenttemperatures:
1. Denaturation at >90°C2. Annealing at 54°C3. Extension at 72°C
1. DENATURATION
2. ANNEALING
3. EXTENSION
PCR
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Exponential Amplication
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Paternity Testing/Forensics
http://en.wikipedia.org/wiki/Image:Pcr_fingerprint.png