repair mechanisms & biotechnology

8/17/2019 Repair Mechanisms & Biotechnology

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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

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