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Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons
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Biologie
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Journal JoVE Biologie
Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

DOI:
10.3791/4168-v

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15:01 min

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August 06, 2012

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, ,
1Department of Pharmacology & Chemical Biology,University of Pittsburgh School of Medicine, 2Hillman Cancer Center,University of Pittsburgh Cancer Institute, 3Department of Experimental Therapy,The Netherlands Cancer Institute, 4Department of Human Genetics,University of Pittsburgh School of Public Health

Chapitres

  • 00:05Titre
  • 02:53Molecular Beacon Design
  • 04:31Nuclear Lysate Preparation
  • 08:31Molecular Beacon Assay
  • 11:51Representative Analysis of Tumor Cell Lysates
  • 13:54Conclusion

Summary

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We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

Tags

Quantitative AnalysisReal-time MeasurementBase Excision RepairDNA GlycosylaseAP EndonucleaseMolecular BeaconsSubstrateDeoxyoligonucleotideCarboxyfluorescein (6-FAM)DabcylStem-loop StructureNucleotidesBase PairsFörster Resonance Energy Transfer (FRET)FluorescenceDNA RepairFluorescence ValuesReal-time AssessmentPCR Instruments

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