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使用DNA纤维测定检测人细胞中复制后间隙的积累和修复

JoVE Journal
Genetics

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JoVE Journal Genetics

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

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使用DNA纤维测定检测人细胞中复制后间隙的积累和修复

DOI:

10.3791/63448-v

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10:32 min

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February 03, 2022

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Davi J. Martins*¹, Stephanie Tirman*², Annabel Quinet, Carlos F. M. Menck

¹Department of Microbiology, Institute of Biomedical Sciences,University of São Paulo, ²Division of Oncology, Department of Internal Medicine,Washington University in St. Louis

Chapters

00:04Introduction
01:27S1 Fiber Experimental Setup
03:51DNA Fiber Preparation by Spreading
04:58Immunostaining of DNA Fibers
06:45Image Acquisition and Analysis
08:39Results: Post-Replicative ssDNA Gaps on Ongoing Forks by S1 Fiber Assay and Post-Replicative Repair by Gap Filling
09:42Conclusion

Summary

Automatic Translation

在这里，我们描述了DNA纤维测定的两种修饰，以研究病变诱导后复制DNA的单链DNA间隙。S1纤维测定能够使用ssDNA特异性S1内切酶检测复制后间隙，而间隙填充测定允许间隙修复的可视化和定量。

Tags

Keywords: DNA Fiber Assay Post-replicative Gaps S1 Nuclease DNA Repair Replication Stress Genome Integrity UV-C Irradiation CldU Incorporation CSK Permeabilization DNA Lesion Detection

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